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https://openalex.org/W3007191987
https://pure.eur.nl/files/48294669/Repub_125109_O-A.pdf
English
null
Comparison of sequencing methods and data processing pipelines for whole genome sequencing and minority single nucleotide variant (mSNV) analysis during an influenza A/H5N8 outbreak
PloS one
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RESEARCH ARTICLE Editor: Ming Zhang, University of Georgia, UNITED STATES Received: January 25, 2019 Accepted: December 30, 2019 Published: February 20, 2020 Copyright: © 2020 Poen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data files are available from the ENA database (project accession number(s) PRJEB9846, PRJEB12582, PRJEB9687). Funding: This work was supported by the European Union’s Horizon 2020 research and innovation program under grant agreements No 643476 (COMPARE), and No 653316 (EVAg). This work was also supported by the NIAID/NIH grant Comparison of sequencing methods and data processing pipelines for whole genome sequencing and minority single nucleotide variant (mSNV) analysis during an influenza A/H5N8 outbreak Marjolein J. PoenID1, Anne Pohlmann2, Clara Amid3, Theo M. Bestebroer1, Sharon M. Brookes4, Ian H. Brown4, Helen Everett4, Claudia M. E. Schapendonk1, Rachel D. Scheuer1, Saskia L. Smits1, Martin Beer2, Ron A. M. Fouchier1, Richard J. Ellis4* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Erasmus MC, Department of Viroscience, Rotterdam, the Netherlands, 2 Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Insel Riems, Germany, 3 European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute (EBI), Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom, 4 Animal and Plant Health Agency (APHA) - Weybridge, Addlestone, Surrey, United Kingdom 1 Erasmus MC, Department of Viroscience, Rotterdam, the Netherlands, 2 Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Insel Riems, Germany, 3 European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute (EBI), Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom, 4 Animal and Plant Health Agency (APHA) - Weybridge, Addlestone, Surrey, United Kingdom * richard.ellis@apha.gov.uk Abstract Citation: Poen MJ, Pohlmann A, Amid C, Bestebroer TM, Brookes SM, Brown IH, et al. (2020) Comparison of sequencing methods and data processing pipelines for whole genome sequencing and minority single nucleotide variant (mSNV) analysis during an influenza A/H5N8 outbreak. PLoS ONE 15(2): e0229326. https://doi. org/10.1371/journal.pone.0229326 As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing tech- nologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed align- ment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment pro- cesses in the different data processing pipelines which affected the consistency of mSNV detec- tion. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study. Editor: Ming Zhang, University of Georgia, UNITED STATES * richard.ellis@apha.gov.uk Comparison of sequencing methods and data processing pipelines HHSN272201400008C to RAMF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. compared to traditional Sanger sequencing [1, 2]. The use of next-generation sequencing (NGS) technologies is continuously expanding and has revolutionized the field of genomics and molecular biology. HHSN272201400008C to RAMF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. gy In many fields of infectious disease research, nucleotide changes in DNA or RNA sequences are used to monitor genetic adaptions indicative of evolution, the emergence of drug resis- tance, immune evasion or as a tool in epidemiological tracing [3]. In clinical settings, sequenc- ing information is used to improve diagnostics and prognosis. NGS technologies play an increasingly important role in these processes as clinically or epidemiologically important nucleotide changes can be present in the minority of DNA or RNA sequences only, which might be missed with more traditional (consensus) sequencing methods which determine the most abundant sequence variants in a population. Nucleotide variants that are present in only a minority of the sequenced virus population are referred to as minority Single Nucleotide Variants (mSNVs). These variants, initially occurring due to replication errors, can become fixed in the population when they have some sort of evolutionary advantage, for instance, mutations related to drug resistance. Furthermore, mSNVs can be also used for high-resoltion molecular epidemiology, which becomes more and more important for outbreak assessment [4, 5]. Traditional Sanger sequencing for instance has been described to detect minority vari- ants provided they are present in at least 10% of the analysed DNA or RNA strands within a sample [6, 7]. Hence, the use of traditional sequencing methods is usually restricted to obtain- ing consensus sequences or to determine heterozygosity in diploid organisms. In contrast, NGS technologies are able to detect low frequency mSNVs in sequence fragments or even whole genomes. Typically, NGS sensitivity for minority sequence variant identification is restricted to a level of variation of 0.1–1%, mainly due to sequencing related background errors [8–10], but sensitivity can be increased using sophisticated approaches like circle sequencing [11] or improved bioinformatic analysis workflows [10]. Introduction Over the past decade, high-throughput sequencing technologies have evolved, providing faster, cheaper, and less laborious alternatives to obtain (whole genome) DNA and RNA sequences 1 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 The reliability of mSNV analysis using NGS methods is influenced by many factors, like the quantity and quality of the input sample, the laboratory procedures, the type of sequencing platform and the software and settings used to analyse the raw sequence data. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Due to the technical improvements, NGS technologies have become more important as diagnostic tools to characterize pathogens in outbreak situations. However, the increasing use of these technologies to address new and important (outbreak related) research and surveil- lance questions emphasizes the need to determine the reproducibility of, and the important technical considerations affecting, outcomes obtained by different laboratories following dif- ferent protocols. Given this, comparative studies focusing on different platforms and data analysis methods are essential to cross-validate different methodologies and determine the reliability of newly obtained data. In addition, there is a growing need (as exemplified by the recent Ebola and Zika virus outbreaks) to share also comprehensive sequencing data as quickly as possible to help with source attribution and developing control strategies. However, the underlying technologies and methods used for NGS are still diverse and there is a strong demand for harmonization of laboratory procedures and approaches for a reliable and opti- mized analysis of the data. This study is part of the European Union’s HORIZON 2020 project “COMPARE” (http:// www.compare-europe.eu/), aiming to improve the analytical tools for emerging zoonotic pathogens and its underpinning research. Here, the comparability of NGS output data obtained from different sequence approaches were evaluated and demonstrated suitable shar- ing strategies for comprehensive NGS data sets. In November 2014, a newly emerging strain of highly pathogenic avian influenza (HPAI) virus was detected in several European countries [12, 13]. In the United Kingdom [14], Germany [15], and The Netherlands [16–18] this sub- type was detected in commercial poultry farms within a few days of one another. In each of PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 2 / 28 Comparison of sequencing methods and data processing pipelines those countries, NGS was used to generate whole-genome sequences rapidly after detection, but as the laboratories in each country were working independently, different approaches were used for both sequencing and data analysis, and the data were shared as part of a wider study to determine the likely source of the outbreak [19]. It is important to determine whether the different analytical approaches have any impact on the outcome. Therefore, the aim of this study was to determine how comparable consensus and minority variant results were between laboratories performing their standard analyses, and whether discrepancies could be attributed to the sequence platform (SP), the data processing platform (DPP) or a combination of both. With the lack of a ground truth/gold standard, all datasets obtained were compared amongst each other. The hypothesis we test in this study is that outputs from NGS analysis of viruses will be comparable irrespective of laboratory, sequencing platform and data analysis platform. Therefore, virus isolates obtained in each of the three countries (United Kingdom, Germany and the Netherlands) were shared between these three partners and subsequently sequenced and analysed in each of the three laboratories according to local procedures. In addition, the use of a specially designed data sharing platform, a COMPARE “Data Hub” at EMBL-EBI, Hinxton UK, was evaluated. This study presents genome coverage data, consensus sequences, the analysis of the comparability of mSNV identifications of the different SPs, and DPPs. Our hypothesis was confirmed at the consensus sequence level, since consensus sequences could be reproduced independent of the combination of SP and DPP used. However, the iden- tification of minority variants appeared to be poorly reproducible, primarily due to the well- known errors in 454 sequencing, and due to differences induced by the alignment processes in the different DPPs. The interpretation of minority variant analysis thus needs a different level of careful standardization and awareness about the possible limitations as shown in this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Samples All samples were obtained from outbreaks in commercial poultry holdings. Isolate A/duck/ England/36254/2014 was obtained from pooled intestinal material from index case ducks (Anas platyrhynchos domesticus). Tissue homogenate material was inoculated into embryo- nated chicken eggs and allantoic fluid was harvested at 1 day post-inoculation [14]. The Dutch isolate (A/chicken/Netherlands/EMC-3/2014) was obtained by passaging lung material of a dead commercial layer hen (Gallus gallus domesticus) in MDCK cells twice and harvesting the supernatant after approximately 40 hours post-inoculation [23]. The German isolate (A/tur- key/Germany/AR2485/2014) originated from lung tissue of a commercially kept turkey (Meleagris gallopavo) and was passaged in embryonated chicken eggs [15]. (Table 1). Experimental design The experimental design is summarized in Fig 1. spreadsheet file per virus to check the reproducibility of mSNV identification when using dif- ferent combinations of SP and DPP. The experimental design is summarized in Fig 1. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Experimental design Three avian influenza A virus isolates that were obtained from three different avian species during the 2014/15 outbreak of HPAI H5N8 virus in Europe were shared among three institu- tions in the United Kingdom (Animal Plant and Health Agency [APHA]), Germany (Fried- rich-Loeffler-Institut [FLI]) and the Netherlands (Erasmus Medical Center [EMC]), later referred to as anonymized institutions I, II and III (Fig 1). All three institutions sequenced all three virus isolates according to their own standard procedures. Adaptors used in the sequenc- ing processes were trimmed off before the raw sequence data files were shared. The sequence data files (.fastq files), alignment files (.bam files), sample metadata and experimental meta- data were shared between the three laboratories and analysed in their own DPPs yielding sequence datasets for each virus (Table 1). This approach enabled to separate the biological features of the viruses from variation introduced by technical methodology. Data sharing was facilitated via a “Data Hub” provided by the EMBL-EBI’s European Nucleotide Archive (ENA) in the framework of the COMPARE collaborative project; all data were stored and subse- quently published in ENA [20] (https://www.ebi.ac.uk/ena, for the accession numbers, see Table 1). ENA is an open repository for sequence and related data and a member of the Inter- national Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org/) [21]. A full description of the COMPARE Data Hub system is provided in a preprint version of Amid et al. [22]. First, consensus sequences derived from a preliminary analysis were compared and one overarching consensus sequence was determined for each gene segment for each virus. This custom-made consensus was used by all three institutions as the reference genome for undertaking mSNV analysis. The resulting nine mSNV reports (originating from three whole- genome raw data sequences times three DPPs) were combined for all three viruses in one PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 3 / 28 Comparison of sequencing methods and data processing pipelines Fig 1. Flowchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. https://doi.org/10.1371/journal.pone.0229326.g001 wchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. Fig 1. Flowchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. Fig 1. Flowchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. https://doi.org/10.1371/journal.pone.0229326.g001 https://doi.org/10.1371/journal.pone.0229326.g001 spreadsheet file per virus to check the reproducibility of mSNV identification when using dif- ferent combinations of SP and DPP. Sequencing Institution I: SP1. RNA was extracted using a Qiagen QIAamp viral RNA mini kit (Qia- gen, Germany) according to the manufacturers’ instructions except that carrier RNA was omitted from the AVL lysis buffer and the sample was eluted in 50μl RNAse-free water. RNA was then processed to double-stranded cDNA (cDNA Synthesis System, Roche) using random hexamers and purified using magnetic beads (AmpureXP, Beckman Coulter, USA). The dou- ble-stranded cDNA was diluted to 0.2 ng/μl and used to produce a sequencing library using the NexteraXT kit (Illumina, USA). Libraries were then sequenced in paired-end mode on an Illumina MiSeq (Illumina, USA), with run lengths varying from 2 x 75 bases (UKDD virus) to 2 x 150 bases (NLCH and DETU viruses) depending on whether time-constraints were imple- mented to provide a rapid response to an outbreak. Demultiplexing and removal of sequencing adapters was done by the MiSeq RTA software to generate raw fastq files. SP1 process included a limited 12-cycle PCR enrichment of the library. Sequencing Post-hoc analysis showed that duplication PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 4 / 28 Comparison of sequencing methods and data processing pipelines p UKDD DETU NLCH Virus strain A/duck/England/36254/2014 A/turkey/Germany/AR2485-L01478/2014 A/chicken/Netherlands/EMC-3/2014 Isolation source Pooled intestines Lung tissue Lung tissue Host Scientific Name Anas platyrhynchos Meleagris gallopavo Gallus gallus domesticus Host Common Name Domestic duck Turkey Chicken Collection Date 14 November 2014 04 November 2014 23 November 2014 Collection Country United Kingdom Germany Netherlands Collection Region East Yorkshire Mecklenburg-Western Pomerania Ter Aar Influenza Test Method MP gene RRT-PCR, H5 RRT-PCR MP gene RRT-PCR, H5 RRT-PCR MP gene RRT-PCR, H5 RRT-PCR Culture Status Sample Egg passage 1 Egg passage 1 MDCK passage 2 Institution I Institution II Institution III Institution I Institition II Institution III Institution I Institition II Institution III Study Accession PRJEB9846 PRJEB12582 PRJEB9687 PRJEB9846 PRJEB12582 PRJEB9687 PRJEB9846 PRJEB12582 PRJEB9687 Run Accession ERR972805 ERR1293054 ERR926712 ERR926713 ERR1354020 ERR1293053 ERR926714 ERR926715 ERR1354021 ERR1293055 ERR926717 ERR926718 DPP1 .bam file run accession ERR3093746 ERR3093752 ERR9033756 ERR3093744 ERR3093753 ERR3093757 ERR3093745 ERR3093754 ERR3093758 DPP2 .bam file run accession ERR2992676 ERR2992677 ERR2992675 ERR2992679 ERR2992680 ERR2992678 ERR2992682 ERR2992683 ERR2992681 DPP3 .bam file run accession ERR2985803 ERR2985804 ERR2985802 ERR2985806 ERR2985807 ERR2985805 ERR2985809 ERR2985810 ERR2985808 Experiment Accession 100k ERX315615 ERX2986848 NA NA ERX315616 ERX2986847 NA NA ERX315617 ERX2986849 NA NA Run Accession 100k  ERR3090788 ERR2984276 NA NA ERR3090789 ERR2984275 NA NA ERR3090790 ERR2984277 NA NA  Using the Study Accession numbers in the European Nucleotide Archive all related data files can be accessed, or accessed directly from https://www.ebi.ac.uk/ena/data/view/accession, e.g.: https:// www.ebi.ac.uk/ena/data/view/PRJEB9846 (Study Accession Institution I), https://www.ebi.ac.uk/ena/data/view/ERR972805 (Run Accession UKDD Institution I). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 5 / 28 Comparison of sequencing methods and data processing pipelines levels were less than 0.02% of the total reads which were considered to have negligible impact on the results. Institution II: SP2. RNA was extracted using a combined approach with TRIzol (Thermo Fisher Scientific, USA) and an RNeasy Kit (Qiagen, Germany). Further concentration and cleaning was done with Agencourt RNAClean XP magnetic beads (Beckman Coulter, USA). RNA was quantified using a Nanodrop UV spectrometer ND-1000 (Peqlab, Germany) and used as template for cDNA synthesis with a cDNA Synthesis System (Roche, Germany) with random hexamers. Fragmentation of the cDNA applying a target size of 300 bp was done with a Covaris M220 ultrasonicator. Sequencing The sonicated cDNA was used for library preparation using Illumina indices (Illumina, USA) on a SPRI-TE library system (Beckman Coulter, USA) using a SPRIworks Fragment Library Cartridge II (for Roche FLX DNA sequencer; Beckman Coul- ter, USA) without automatic size selection. Subsequently, upper and lower size exclusion of the library was done with Ampure XP magnetic beads (Beckman Coulter, USA). The libraries were quality checked using High Sensitivity DNA Chips and reagents on a Bioanalyzer 2100 (Agilent Technologies, Germany) and quantified via qPCR with a Kapa Library Quantification Kit (Kapa Biosystems, USA) on a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories, USA). SP2 did not amplify sample nor library. Sequencing was done on an Illumina MiSeq using MiSeq reagent kit v3 (Illumina, USA) resulting in paired end sequences with a read length of 300. Demultiplexed and adapter-trimmed reads were used to generate raw fastq files. Institution III: SP3. RNA was extracted using the High Pure RNA isolation kit (Roche Diagnostics, Germany) according to manufacturer’s instructions. RNA was converted to cDNA using the SuperScript III Reverse Transcriptase kit (Invitrogen, Thermo Fisher, USA) as described previously [24], and amplified by PCR using primers covering the full viral genome (S1 Table). All 32 PCR fragments from approximately 400–600 nucleotides in length, were sequenced using the 454/Roche GS-FLX sequencing platform. The PCR fragments were pooled in equimolar ratio and purified using the MinElute PCR Purification kit (Qiagen, Germany) according to the manufacturer’s instructions. Rapid Library preparation, Emulsion PCR and Next Generation 454-sequencing were performed according to instructions of the manufacturer (Roche Diagnostics, Germany). Protocols are described in the following manuals: Rapid Library Preparation Method Manual (Roche; May 2010), emPCR Amplification Method Manual–Lib-L (Roche; May 2010) and Sequencing Method Manual (Roche; May 2010). All three samples were sequenced in one run. Samples were pooled using MID adaptors to determine which sequences came from which sample, each sample was assigned two different MID’s. Demultiplexing and basic trimming was done by CLC-bio software to generate raw fastq files (S1 File). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Data processing Institution I: DPP1. In the FluSeqID script (https://github.com/ellisrichardj/FluSeqID) the following steps are run automatically: the mapping of raw sequence data to the host genome (BWA v0.7.12-r1039 [25]), extracting reads that do not map to the host (Samtools v1.2 [26]), assembling non-host reads (Velvet v1.2.10 [27]), identification of the closest match for each genome segment (BLAST v2.2.28 [28] using the custom databases generated from the Influenza Research Database as indicated in the GitHub repository), mapping original data to the top reference segments (BWA), calling new consensus sequences (vcf2consensus.pl), per- forming further iterations of the last two steps to improve new consensus (IterMap), and finally outputting the genome consensus sequence. The data processing pipeline has in-build defaults for k-mer and coverage cut-off for de novo assembly, and the e-value cut-off for BLAST, which can be changed via command line options (see https://github.com/ellisrichardj/ FluSeqID). Since the aligner (BWA-MEM) used performs soft-clipping and ignores low PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 6 / 28 Comparison of sequencing methods and data processing pipelines quality data, quality trimming is unnecessary. For mSNV analysis, the reads were mapped to the unified consensus using BWA. Samtools was used to generate a pileup file which was then analysed using custom python and R scripts to determine the depth of coverage and basecalls at each position (available at https://github.com/ellisrichardj/MinorVar). The combination of BWA-MEM and samtools was shown to be accurate for SNV identification [29]. In order to be included in the final output the minimum basecall quality was 20 and the minimum mapping quality was 50. Institution II: DPP2. Raw sequence data were analysed and mapped using the Genome Sequencer software suite (v. 3.0; Roche, Mannheim, Germany) and the Geneious software suite (v. 9.0.5; Biomatters, Auckland, New Zealand). Raw reads were trimmed and subsets of each trimmed dataset were assembled de novo to generate reference sequences for each data set (Newbler Assembler of Genome Sequencer software suite v. 3.0; Roche, Mannheim, Ger- many). The trimmed raw influenza virus reads were mapped to the reference sequences (New- bler Mapper of Genome Sequencer software suite v. 3.0; Roche, Mannheim, Germany). The output assemblies were imported into the Geneious software suite (v. 9.0.5; Biomatters, Auck- land, New Zealand) for further analysis and processing. Data processing Regions of low and high coverage (threshold was 2 x standard deviations from the mean for low and high coverage) and regions of low quality (minimum quality/phred score 20) were evaluated and if necessary, excluded from further analyses. Consensus sequences were generated and annotated using annotated reference sequences. Sequences were compared, and annotations that matched with a similar- ity (> 90%) were copied. This was done on nucleotide sequences and also for translation in all six reading frames. Annotations were manually inspected and curated. Trimmed raw reads of the datasets or subsets thereof were mapped to the consensus, mapping was fine-tuned and mSNVs were determined using generic SNP finder of the Geneious software suite, applying parameters of maximum p-value of 10−5 and filter for strand bias. The threshold for SNP iden- tification was set at 1%, and variants were checked manually for accuracy. Institution III: DPP3. Raw sequence data were analysed and mapped using the CLC Genomics software package, workbench 8 (CLC Bio). Reads obtained by 454 sequencing were sorted by MID adaptor, quality-trimmed, and analysed using the parameters as shown in S1 File. In short, after sorting by MID, the sequence reads were trimmed at 30 nucleotides from the 30 and 50 ends to remove all primer sequences. Data from the shared Illumina sequence files had already been trimmed and were imported in CLC Bio without additional processing steps (S1 File). Reads were initially aligned to their own reference sequences that were uploaded during the H5N8 outbreak (Gisaid accession numbers EPI-ISL-169282 (NLCH), EPI-ISL-167904 (UKDD) and EPI-ISL-169273 (DETU)). Consensus sequences were automati- cally generated by CLC after alignment to the reference, for detailed settings see S1 File. For the mSNV analysis the raw data were mapped to the new custom-made consensus sequences per gene segment per sample. Fastq files of these alignments were shared with the other institu- tions. The threshold for mSNV identification was set at 1%, and registered minority variants were checked manually for accuracy (minimal quality/phred score 20). Determining the influence of the DPP alignment steps versus DPPs mSNV identifica- tion methods. Data processing pipelines process raw data in several steps, roughly divided into trimming, aligning data to a reference sequence, and variant calling (the mSNV identifica- tion procedure). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Data processing In order to determine to what extent the trimming and subsequent alignment processes contributed to the observed differences the nucleotide coverage results obtained by the three DPPs when aligning the same SP raw datasets were compared. To study the influence of the mSNV identification process, quality-trimmed alignment files that had been generated by each DPP and shared as .bam files were subjected to the mSNV identification process used in DPP3 to determine the differences in mSNV detection output when only the alignment PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 7 / 28 Comparison of sequencing methods and data processing pipelines processes differed. DPP3 was randomly picked for this analyses, mSNV detection parameters were set to the institutions default settings for mSNV identification using CLC-bio software and can be seen in the S1 File. Data sharing To test the applicability of real-time sequence data sharing within the COMPARE network, all raw sequence data used in this study were uploaded to and shared via a “Data Hub” in the environment of the European Nucleotide Archive (ENA). Each institution received its own study accession in which all raw sequence data files and metadata files were assigned with individual experimental accession numbers (Table 1). In addition to the sequence data, all trimmed alignment files (.bam) have been uploaded to the ENA. Using these hubs, sharing between institutions was facilitated and immediate access to the data prior to the public release was possible to enable joint evaluation and comparison. All data files have been made publicly available via the ENA (https://www.ebi.ac.uk/ena). Designing the custom-made consensus sequences Each institution produced a consensus sequence for the 8 influenza gene segments (PB2, PB1, PA, HA, NP, NA, MP, NS) for each of the three viruses. The obtained consensus sequences were aligned using the BioEdit sequence alignment editor (version 7.2.0) [30]. Raw sequence data from each SP were initially aligned to their own reference sequences that were uploaded during the H5N8 outbreak (Gisaid accession numbers EPI-ISL-169282 (NLCH), EPI-ISL- 167904 (UKDD) and xxx (DETU)). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Results In order to determine the comparability of consensus sequences and mSNV identification the biological samples, the sequence data from 3 SPs and the .bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. All data sets were subsequently analysed in 3 different DPPs. The resulting 9 mSNV reports per virus (3 SP data sets each ana- lysed in 3 DPPs) were evaluated for comparability of mSNV identification using different com- binations of SP and DPP. Data sharing Data sharing using the COMPARE “Data Hub” provided by ENA proved to be easy, quick and successful. The “Data Hub” enables the File Transport Protocol (FTP) protected upload and download of large data files and facilitates sharing between collaborators with the possibility to evaluate and compare all data prior to their public release by generating and specifically shar- ing accession numbers using standard ENA procedures. The Data Hub used an influenza virus sample checklist. In addition, data sets are ultimately made publicly and through the INSDC network globally available and accessible in real-time as required without further upload to a different repository. Full details of the COMPARE Data Hub system are available in a submit- ted manuscript [22]. In summary, this process was suitable for quick data sharing in an out- break scenario. Designing the custom-made consensus sequences For each of the 8 gene segments of the 3 viruses separately, 9 initial consensus sequences (3 SPs x 3 DPPs) were generated, resulting in 72 consensus sequences per virus. The custom-made consensus sequence per virus and per gene segment was 1) trimmed to a length represented by all 9 initial consensus sequences and 2) nucleotides had to be identical to at least 6/9 consensus sequences to be included. Although some sequences contained insertions or deletions, those could always be corrected for using the other SP sequences following the criteria mentioned previously. This resulted in a unique custom-made consensus for each gene segment for all three viruses. mSNV analysis comparison For the mSNV analyses the custom-made consensus for each virus isolate was used as a refer- ence for mapping, thereby standardizing positions within the genome to make comparison between institutions easier. To avoid unnecessary increases in analytical time and memory, datasets were down-sampled to 100.000 reads per sample when needed. Each DPP produced a report on the identified mSNVs in a tabulated format. The analysis output files were filtered for mSNVs only, thereby ignoring detected nucleotide insertions and deletions (InDels). There is a current lack of data or evidence-based approaches on how to calculate the required sequence depth (i.e. coverage) for mSNV analyses an evidence-based. In this study, a mini- mum coverage threshold for the identification of mSNVs was applied. This minimum nucleo- tide coverage (i.e. number of reads per nucleotide after trimming) was determined using a basic sample size calculation method, n = log β / log p’ [31]. Here β represents the required power (e.g. for 95% chance of detecting something β = 0.05), and p’ is 1—the proportion of events that you want to detect. For a 95% certainty of detecting a variant at 1%, a minimum coverage of 298 reads per position is needed. For variants that occur in 5% of reads, the number of reads required is >58, and for variants that occur in 10% of the reads the mini- mum coverage is >28. For the mSNV identification literature commonly uses the mSNV cut- off frequencies of 10%, 5% and 1%. However, it needs to be noted that these cut-off values are arbitrary. Therefore, where depth of coverage was sufficient, this study will report mSNV detected with a frequency of 1%, but initial comparisons started with positions show- ing mSNVs with frequencies of 10% in at least one of the SP/DPP combinations, followed by those with mSNV of 5% -<10%, and lastly those 1%-<5%. For all those positions identi- fied, the number of reads and number of variant nucleotides in all other SP/DPP combinations for that position will be noted regardless of frequencies. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 8 / 28 Comparison of sequencing methods and data processing pipelines PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Consensus sequences When ignoring insertions and deletions in the homopolymer regions of the 454 data for most gene segments the identified consensus sequences were identical regardless of the SP and DPP combinations used with the exemption of the differences mentioned in Table 2. However, the number of insertions and deletions in homopolymer regions of the SP3 sequences were con- siderable in all 3 viruses. There was no clear difference in the number of insertions and dele- tions related to homopolymer regions between the different DPPs (20, 17 and 18 for DPP 1, 2 and 3 respectively). Nucleotide differences that were not related to homopolymer regions were only observed for sequences obtained in SP3 and SP2 data when processed in DPP1. In summary, the homopolymer errors inherent in the 454 dataset caused problems for all DPPs, as expected. Consensus sequences generated by DPP1 from SP3 (454) data showed some unexpected differences, but it performed well with the SP1 data formats it was designed for and reasonably well with SP2 data. Overall, the consensus sequences can be reproduced by all DPPs using Illumina data but that the analysis of the 454 data from SP3 was more prob- lematic, as it would require editing of the sequences at homopolymer regions. Consensus sequences from this study can be found in the S2 Table. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 9 / 28 Comparison of sequencing methods and data processing pipelines Table 2. The differences in consensus sequences obtained from each SP/DPP combination, sorted per virus and per gene segment. https://doi.org/10.1371/journal.pone.0229326.t002 mutations were detected. InDel: insertions or deletion; SP: Sequence platform; DPP: Data processing pipeline; ND: not detected. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or The mSNV analysis comparison Nucleotide coverage and the influence of DPP-dependent alignment. The observed number of reads per nucleotide (referred to as nucleotide coverage) differed depending on the SP/DPP combination. All DPPs handled both 454 and Illumina data formats, although some modifications (settings for the bwa mapper to handle single end 454 data) were required for DPP1, which was specifically designed for Illumina paired-end reads. The observed nucleotide coverages showed near to identical profiles for all three viruses. The coverage results obtained from the three different SPs and DPPs for the NLCH virus (Fig 2) and for the other two viruses (S1 Fig) were plotted. In general, lower nucleotide coverage was observed at the termini of each gene segment. The SP3 data showed more variation in nucleotide coverage within gene segments compared to SP1 and SP2 data, due to the sequencing of 32 PCR amplicons. The non-normalised number of raw sequence reads and influenza virus reads per virus per SP can be found in the S3 Table. The differences in nucleotide coverage were visualized for the three different SP raw datasets analysed with the same DPP (Fig 2A). Overall, SP3 data (green lines) showed a lower coverage compared to SP1 (purple) and SP2 data (yellow). The overall coverage for SP1 and SP2 data was similar with small variations for different viruses and DPPs. The shorter read lengths in SP1 virus data did not appear to have influenced the overall nucleotide coverage substantially. The differences in nucleotide coverage introduced by different alignment procedures were also assessed, by comparing the coverage results for each SP raw dataset analysed with the three different DPPs (Fig 2B). DPP2 (orange lines) generally retained the highest nucleotide coverage for data from the different SPs. However, DPP3 (grey lines) generally also retained high coverage for SP3 data, for which it was optimized. The nucleotide coverage of SP3 data showed larger variation between the three different DPPs, leading to differences in nucleotide coverage up to 50% depending on the DPP, because DPP1 and DPP2 were not optimized for this SP. Data from SP2 were handled very similar by all three DPPs. In conclusion, both the SP and the DPP influenced the number of reads per nucleotide position. SP3 showed the lowest output in number of reads compared to SP1 and SP2 Illumina data. The mSNV analysis comparison The influence of the DPP depended highly on the data input, with best DPP performance for the SP dataset for which it was optimized. The mSNV identification. The mSNV identification thresholds were set to 1% in all DPPs. Because of the high number of mSNVs identified, the comparison of these mSNVs started with a manually set arbitrary threshold of 10% that was subsequently decreased to 5%, and 1%. A mSNV position was identified when at least 1 of the SP/DPP combinations showed a variant that exceeded the frequency threshold, and when the coverage at that posi- tion exceeded the minimum number of reads needed to detect that variant with a 95% proba- bility, as described previously. The presence of mSNV and coverage for all SP/DPP The mSNV identification. The mSNV identification thresholds were set to 1% in all DPPs. Because of the high number of mSNVs identified, the comparison of these mSNVs started with a manually set arbitrary threshold of 10% that was subsequently decreased to 5%, and 1%. A mSNV position was identified when at least 1 of the SP/DPP combinations showed a variant that exceeded the frequency threshold, and when the coverage at that posi- tion exceeded the minimum number of reads needed to detect that variant with a 95% proba- bility, as described previously. The presence of mSNV and coverage for all SP/DPP combinations were compared for each of the positions in which a mSNV had been detected in at least one of the combinations. The coverages indicated for those positions where no mSNVs were detected were derived from the alignment files and were not subjected to possible addi- tional read filtering parameters in the mSNV identification process. The average quality (Q- score/phred score) was set to or exceeding 20. combinations were compared for each of the positions in which a mSNV had been detected in at least one of the combinations. The coverages indicated for those positions where no mSNVs were detected were derived from the alignment files and were not subjected to possible addi- tional read filtering parameters in the mSNV identification process. The average quality (Q- score/phred score) was set to or exceeding 20. Ten positions across the three virus genomes were identified with mSNVs occurring in 10% of reads. Three of the mSNVs (NLCH:PB2 G1879A, NLCH:PB2 G2101A and DETU:HA T963C) were detected in all SP/DPP combinations but with slightly different relative abundance. Consensus sequences Virus Segment Start End Number of InDels at homoplymer regions Other nucleotide differences NLCH PB2 1 2280 2 (DPP1) C506A (SP3) 2 (DPP3) G2101R (SP3) PB1 1 2277 1 (DPP1/DPP2/DPP3) 1 (DPP1/DPP2) A949W (SP3) 1 (DPP2/DPP3) 2272 ins AAG (SP2) 1 (DPP3) PA -6# 2190 1 (DPP1/DPP2) ND 2 (DPP1) HA 7 1704 1 (DPP2/DPP3) A427W (SP2) NP 1 1497 1 (DPP1) C420Y (SP3) NA 4 1419 ND ND MP -5# 982 ND ND NS 1 838 ND ND DETU PB2 1 2287 1 (DPP1/DPP2/DPP3) 2272 Del A (SP3) 3 (DPP1) PB1 1 2277 1 (DPP1/DPP2/DPP3) T956C (SP3) 1 (DPP1) 1 (DPP2) 1 (DPP3) PA 7 2189 1 (DPP1/DPP2) ND HA 1 1728 1 (DPP2/DPP3) ND NP 1 1497 2 (DPP3) ND NA 1 1413 1 (DPP1) 778 ins CCA (SP3) MP -1# 982 1 (DPP2) ND NS 2 838 ND ND UKDD PB2 1 2298 1 (DPP1/DPP2/DPP3) C504T (SP3) 1 (DPP3) C506M (SP3) PB1 1 2277 1 (DPP1/DPP2/DPP3) T951W (SP3) 1 (DPP2/DPP3) PA 1 2151 2 (DPP1) ND 1 (DPP2) HA 1 1704 1 (DPP2/DPP3) ND NP 1 1497 1 (DPP3) T1003Y (SP2) NA 4 1420 ND 782 del TA (SP3) MP -5# 982 1 (DPP2) ND NS -5# 849 ND ND The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or Table 2. The differences in consensus sequences obtained from each SP/DPP combination, sorted per virus and per gene segment. The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or mutations were detected. InDel: insertions or deletion; SP: Sequence platform; DPP: Data processing pipeline; ND: not detected.  Start is counted from the ATG start codon;  Exclusively identified in SP3 sequence data, InDels related to homopolymer regions;  E l i l id tifi d i DPP1  Exclusively identified in SP3 sequence data, InDels related to homopolymer regions;  Exclusively identified in DPP1; # ’-’ indicates the number of nucleotides before the ATG start codon included in the consensus 10 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Comparison of sequencing methods and data processing pipelines PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 The mSNV analysis comparison The other mSNVs were identified in only one (n = 6) or two (n = 1) of the SP/DPP combinations (Table 3). Thirty-seven positions were identified with mSNVs occurring in 5% of reads. Of those, the same mSNV was identified in all SP/DPP combinations for 9 positions (24,3%), in seven or Thirty seven positions were identified with mSNVs occurring in 5% of reads. Of those, the same mSNV was identified in all SP/DPP combinations for 9 positions (24,3%), in seven or PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 11 / 28 Comparison of sequencing methods and data processing pipelines Fig 2. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position for full genome sequences of the NLCH virus reads mapped to the NLCH reference sequences. Panel A shows the coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs (SP1: lilac; SP2: yellow; SP3: green) for each of the DPPs. The X-axis represents the position in the genome, the Y-axis represents the number of sequence reads per position. https://doi.org/10.1371/journal.pone.0229326.g002 Fig 2. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position for full genome sequences of the NLCH virus reads mapped to the NLCH reference sequences. Panel A shows the coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs (SP1: lilac; SP2: yellow; SP3: green) for each of the DPPs. The X-axis represents the position in the genome, the Y-axis represents the number of sequence reads per position. Fig 2. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position for full genome sequences of the NLCH virus reads mapped to the NLCH reference sequences. Panel A shows the coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs (SP1: lilac; SP2: yellow; SP3: green) for each of the DPPs. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 The mSNV analysis comparison The X-axis represents the position in the genome, the Y-axis represents the number of sequence reads per position. https://doi.org/10.1371/journal.pone.0229326.g002 12 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Comparison of sequencing methods and data processing pipelines The minority variants occurring in at least one of the sequence platform—Data processing pipelines as a 5% variant. Position Sequence platform Data processing pipeline 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage PB2.1879 G!A 1 81/1301 6,2% 246/2716 9,1% 112/1203 9,3% 2 47/956 4,9% 117/1137 10,3% 114/1064 10,7% 3 49/530 9,2% 131/1341 9,8% 129/1338 9,6% PB2.2101 G!A 1 53/1118 4,7% 261/2704 9,7% 110/897 12,3% 2 21/1578 1,3% 125/1875 6,7% 121/1463 8,3% 3 13/542 2,4% 199/1433 13,9% 199/1435 13,9% PB2.2277 T!G 1 ND/479 <1% 86/1008 8,5% 33/190 17,4% 2 ND/557 <1% ND/623 <1% ND/534 <1% 3 ND/680 <1% ND/1117 <1% ND/1024 <1% PB1.87 A!G 1 ND/818 <1% ND/1754 <1% ND/1114 <1% 2 25/230 10,9% ND/376 <1% ND/328 <1% 3 ND/275 <1% ND/537 <1% ND/537 <1% PB1.2240 G!C 1 ND/664 <1% 54/1341 4,0% 38/418 9,1% 2 ND/1231 <1% ND/1271 <1% ND/1233 <1% 3 ND/161 <1% ND/277 <1% ND/276 <1% PB1.2268 A!G 1 ND/336 <1% 29/641 4,5% 11/176 6,3% 2 ND/993 <1% ND/1026 <1% ND/1002 <1% 3 ND/53 <1% ND/159 <1% ND/148 <1% HA.104 A!G 1 ND/733 <1% ND/1761 <1% ND/1151 <1% 2 ND/437 <1% ND/1370 <1% ND/1156 <1% 3 ND/1 <1% ND/105 <1% 12/105 11,4% HA.1689 T!C 1 ND/390 <1% ND/694 <1% 11/217 5,1% 2 ND/2018 <1% ND/4083 <1% ND/3979 <1% 3 ND/937 <1% ND/1669 <1% ND/1680 <1% NP.105 A!G 1 ND/182 <1% ND/449 <1% ND/343 <1% 2 83/1507 5,5% ND/1890 <1% ND/1804 <1% 3 ND/89 <1% ND/704 <1% ND/702 <1% NP.1239 A!T 1 32/2428 1,3% 279/5410 5,2% ND/3092 <1% 2 ND/2345 <1% ND/2643 <1% ND/2453 <1% 3 ND/1711 <1% ND/2111 <1% ND/2117 <1% NP.1489 G!A 1 ND/182 <1% 26/336 7,7% ND/172 <1% 2 ND/436 <1% ND/452 <1% ND/444 <1% 3 ND/1320 <1% ND/1799 <1% ND/1799 <1% NS.833 A!T 1 ND/187 <1% ND/287 <1% 5/88 5,7% 2 ND/1224 <1% ND/1327 <1% ND/1284 <1% 3 ND/1367 <1% ND/2430 <1% ND/2333 <1% PB2.1054 T!C 1 69/1369 5,0% 168/2637 6,4% 97/1304 7,4% 2 60/1477 4,1% 115/1836 6,3% 99/1605 6,2% 3 6/392 1,5% 94/2038 4,6% 48/1054 4,6% PB2.2257 A!C 1 ND/867 <1% ND/1563 <1% 24/463 5,2% 2 ND/531 <1% ND/581 <1% ND/378 <1% 3 ND/893 <1% ND/2286 <1% ND/1346 <1% PB2.2277 T!G 1 ND/644 <1% 52/1150 4,5% 27/307 8,8% 2 ND/418 <1% ND/472 <1% ND/284 <1% 3 ND/1208 <1% ND/1948 <1% ND/1209 <1% (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 13 / 28 Comparison of sequencing methods and data processing pipelines Table 3. (Continued) Virus Position Sequence platform Data processing pipeline 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage UKDD PB2.2277 T!G 1 ND/415 <1% 28/507 5,5% ND/475 <1% 2 ND/589 <1% ND/620 <1% ND/601 <1% 3 ND/1140 <1% ND/1996 <1% ND/2065 <1% PB1.87 A!G 1 ND/387 <1% ND/440 <1% ND/439 <1% 2 26/327 8,0% 32/395 8,1% ND/351 <1% 3 ND/617 <1% ND/1133 <1% ND/1136 <1% PB1.728 C!A 1 ND/750 <1% ND/832 <1% ND/836 <1% 2 ND/776 <1% 52/928 5,6% ND/829 <1% 3 ND/2459 <1% ND/4290 <1% ND/4293 <1% PB1.730 C!T 1 ND/742 <1% ND/824 <1% ND/826 <1% 2 ND/767 <1% 57/1008 5,7% ND/832 <1% 3 ND/2339 <1% ND//4286 <1% ND/4289 <1% PB1.883 G!C 1 ND/942 <1% ND/997 <1% ND/997 <1% 2 ND/1689 <1% ND/1865 <1% ND/1760 <1% 3 ND/2479 <1% 47/690 6,8% ND/3681 <1% PA.49 G!C 1 ND/103 <1% 6/117 5,1% ND/115 <1% 2 ND/337 <1% ND/435 <1% ND/392 <1% 3 ND/111 <1% ND/207 <1% ND/204 <1% PA.82 C!T 1 ND/155 <1% ND/180 <1% ND/177 <1% 2 ND/695 <1% ND/809 <1% ND/745 <1% 3 ND/64 <1% ND/247 <1% 30/248 12,1% NS.811 G!T 1 ND/221 <1% 17/270 6,3% ND/249 <1% 2 ND/2452 <1% ND/2725 <1% ND/2557 <1% 3 ND/3117 <1% ND/4125 <1% ND/4139 <1% Colours display the variant frequency with 10% (green), 5–10% (purple) and <5% (pink). ND: not detected. https://doi org/10 1371/journal pone 0229326 t003 Colours display the variant frequency with 10% (green), 5–10% (purple) and <5% (pink). ND: not detected. https://doi.org/10.1371/journal.pone.0229326.t003 eight of the SP/DPP combinations for 2 positions (5,4%) and in at least two SP/DPP combina- tions for 19 positions (51.4%), although not always in a frequency of 5%. However, for 18 positions (48.6%) the mSNV was not reproduced at a 1% frequency in any of the other SP/ DPP combinations (Table 3). Focussing on the separate SP data analysed in the 3 DPPs, most of the identified positions with 5% mSNVs in at least 1 SP/DPP combination were identified in SP1 data (47%) followed by SP2 (29%) and SP3 (24%) data. Looking at the 5% mSNV reproducibility per SP dataset in all three DPPs within these thirty-seven positions, forty-eight SP datasets showed a 5% mSNV in at least one of the DPP outputs. Comparison of sequencing methods and data processing pipelines (Continued) Position Sequence platform Data processing pipeline 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage PB1.14 C!T 1 ND/144 <1% 48/433 11,1% ND/239 <1% 2 ND/90 <1% ND/355 <1% ND/304 <1% 3 ND/562 <1% ND/792 <1% ND/496 <1% PB1.23 T!G 1 ND/207 <1% 30/535 5,6% ND/315 <1% 2 ND/103 <1% ND/365 <1% ND/319 <1% 3 ND/699 <1% ND/950 <1% ND/609 <1% PB1.87 A!G 1 ND/744 <1% ND/1644 <1% ND/1076 <1% 2 49/365 13,4% ND/677 <1% ND/576 <1% 3 ND/721 <1% ND/1156 <1% ND/793 <1% PB1.2240 G!C 1 ND/757 <1% 23/1517 1,5% 26/515 5,0% 2 ND/944 <1% ND/985 <1% ND/806 <1% 3 ND/274 <1% ND/439 <1% ND/253 <1% PB1.2268 A!G 1 5/470 1,1% 33/928 3,6% 22/278 7,9% 2 ND/798 <1% ND/829 <1% ND/671 <1% 3 ND/109 <1% ND/259 <1% ND/123 <1% PB1.2271 A!G 1 12/446 2,7% 59/901 6,5% 16/263 6,1% 2 ND/729 <1% 47/810 5,8% 40/649 6,2% 3 1/32 3,1% ND/123 <1% 2/83 2,4% HA.867 C!T 1 59/1533 3,8% 206/3183 6,5% 104/1537 6,8% 2 59/2031 2,9% 150/2525 5,9% 127/2253 5,6% 3 11/180 6,1% 48/647 7,4% 28/385 7,3% HA.963 T!C 1 122/1401 8,7% 446/3071 14,5% 189/1419 13,3% 2 90/1517 5,9% 318/2189 14,5% 247/1828 13,5% 3 5/69 7,2% 107/606 17,7% 47/293 16,0% NP.1491 C!A 1 ND/278 <1% 71/583 12,2% ND/206 <1% 2 ND/723 <1% ND/769 <1% ND/692 <1% 3 ND/799 <1% ND/2031 <1% ND/1206 <1% NA.65 T!C 1 19/503 3,8% 52/1229 4,2% 16/467 3,4% 2 20/662 3,0% 50/1104 4,5% 45/992 4,5% 3 24/557 4,3% 53/1099 4,8% 37/727 5,1% NA.78 T!C 1 23/599 3,8% 57/1403 4,1% 20/557 3,6% 2 21/692 3,0% 55/1147 4,8% 50/1033 4,8% 3 23/580 4,0% 51/1124 4,5% 37/735 5,0% NA.89 T!C 1 23/713 3,2% 55/1670 3,3% 22/651 3,4% 2 23/798 2,9% 56/1261 4,4% 50/1134 4,4% 3 24/580 4,1% 55/1196 4,6% 40/775 5,2% NA.117 T!C 1 37/908 4,1% 87/2140 4,1% 36/818 4,4% 2 28/1102 2,5% 67/1631 4,1% ND/1459 <1% 3 22/531 4,1% 57/1276 4,5% 42/812 5,2% NA.126 T!C 1 37/983 3,8% 83/2294 3,6% 36/876 4,1% 2 31/1126 2,8% 72/1676 4,3% 65/1502 4,3% 3 26/519 5,0% 62/1395 4,4% 43/812 5,3% (C ti d) (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 14 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Positions with 1% variants that were identified in more than one of the SPs or DPPs respectively are displayed in the overlapping coloured areas, the centre part representing the number of 1% variants that were detected with all three DPPs (top row) or SPs (bottom row). The total number of positions with 1% variants detected was 250in SP1, 213 in SP2, 45 in SP3, and 50 in DPP1, 353 in SP2, and 93 in SP3. This figure was produced using Venny 2.1. Fig 3. The reproducibility of 1% variants with sufficient coverage (>298) for all sequence data combined. Each figure shows the number of 1% variants detected per sequence platform (SP, top row) and data processing pipeline (DPP, bottom row) for SP1/DPP1 (left column), SP2/DPP2 (middle column), and SP3/DPP3 (right column). The colours represent the different DPPs and SPs respectively, in which the >1% variants were detected: SP1/ DPP1 (purple), SP2/DPP2 (yellow) and SP3/DPP3 (green). Positions with 1% variants that were identified in more than one of the SPs or DPPs respectively are displayed in the overlapping coloured areas, the centre part representing the number of 1% variants that were detected with all three DPPs (top row) or SPs (bottom row). The total number of positions with 1% variants detected was 250in SP1, 213 in SP2, 45 in SP3, and 50 in DPP1, 353 in SP2, and 93 in SP3. This figure was produced using Venny 2.1. https://doi.org/10.1371/journal.pone.0229326.g003 and per DPP analysing data from the three SPs (influence of SP). The genome positions with 1% variants were listed per SP/DPP combination and entered in the program Venny 2.1 that calculated the overlapping positions as a fraction of the total number of positions between the SP/DPP combinations as compared to the total number of positions, resulting in Fig 3. It needs to be noted that especially SP3 did not always reach the minimum coverage require- ments and may therefore not be suitable to detect low-frequency variants with (see also Table 4). Positions where the coverage in one or more of the nine SP/DPP combinations didn’t meet the minimum required coverage of 298 were not included in the comparison in Fig 3. The reproducibility of 1% variants using one SP dataset in all three DPPs was 10%, 9.4% and 31.1% for SP1, SP2 and SP3 sequences, respectively. Additionally, for eleven positions, all in the DETU virus, the variant was reproduced by all DPPs, however at a <5% frequency (for instance SP3 data at PB2.1054, and SP1 and SP2 data at NA.65) In 53% (31/59) of cases the same mSNVs from 1 SP dataset was reproduced in all three DPP’s in at least a 1% frequency, in 31% (18/59) of cases the variant was only detected in 1 DPP even though coverage in the other DPPs was theoretically high enough to detect variants at a 1% level. Lowering the threshold value to a mSNV frequency of 1% resulted in a large increase in the number of positions identified with mSNVs. To investigate the reproducibility of these mSNVs, the data for all 3 viruses was combined per SP in the three DPPs (influence of DPP), PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 15 / 28 Comparison of sequencing methods and data processing pipelines Fig 3. The reproducibility of 1% variants with sufficient coverage (>298) for all sequence data combined. Each figure shows the number of 1% variants detected per sequence platform (SP, top row) and data processing pipeline (DPP, bottom row) for SP1/DPP1 (left column), SP2/DPP2 (middle column), and SP3/DPP3 (right column). The colours represent the different DPPs and SPs respectively, in which the >1% variants were detected: SP1/ DPP1 (purple), SP2/DPP2 (yellow) and SP3/DPP3 (green). Positions with 1% variants that were identified in more than one of the SPs or DPPs respectively are displayed in the overlapping coloured areas, the centre part representing the number of 1% variants that were detected with all three DPPs (top row) or SPs (bottom row). The total number of positions with 1% variants detected was 250in SP1, 213 in SP2, 45 in SP3, and 50 in DPP1, 353 in SP2, and 93 in SP3. This figure was produced using Venny 2.1. https://doi.org/10.1371/journal.pone.0229326.g003 Fig 3. The reproducibility of 1% variants with sufficient coverage (>298) for all sequence data combined. Each figure shows the number of 1% variants detected per sequence platform (SP, top row) and data processing pipeline (DPP, bottom row) for SP1/DPP1 (left column), SP2/DPP2 (middle column), and SP3/DPP3 (right column). The colours represent the different DPPs and SPs respectively, in which the >1% variants were detected: SP1/ DPP1 (purple), SP2/DPP2 (yellow) and SP3/DPP3 (green). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 The reproducibility of 1% vari- ants using raw data of a virus sequenced in three different SPs was 20%, 7.4% and 22.6% for DPP1, DPP2 and DPP3 respectively (Fig 3). Most 1% variants were not reproduced by any of the other DPPs processing the same SP data (~75%) for SP1 and SP2 data. This was less for PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 16 / 28 Comparison of sequencing methods and data processing pipelines Table 4. The minority variants occurring in at least one of the sequence platform—Data processing pipelines as a 1% variant in the HA segment of the DETU sample with a minimum coverage of 298 reads at that position. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Position Sequence platform Data processing pipeline 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage HA.170 T!A 1 ND/935 <1% ND/2191 <1% ND/1348 <1% 2 ND/300 <1% 11/693 1,59% ND/551 <1% 3 ND/82 <1% ND/245 <1% ND/210 <1% HA.170 T!C 1 ND/935 <1% ND/2191 <1% ND/1348 <1% 2 ND/300 <1% 18/693 2,60% ND/551 <1% 3 ND/82 <1% ND/245 <1% ND/210 <1% HA.171 C!A 1 ND/931 <1% ND/2184 <1% ND/1339 <1% 2 ND/323 <1% 12/698 1,72% ND/558 <1% 3 ND/82 <1% ND/245 <1% ND/210 <1% HA.194 C!A 1 ND/991 <1% ND/2397 <1% ND/1455 <1% 2 ND/353 <1% 22/701 3,14% ND/553 <1% 3 ND/58 <1% ND/250 <1% ND/212 <1% HA.195 C!A 1 ND/995 <1% ND/2390 <1% ND/1464 <1% 2 ND/356 <1% 20/701 2,85% ND/553 <1% 3 ND/55 <1% ND/250 <1% ND/212 <1% HA.268 C!T 1 ND/1140 <1% ND/2580 <1% ND/1626 <1% 2 ND/1293 <1% 25/1563 1,60% ND/1338 <1% 3 ND/88 <1% ND/252 <1% ND/212 <1% HA.272 A!T 1 ND/1156 <1% ND/2593 <1% ND/1639 <1% 2 17/1424 1,19% 20/1563 1,28% ND/1404 <1% 3 ND/81 <1% ND/253 <1% ND/213 <1% HA.407 G!T 1 ND/1144 <1% ND/2364 <1% ND/1553 <1% 2 ND/1773 <1% 31/2121 1,46% ND/1855 <1% 3 ND/74 <1% ND/237 <1% ND/212 <1% HA.407 G!A 1 ND/1144 <1% 27/2364 1,14% ND/1553 <1% 2 ND/1773 <1% ND/2121 <1% ND/1856 <1% 3 ND/74 <1% ND/237 <1% ND/212 <1% HA.418 A!G 1 ND/1111 <1% ND/2319 <1% ND/1492 <1% 2 29/2195 1,32% 38/2513 1,51% ND/2197 <1% 3 ND/69 <1% ND/237 <1% ND/212 <1% HA.453 T!G 1 ND/1339 <1% 29/2736 1,06% ND/1811 <1% 2 ND/2342 <1% ND/2695 <1% ND/2384 <1% 3 ND/91 <1% ND/193 <1% ND/179 <1% HA.560 A!G 1 43/1587 2,71% 113/3385 3,34% 55/1517 3,63% 2 56/2397 2,34% 145/2912 4,98% 113/2495 4,53% 3 21/884 2,38% 72/1754 4,10% 43/1245 3,45% HA.715 C!T 1 ND/1663 <1% 62/3832 1,62% 24/1582 1,52% 2 26/2283 1,14% 55/2722 2,02% 50/2420 2,07% 3 ND/531 <1% 20/1883 1,06% 15/1245 1,20% HA.867 C!T 1 59/1533 3,85% 206/3183 6,47% 104/1537 6,77% 2 59/2031 2,90% 150/2525 5,94% 127/2253 5,64% 3 11/180 6,11% 48/647 7,42% 28/385 7,27% (Continued) Table 4. The minority variants occurring in at least one of the sequence platform—Data processing pipelines as a 1% variant in the HA segment of the DETU sample with a minimum coverage of 298 reads at that position. (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 17 / 28 Comparison of sequencing methods and data processing pipelines Table 4. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 (Continued) (Continued) Position Sequence platform Data processing pipeline 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage HA.963 T!C 1 122/1401 8,71% 446/3071 14,52% 189/1419 13,32% 2 90/1517 5,93% 318/2189 14,53% 247/1828 13,51% 3 5/69 7,25% 107/606 17,66% 47/293 16,04% HA.1000 A!C 1 ND/1409 <1% 48/2962 1,62% ND/1873 <1% 2 ND/1629 <1% ND/1919 <1% ND/1645 <1% 3 ND/84 <1% ND/614 <1% ND/293 <1% HA.1177 G!A 1 ND/1222 <1% ND/2224 <1% ND/1597 <1% 2 ND/1652 <1% 34/1901 1,79% ND/1724 <1% 3 ND/289 <1% ND/549 <1% ND/270 <1% HA.1183 A!G 1 ND/1210 <1% ND/2226 <1% ND/1589 <1% 2 ND/1770 <1% ND/1892 <1% ND/1723 <1% 3 ND/280 <1% 6/547 1,10% ND/268 <1% HA.1199 T!G 1 ND/1182 <1% ND/2124 <1% ND/1518 <1% 2 ND/1615 <1% 27/1899 1,42% ND/1732 <1% 3 ND/296 <1% ND/545 ND/266 <1% HA.1263 A!G 1 16/963 1,66% 57/1841 3,10% 26/954 2,73% 2 26/1924 1,35% 56/2207 2,54% 41/1967 2,08% 3 ND/1161 <1% 63/2226 2,83% 33/1350 2,44% HA.1430 A!G 1 ND/1311 <1% ND/2870 <1% ND/1827 <1% 2 ND/1498 <1% 36/1924 1,87% ND/1659 <1% 3 ND/955 <1% ND/2391 <1% ND/1452 <1% HA.1455 C!T 1 ND/1333 <1% ND/2753 <1% 14/1233 1,14% 2 ND/1846 <1% ND/2242 <1% ND/1895 <1% 3 ND/1093 <1% ND/2373 <1% ND/1449 <1% HA.1543 A!G 1 25/1209 2,07% 94/2757 3,41% 37/1142 3,24% 2 ND/1660 <1% 56/1857 3,02% 41/1585 2,59% 3 ND/1182 <1% ND/3324 <1% ND/1972 <1% HA.1624 C!A 1 ND/998 <1% ND/2174 <1% ND/1478 <1% 2 ND/1173 <1% 25/1291 1,94% ND/1120 <1% 3 ND/2218 <1% ND/3654 <1% ND/2244 <1% HA.1634 C!A 1 ND/930 <1% ND/2032 <1% ND/1388 <1% 2 ND/1091 <1% 16/1218 1,31% ND/1048 <1% 3 ND/2616 <1% ND/3704 <1% ND/2269 <1% HA.1638 C!A 1 ND/932 <1% ND/1991 <1% ND/1368 <1% 2 ND/1083 <1% 15/1180 1,27% ND/1010 <1% 3 ND/2600 <1% ND/3709 <1% ND/2276 <1% HA.1643 T!A 1 ND/875 <1% ND/1892 <1% ND/1291 <1% 2 ND/1028 <1% 13/1110 1,17% ND/944 <1% 3 ND/2612 <1% ND/3703 <1% ND/2278 <1% HA.1643 T!G 1 ND/875 <1% ND/1892 <1% ND/1291 <1% 2 ND/1028 <1% 12/1110 1,08% ND/944 <1% 3 ND/2612 <1% ND/3703 <1% ND/2278 <1% (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 18 / 28 Comparison of sequencing methods and data processing pipelines Table 4. (Continued) Position Sequence platform Data processing pipeline 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage HA.1691 G!A 1 ND/596 <1% ND/1110 <1% 7/404 1,73% 2 ND/767 <1% ND/873 <1% ND/696 <1% 3 ND/2499 <1% ND/3575 <1% ND/2222 <1% HA.1693 A!T 1 ND/582 <1% ND/1081 <1% 7/391 1,79% 2 ND/751 <1% ND/864 <1% ND/690 <1% 3 ND/2310 <1% ND/3569 <1% ND/2219 <1% HA.1695 T!C 1 ND/555 <1% ND/1030 <1% 7/366 1,91% 2 ND/779 <1% ND/3557 <1% ND/688 <1% 3 ND/1767 <1% ND/3557 <1% ND/2220 <1% HA.1698 C!T 1 ND/537 <1% ND/977 <1% ND/601 <1% 2 ND/758 <1% 11/852 1,29% ND/681 <1% 3 ND/2260 <1% ND/3520 <1% ND/2113 <1% HA.1705 A!G 1 ND/492 <1% ND/883 <1% ND/528 <1% 2 ND/733 <1% 11/832 1,32% ND/660 <1% 3 ND/1709 <1% ND/3300 <1% ND/2016 <1% Positions with a too low coverage (<298 reads/position) to detect 1% variants are marked with an asterisk (). Numbers are displayed as [number of variants]/[number of reads on that position]. ND: not detected. https://doi.org/10.1371/journal.pone.0229326.t004 SP3 data but this might be due to the fact that many positions identified in SP3 data did not meet the minimum coverage criteria and were therefore discarded. For brevity, the detailed results for the HA gene segment of the DETU virus are shown in Table 4. This virus segment was chosen because it showed the best reproducibility of results for 5% minority variants in all SP/DPP combinations. In the DETU HA segment, 33 posi- tions containing a mSNV occurring in 1% of reads with sufficient coverage (298 reads) were identified. Only 3 of these positions (9%) were identified in all SP/DPP combinations. The majority of the positions (25/33, 76%) were only identified in one of the nine SP/DPP combinations. However, it needs to be noted that the SP3 data coverage was insufficient in all three DPPs to detect 1% variants for 11 of those positions (Table 4). Although a comparison between the frequencies of the detected mSNVs might be appropri- ate, based on these results where even absence vs. presence of the mSNVs is poorly comparable further in-depth analyses on these frequencies is not performed because of its limited value. Determining the influence of the minor variant detection method. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Positions with a too low coverage (<298 reads/position) to detect 1% variants are marked with an asterisk (). Numbers are displayed as [number of variants]/[number of reads on that position]. ND: not detected. Comparison of sequencing methods and data processing pipelines le 5. The reproducibility of positions with at least one 5% variant when alignment files from the respective DPPs are all uploaded into DPP3 for only the NV identification process versus when the mSNV identifications are fully performed by the respective DPPs. To isolate the effect of just the mSNV identification step in the DPP, independent of the alignment step, quality- trimmed alignment files (.bam files) of the data (subdivided per virus, per SP and per DPP) were shared and subjected to the same DPP mSNV detection process (in this case DPP3) and compared to the original outcomes from DPP1 and DPP2 (Table 5). In the majority of posi- tions, the different mSNV identification processes did not influence the results, as 84% (119/ 142) of the mSNVs were identified regardless of the mSNV identification process. Twenty- three mSNVs that were not reproduced by DPP3 mSNV identification analysis, were repro- duced when the ‘Direction and position Filters’ in DPP3 were ignored (Table 5, marked with # of ##). These parameters filter out mSNVs when the set criteria for the read direction (variant must occur in both forward and reverse reads), relative read direction (statistical approach of PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 19 / 28 Comparison of sequencing methods and data processing pipelines le 5. s Position Sequence platform Data Processing pipeline Bam file generating processing pipeline 1 2 3 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage H PB2.1879 G!A 1 81/1301 6,2% 246/2716 9,1% 112/1203 9,3% 132/1375 9,6% 246/2716 9,1% 121/1301 9,3% 2 47/956 4,9% 117/1137 10,3% 114/1064 10,7% 119/1122 10,6% 117/1137 10,3% 114/1064 10,7% 3 49/530 9,2% 131/1341 9,8% 129/1338 9,6% 54/542 10,0% 131//1341 9,8% 129/1338 9,6% PB2.2101 G!A 1 53/1118 4,7% 261/2704 9,7% 110/897 12,3% 138/1180 11,7% 261/2704 9,7% 121/1086 11,1% 2 21/1578 1,3% 125/1875 6,7% 121/1463 8,3% ND/1856## <1% ND/1850# <1% 121/1463 8,3% 3 13/542 2,4% 199/1433 13,9% 199/1435 13,9% 87/625 13,9% 199/1433 13,9% 199/1435 13,9% PB2.2277 T!G 1 ND/479 <1% 86/1008 8,5% 33/190 17,4% ND/849 <1% ND/1008## <1% 37/281 13,2% 2 ND/557 <1% ND/623 <1% ND/534 <1% ND/619 <1% ND/623 <1% ND/534 <1% 3 ND/680 <1% ND/1117 <1% ND/1024 <1% ND/708 <1% ND/1117 <1% ND/1027 <1% PB1.87 A!G 1 ND/818 <1% ND/1754 <1% ND/1114 <1% ND/1264 <1% ND/1753 <1% ND/1114 <1% 2 25/230 10,9% ND/376 <1% ND/328 <1% ND/368## <1% ND/376 <1% ND/328 <1% 3 ND/275 <1% ND/537 <1% ND/537 <1% ND/278 <1% ND/537 <1% ND/537 <1% PB1.2240 G!C 1 ND/664 <1% 54/1341 4,0% 38/418 9,1% ND/1004 <1% ND/1341# <1% 46/486 9,5% 2 ND/1231 <1% ND/1271 <1% ND/1233 <1% ND/1277 <1% ND/1271 <1% ND/1235 <1% 3 ND/161 <1% ND/277 <1% ND/276 <1% ND/163 <1% ND/277 <1% ND/276 <1% PB1.2268 A!G 1 ND/336 <1% 29/641 4,5% 11/176 6,3% 15/322 4,66% 37/641 5,8% 13/213 6,1% 2 ND/993 <1% ND/1026 <1% ND/1002 <1% ND/1025 <1% ND/1026 <1% ND/1002 <1% 3 ND/53 <1% ND/159 <1% ND/148 <1% ND/90 <1% ND/159 <1% ND/151 <1% PA.2167 T!G 1 ND/141 <1% ND/288 <1% ND/154 <1% ND/235 <1% 21/288 7,29% ND/154 <1% 2 ND/757 <1% ND/807 <1% ND/773 <1% ND/812 <1% ND/807 <1% ND/733 <1% 3 ND/704 <1% ND/1070 <1% ND/1077 <1% ND/714 <1% ND/1070 <1% ND/1078 <1% HA.104 A!G 1 ND/733 <1% ND/1761 <1% ND/1151 <1% ND/1175 <1% ND/1761 <1% ND/1135 <1% 2 ND/437 <1% ND/1370 <1% ND/1156 <1% ND/1326 <1% ND/1369 <1% ND/1142 <1% 3 ND/1 <1% ND/105 <1% 12/105 11,4% ND/6 <1% ND/105 <1% 12/105 11,4% HA.1689 T!C 1 ND/390 <1% ND/694 <1% 11/217 5,1% ND/610 <1% ND/694 <1% 13/260 5,0% 2 ND/2018 <1% ND/4083 <1% ND/3979 <1% ND/4045 <1% ND/4081 <1% ND/3979 <1% 3 ND/937 <1% ND/1669 <1% ND/1680 <1% ND/1106 <1% ND/1669 <1% ND/1680 <1% NA.3 T!C 1 ND/32 <1% ND/105 <1% ND/49 <1% ND/92 <1% 7/105 6,67% ND/49 <1% 2 ND/6 <1% ND/313 <1% ND/297 <1% ND/305 <1% ND/313 <1% ND/297 <1% 3 ND/2 <1% ND/25 <1% ND/25 <1% ND/6 <1% ND/25 <1% ND/25 <1% NP.105 A!G 1 ND/182 <1% ND/449 <1% ND/343 <1% ND/374 <1% 6/449 1,34% ND/343 <1% 2 83/1507 5,5% ND/1890 <1% ND/1804 <1% ND/1866## <1% ND/1890 <1% ND/1805 <1% 3 ND/89 <1% ND/704 <1% ND/702 <1% ND/246 <1% ND/704 <1% ND/703 <1% NP.1239 A!T 1 32/2428 1,3% 279/5410 5,2% ND/3092 <1% ND/3372## <1% ND/5410# <1% ND/3092 <1% 2 ND/2345 <1% ND/2643 <1% ND/2453 <1% ND/2626 <1% ND/2643 <1% ND/2453 <1% 3 ND/1711 <1% ND/2111 <1% ND/2117 <1% ND/1712 <1% ND/2111 <1% ND/2117 <1% NP.1489 G!A 1 ND/182 <1% 26/336 7,7% ND/172 <1% ND/242 <1% 26/376 6,9% ND/172 <1% 2 ND/436 <1% ND/452 <1% ND/444 <1% ND/451 <1% ND/451 <1% ND/444 <1% 3 ND/1320 <1% ND/1799 <1% ND/1799 <1% ND/1325 <1% ND/1799 <1% ND/1799 <1% NS.827 C!T 1 ND/249 <1% 19/419 4,5% ND/205 <1% ND/365 <1% 21/412 5,3% ND/205 <1% 2 ND/1316 <1% ND/1423 <1% ND/1375 <1% ND/1427 <1% ND/1422 <1% ND/1375 <1% 3 ND/2091 <1% ND/2901 <1% ND/2757 <1% ND/2293 <1% ND/2898 <1% ND/2929 <1% NS829 G!T 1 ND/221 <1% 19/380 5,0% ND/179 <1% ND/328 <1% 19/376 5,4% ND/179 <1% 2 ND/1302 <1% ND/1391 <1% ND/1341 <1% ND/1388 <1% ND/1389 <1% ND/1341 <1% 3 ND/2117 <1% ND/2852 <1% ND/2727 <1% ND/2279 <1% ND/2852 <1% ND/2880 <1% NS.833 A!T 1 ND/187 <1% ND/287 <1% 5/88 5,7% ND/259 <1% 11/257 4,28% 5/96 5,2% 2 ND/1224 <1% ND/1327 <1% ND/1284 <1% ND/1314 <1% ND/1322 <1% ND/1284 <1% 3 ND/1367 <1% ND/2430 <1% ND/2333 <1% ND/1779 <1% ND/2430 <1% ND/2360 <1% (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 20 / 28 (Continued) us Position Sequence platform Data Processing pipeline Bam file generating processing pipeline 1 2 3 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage U PB2.900 A!G 1 38/1335 2,9% 136/2740 5,0% 61/1231 5,0% 68/1328 5,12 136/2740 4,96 65/1322 4,92 2 35/1645 2,1% 77/1800 4,3% 66/1629 4,1% 70/1775 4,0% 77/1800 4,3% 66/1629 4,1% 3 30/861 3,5% 86/2308 3,7% 47/1245 3,8% ND/1001## <1% ND/2308# <1% 47/1245 3,8% PB2.1054 T!C 1 69/1369 5,0% 168/2637 6,4% 97/1304 7,4% 105/1393 7,5% 168/2637 6,4% 100/1376 7,3% 2 60/1477 4,1% 115/1836 6,3% 99/1605 6,2% 113/1810 6,2% 115/1836 6,3% 99/1605 6,2% 3 6/392 1,5% 94/2038 4,6% 48/1054 4,6% 32/524 6,1% 94/2038 4,6% 48/1054 4,6% PB2.2257 A!C 1 ND/867 <1% ND/1563 <1% 24/463 5,2% ND/1447 <1% ND/1562 <1% 26/472 5,5% 2 ND/531 <1% ND/581 <1% ND/378 <1% ND/588 <1% ND/580 <1% ND/378 <1% 3 ND/893 <1% ND/2286 <1% ND/1346 <1% ND/1341 <1% ND/2185 <1% ND/1347 <1% PB2.2277 T!G 1 ND/644 <1% 52/1150 4,5% 27/307 8,8% ND/1062 <1% ND/1150# <1% 28/381 7,4% 2 ND/418 <1% ND/472 <1% ND/284 <1% ND/474 <1% ND/472 <1% ND/284 <1% 3 ND/1208 <1% ND/1948 <1% ND/1209 <1% ND/1251 <1% ND/1948 <1% ND/1214 <1% PB1.14 C!T 1 ND/144 <1% 48/433 11,1% ND/239 <1% ND/362 <1% 48/433 11,1% ND/239 <1% 2 ND/90 <1% ND/355 <1% ND/304 <1% ND/345 <1% ND/351 <1% ND/304 <1% 3 ND/562 <1% ND/792 <1% ND/496 <1% ND/633 <1% ND/655 <1% ND/504 <1% PB1.23 T!G 1 ND/207 <1% 30/535 5,6% ND/315 <1% ND/470 <1% 30/535 5,6% ND/315 <1% 2 ND/103 <1% ND/365 <1% ND/319 <1% ND/365 <1% 4/365 1,96% ND/319 <1% 3 ND/699 <1% ND/950 <1% ND/609 <1% ND/702 <1% ND/950 <1% ND/609 <1% PB1.87 A!G 1 ND/744 <1% ND/1644 <1% ND/1076 <1% ND/1218 <1% ND/1644 <1% ND/1076 <1% 2 49/365 13,4% ND/677 <1% ND/576 <1% 13/638 2,0% ND/674 <1% ND/576 <1% 3 ND/721 <1% ND/1156 <1% ND/793 <1% ND/731 <1% ND/1156 <1% ND/793 <1% PB1.2240 G!C 1 ND/757 <1% 23/1517 1,5% 26/515 5,0% ND/1266 <1% ND/1515# <1% 28/631 4,4% 2 ND/944 <1% ND/985 <1% ND/806 <1% ND/994 <1% ND/984 <1% ND/806 <1% 3 ND/274 <1% ND/439 <1% ND/253 <1% ND/301 <1% ND/439 <1% ND/253 <1% PB1.2268 A!G 1 5/470 1,1% 33/928 3,6% 22/278 7,9% 28/420 6,7% ND/928## <1% 23/354 6,5% 2 ND/798 <1% ND/829 <1% ND/671 <1% ND/839 <1% ND/829 <1% ND/671 <1% 3 ND/109 <1% ND/259 <1% ND/123 <1% ND/193 <1% ND/259 <1% ND/126 <1% PB1.2271 A!G 1 12/446 2,7% 59/901 6,5% 16/263 6,1% 29/413 7,0% 59/901 6,6% 21/336 6,3% 2 ND/729 <1% 47/810 5,8% 40/649 6,2% 43/750 5,73% 47/810 5,8% 40/649 6,2% 3 1/32 3,1% ND/123 <1% 2/83 2,4% 5/75 6,7% 5/124 4,03% 2/83 2,4% HA.867 C!T 1 59/1533 3,8% 206/3183 6,5% 104/1537 6,8% 112/1584 7,1% 206/3183 6,5% 109/1573 6,9% 2 59/2031 2,9% 150/2525 5,9% 127/2253 5,6% 144/2502 5,8% 150/2525 5,9% 127/2253 5,6% 3 11/180 6,1% 48/647 7,4% 28/385 7,3% 13/182 7,1% 48/647 7,4% 28/385 7,3% HA.963 T!C 1 122/1401 8,7% 446/3071 14,5% 189/1419 13,3% 200/1468 13,6% 446/3071 14,5% 193/1455 13,3% 2 90/1517 5,9% 318/2189 14,5% 247/1828 13,5% 308/2165 14,2% 318/2189 14,5% 247/1828 13,5% 3 5/69 7,2% 107/606 17,7% 47/293 16,0% 12/81 14,8% 107/606 17,7% 47/293 16,0% NP.1491 C!A 1 ND/278 <1% 71/583 12,2% ND/206 <1% ND/390 <1% ND/579# <1% ND/206 <1% 2 ND/723 <1% ND/769 <1% ND/692 <1% ND/766 <1% ND/769 <1% ND/692 <1% 3 ND/799 <1% ND/2031 <1% ND/1206 <1% ND/858 <1% ND/2031 <1% ND/1206 <1% NA.65 T!C 1 19/503 3,8% 52/1229 4,2% 16/467 3,4% 22/535 4,1% 52/1229 4,2% 20/540 3,7% 2 20/662 3,0% 50/1104 4,5% 45/992 4,5% 52/1063 4,9% 50/1104 4,5% 45/992 4,5% 3 24/557 4,3% 53/1099 4,8% 37/727 5,1% 28/584 4,8% 53/1099 4,8% 37/727 5,1% NA.78 T!C 1 23/599 3,8% 57/1403 4,1% 20/557 3,6% 23/622 3,7% 57/1403 4,1% 24/638 3,8% 2 21/692 3,0% 55/1147 4,8% 50/1033 4,8% 54/1109 4,9% 55/1147 4,8% 50/1033 4,8% 3 23/580 4,0% 51/1124 4,5% 37/735 5,0% 27/585 4,6% ND/1124# <1% 37/735 5,0% NA.89 T!C 1 23/713 3,2% 55/1670 3,3% 22/651 3,4% 26/731 3,6% 55/1670 3,3% 26/751 3,5% 2 23/798 2,9% 56/1261 4,4% 50/1134 4,4% 54/1224 4,4% 56/1261 4,4% 50/1134 4,4% 3 24/580 4,1% 55/1196 4,6% 40/775 5,2% 28/587 4,8% 55/1196 4,6% 40/775 5,2% NA.117 T!C 1 37/908 4,1% 87/2140 4,1% 36/818 4,4% 40/914 4,4% 87/2140 4,7% 43/922 4,7% 2 28/1102 2,5% 67/1631 4,1% ND/1459 <1% 70/1586 4,4% 67/1631 4,1% ND/1459 <1% 3 22/531 4,1% 57/1276 4,5% 42/812 5,2% 28/544 5,2% ND/1276# <1% 42/812 5,2% NA.126 T!C 1 37/983 3,8% 83/2294 3,6% 36/876 4,1% 39/973 4,0% 83/2294 3,6% 43/981 4,4% 2 31/1126 2,8% 72/1676 4,3% 65/1502 4,3% 75/1616 4,6% 72/1676 4,3% 65/1502 4,3% 3 26/519 5,0% 62/1395 4,4% 43/812 5,3% 30/537 5,6% 62/1395 4,4% 43/812 5,3% (Continued) Table 5. Comparison of sequencing methods and data processing pipelines Table 5. (Continued) Locations containing mSNV detections in the DPP3 mSNV analysis of the bam files but not in the original DPPs; Locations containing 1% mSNVs that could be reproduced by deleting DPP3s default ‘Direction and position filters’ with those exactly reproduced (#) and those approximately reproduced but with different coverages and/or variants (##). https://doi.org/10.1371/journal.pone.0229326.t005 forward/reverse balance) and read position (removal of systemic errors) are not met. However, DPP1 and DPP2 contain similar quality parameters in their mSNV identification process, indicating that different DPPs deal differently with quality parameters, and data could be excluded or included based on the DPP used. In addition, 9 additional mSNVs were identified in the .bam files compared to the original mSNV outputs. It needs to be noted that the cover- age of SP data analysed by DPP1 for positions identified with mSNVs was considerably lower compared to the coverage at that position in the input .bam files, suggesting additional quality filtering in the mSNV detection step of DPP1. However, the influence on mSNV identification was limited most likely due to the initial high nucleotide coverage. To better visualise the differences in coverages and allele counts a graphical display of the data for four positions showing mSNVs in different frequencies for each SP/DPP combination is included in S2 Fig. In general, SNVs were rarely missed due to low coverage, as also high coverage SP/DPP combinations display discrepancies (Tables 3 and 4). (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 21 / 28 https://doi.org/10.1371/journal.pone.0229326.t005 (Continued) Virus Position Sequence platform Data Processing pipeline Bam file generating processing pipeline 1 2 3 1 2 3 Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage Minor variants Percentage UKDD PB2.2277 T!G 1 ND/415 <1% 28/507 5,5% ND/475 <1% ND/503 <1% ND/507# <1% ND/475 <1% 2 ND/589 <1% ND/620 <1% ND/601 <1% ND/627 <1% ND/620 <1% ND/601 <1% 3 ND/1140 <1% ND/1996 <1% ND/2065 <1% ND/1186 <1% ND/1996 <1% ND/2071 <1% PB2.2278 T!G 1 ND/367 <1% ND/471 <1% ND/464## <1% ND/465 <1% ND/471 <1% 17/268 6,3% 2 ND/581 <1% ND/613 <1% ND/581 <1% ND/621 <1% ND/588 <1% ND/581 <1% 3 ND/1141 <1% ND/1985 <1% ND/1993 <1% ND/1184 <1% ND/1975 <1% ND/2004 <1% PB1.87 A!G 1 ND/387 <1% ND/440 <1% ND/439 <1% ND/451 <1% ND/417 <1% ND/439 <1% 2 26/327 8,0% 32/395 8,1% ND/351 <1% 33/385 8,6% ND/395# <1% ND/351 <1% 3 ND/617 <1% ND/1133 <1% ND/1136 <1% ND/622 <1% ND/1133 <1% ND/1136 <1% PB1.728 C!A 1 ND/750 <1% ND/832 <1% ND/836 <1% ND/853 <1% ND/832 <1% ND/836 <1% 2 ND/776 <1% 52/928 5,6% ND/829 <1% ND/888 <1% ND/912## <1% ND/829 <1% 3 ND/2459 <1% ND/4290 <1% ND/4293 <1% ND/2471 <1% ND/4287 <1% ND/4292 <1% PB1.730 C!T 1 ND/742 <1% ND/824 <1% ND/826 <1% ND/844 <1% ND/824 <1% ND/826 <1% 2 ND/767 <1% 57/1008 5,7% ND/832 <1% ND/893 <1% ND/1008# <1% ND/832 <1% 3 ND/2339 <1% ND//4286 <1% ND/4289 <1% ND/2464 <1% ND/4285 <1% ND/4284 <1% PB1.883 G!C 1 ND/942 <1% ND/997 <1% ND/997 <1% ND/1016 <1% ND/997 <1% ND/997 <1% 2 ND/1689 <1% ND/1856 <1% ND/1760 <1% ND/1867 <1% ND/1856 <1% ND/1760 <1% 3 ND/2479 <1% 47/690 6,8% ND/3681 <1% ND/2635 <1% ND/690## <1% ND/3697 <1% PA.49 G!C 1 ND/103 <1% 6/117 5,1% ND/115 <1% ND/113 <1% ND/117# <1% ND/115 <1% 2 ND/337 <1% ND/435 <1% ND/392 <1% ND/441 <1% ND/434 <1% ND/392 <1% 3 ND/111 <1% ND/207 <1% ND/204 <1% ND/113 <1% ND/206 <1% ND/206 <1% PA.82 C!T 1 ND/155 <1% ND/180 <1% ND/177 <1% ND/179 <1% ND/180 <1% ND/177 <1% 2 ND/695 <1% ND/809 <1% ND/745 <1% ND/797 <1% ND/809 <1% ND/745 <1% 3 ND/64 <1% ND/247 <1% 30/248 12,1% ND/74 <1% ND/247 <1% 30/248 12,1% NS.811 G!T 1 ND/221 <1% 17/270 6,3% ND/249 <1% ND/261 <1% ND/270# <1% ND/249 <1% 2 ND/2452 <1% ND/2725 <1% ND/2557 <1% ND/2742 <1% ND/2725 <1% ND/2557 <1% 3 ND/3117 <1% ND/4125 <1% ND/4139 <1% ND/3188 <1% ND/4124 <1% ND/4142 <1% Table 5. Discussion NGS data are used for different applications. Although sequence technologies and the accom- panying analysis tools are subjected to rapid development, a lot of follow-up research is based 22 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Comparison of sequencing methods and data processing pipelines on initial findings. Accuracy and repeatability are key values for proper scientific research but the impact of NGS results also reaches beyond science to clinical settings where important clinical management and treatment decisions are based on such results. In this study the com- parability of NGS data analyses were analysed using identical input material per virus but dif- ferent laboratory workflows from nucleic acid extraction and sequencing to data analysis. In addition, the COMPARE “Data Hub” platform was tested for the purpose of sharing large raw datafiles between institutions in an outbreak situation. Using this platform, raw sequence data files up to the size of 8 Gigabytes, alignment files and metadata files of three influenza A/H5N8 viruses were successfully shared in real-time among 3 institutions to allow independent sequencing and analysis procedures, including mSNV identification, to be performed. The Data Hub is available to all institutions. The aim of this study was to determine how comparable consensus and minority variant results were between laboratories performing their standard analyses, and whether discrepan- cies could be attributed to the SP, DPP or a combination of both. With the lack of a ground truth/gold standard, all data obtained were compared amongst each other. Importantly, reli- able consensus sequences were generated independently of the SP/DPP combination used, although the well-known artefactual InDels in homopolymer regions in SP3 (Roche 454 genome sequencer) sequence data required manual editing. Such consensus sequences rou- tinely form the basis for a detailed characterization of the influenza strain in an outbreak situa- tion, as they are used for the prediction of pathogenicity and pandemic potential of influenza strains. In contrast to the reproducible generation of consensus genome sequences, the hypothesis that minority variants could be identified reproducibly has to be rejected. The observed differ- ences were mainly attributed to the alignment processes in the different DPPs. The interpreta- tion of minority variant analysis thus needs a different level of careful standardization and awareness about the possible limitations as shown in this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Discussion Reproducibility of mSNV results appeared to be influenced by both the different SPs (resulting in different sequence depths Fig 2) and DPPs (resulting in differences in alignment and mSNV identification of the same input data, Fig 2 and Table 5). There was limited reproducibility of mSNV identification data, even for relative high frequency mSNVs. As expected, the reproducibility was best (30%) for mSNVs occurring in high frequency (10%), and least for the low frequent (1%) mSNVs (9.4% to 31.1%). Also, the number of positions with 1–5% mSNVs (with sufficient coverage) was much higher (250 in SP1 data, 213 in SP2 data, and 45 in SP3 data) than the number of positions with >5–10% mSNVs (n = 27) or >10% mSNVs (n = 10). The set-up of this study allowed many variables to influence the final result. The differences from first laboratory procedures and sample preparations up to the final analysis methods can all have contributed to the observed differences in mSNV identification. At this level, especially with lacking an NGS gold standard, it becomes difficult to determine which identified mSNVs are ‘true variants’ and which could be due to systematic errors introduced by RNA isolation methods, amplification, sequencing or manipulated by data processing pipeline settings. Unsurprisingly, the results of this study imply that the choice of SP influences the final output, but the results from this study also indicate that the DPP, especially the alignment process, influences coverage. The SP and DPP derived differences in coverage are of importance because up to a certain (currently unknown, probably SP/DPP dependent) threshold, a higher coverage will provide a more reliable result about the presence of mSNVs. Although the aim of this study was to explicitly compare the three institutions own standard workflows, some parameters (like the phred score and detection limit) were synchronized between the different DPPs. Moreover, the data from each SP were re-processed in each DPP. However, all DPPs use different underlying algorithms and interpret the set parameters differently which might PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 23 / 28 Comparison of sequencing methods and data processing pipelines all contribute to the observed differences. Discussion These results are partly in line with previous research that showed the need of NGS result validation and concluded that only those mSNVs with a coverage >100 and a frequency of >40% could be identified by NGS methods without second- ary confirmation [32], however, this conclusion was based on using the same sample prepara- tion method within a single laboratory. Another recent study sets the cut-off for intrahost virus diversity at 3% with input of at least 1000 RNA copies and a read depth of at least 400x at each genome position for Illumina sequencing [33]. Although some studies have been published on SP error rates [34–37] and PCR amplifica- tion induced variants [38–41], a gold standard system for mSNV analysis is lacking. In addi- tion, the DPPs can alter the data due to elimination or inclusion of certain sequences based on the set quality parameters. Allowing too many low-quality reads or being too stringent on the data will influence the coverage per position and might also influence the accuracy of the mSNV identification rate, especially when the coverage is low [42, 43]. Although a low comparability of mSNVs identified in the different SP and DPP combinations was observed, it can be concluded that 454 (SP3) sequencing has approximately the same accuracy as Illu- mina (SP1 and 2) sequencing based on the number and percentage of reproducible mSNVs in this dataset when ignoring InDel errors in homopolymer regions. Although, Roche 454 sequencing machines are no longer in production, it added value to include 454 sequencing as an alternative sequence platform with alternative chemistry to Illumina. In addition, because Roche 454 was the first commercially successful next generation sequencing system, it was used in research that served as a fundament for follow-up studies [44]. A comparison of Illumina with newer third or fourth generation sequencing platforms (e.g. Nanopore or Pac Bio) would be interesting in the future. However, the overall error rate remains higher than the shorter read technologies and recent work concludes that these new platforms are currently not suitable for the detection of minor variants [33]. In addition, it would be inter- esting to compare mSNV results of SPs outputting small sequence reads (like Illumina, 454 and Ion Torrent) to new sequencing techniques that output full-length sequence data (e.g. Nanopore [45]). Discussion The latter might be less vulnerable to quality trimming parameters com- pared to small reads and might provide a more consistent nucleotide coverage over complete gene segment. Although some studies have been published on SP error rates [34–37] and PCR amplifica- tion induced variants [38–41], a gold standard system for mSNV analysis is lacking. In addi- tion, the DPPs can alter the data due to elimination or inclusion of certain sequences based on the set quality parameters. Allowing too many low-quality reads or being too stringent on the data will influence the coverage per position and might also influence the accuracy of the mSNV identification rate, especially when the coverage is low [42, 43]. Although a low comparability of mSNVs identified in the different SP and DPP combinations was observed, it can be concluded that 454 (SP3) sequencing has approximately the same accuracy as Illu- mina (SP1 and 2) sequencing based on the number and percentage of reproducible mSNVs in this dataset when ignoring InDel errors in homopolymer regions. Although, Roche 454 sequencing machines are no longer in production, it added value to include 454 sequencing as an alternative sequence platform with alternative chemistry to Illumina. In addition, For mSNV analyses by different labs, very stringent SP/DPP protocols need to be evalu- ated, for instance by cross-validating results. To allow a better comparison it would be rec- ommended to create some kind of gold standard by for instance evaluating parameters based on sequencing of technical replicates, and controlled mixes of clones. The mSNV anal- ysis can be valuable for epidemiological tracing, to monitor early evolutionary events, or drug resistance, possibly host adaptation, but this would require reproducibility of study outcomes within and between laboratories. As this is currently not that case, more under- standing of biases and errors generated by sample processing (enrichment procedures), sequencing strategy (amplicons, shotgun), sequencing chemistry (each of which have their own internal error rates) and the approach to data processing and analysis is needed. Under- standing the parameters and thresholds in the software can be difficult and a systematic study using a pipeline where the effect of changing each of these parameters both individu- ally and in combination is required to determine the optimal settings for minor variant analysis. As alternate high-throughput sequencing technologies arise there will be a need to under- stand inherent error profiles and how those are handled in data processing approaches. Acknowledgments The authors would like to thank the staff of the European Nucleotide Archive and all technical staff involved in the supporting laboratory and avian surveillance work. S1 File. DPP3 Sequence analysis protocol. (PDF) S1 Fig. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position for full genome sequences of the UKDD and DETU virus reads mapped to the corresponding reference sequences. Panel A shows the coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs (SP1: lilac; SP2: yellow; SP3:green) for each of the DPPs. The X-axis rep- resents the position in the genome, the Y-axis represents the number of sequence reads per position. S2 Fig. Graphical display of the coverage and allele counts for four positions, showing mSNVs in different frequencies for each SP/DPP combination. Arrows indicate the approx- imate percentages in which the mSNVs were detected; 1–5% (orange), 5–10% (purple) and >10% (green). (TIF) Discussion Cross- validation should be supported by international proficiency tests on NGS techniques including mSNV analyses that would be instrumental in validation of results and may foster the trust in NGS-based diagnostics. 24 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326 February 20, 2020 Comparison of sequencing methods and data processing pipelines Supporting information S1 Table. PCR primers used in SP3 to cover the influenza A H5N8 gene segments. (PDF) S2 Table. SP/DPP overarching consensus sequences. (PDF) S3 Table. Number of raw sequences and influenza virus reads per SP per virus. (PDF) S1 File. DPP3 Sequence analysis protocol. (PDF) S1 Fig. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position for full genome sequences of the UKDD and DETU virus reads mapped to the corresponding reference sequences. Panel A shows the coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs (SP1: lilac; SP2: yellow; SP3:green) for each of the DPPs. The X-axis rep- resents the position in the genome, the Y-axis represents the number of sequence reads per position. (TIF) S2 Fig. Graphical display of the coverage and allele counts for four positions, showing mSNVs in different frequencies for each SP/DPP combination. Arrows indicate the approx- imate percentages in which the mSNVs were detected; 1–5% (orange), 5–10% (purple) and >10% (green). (TIF) References 1. Heather J. and Chain B, The sequence of sequencers: The history of sequencing DNA. Genomics, 2016. 107(1): p. 1–8. https://doi.org/10.1016/j.ygeno.2015.11.003 PMID: 26554401 2. 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Практические рекомендации по общим принципам проведения противоопухолевой лекарственной терапии
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ПРАКТИЧЕСКИЕ РЕКОМЕНДАЦИИ ПО ОБЩИМ ПРИНЦИПАМ ПРОВЕДЕНИЯ ПРОТИВООПУХОЛЕВОЙ ЛЕКАРСТВЕННОЙ ТЕРАПИИ Коллектив авторов: Трякин А. А., Бесова Н. С., Волков Н. М., Гладков О. А., Карасева В. Сакаева Д. Д., Фадеева Н. В., Федянин М. Ю. Коллектив авторов: Трякин А. А., Бесова Н. С., Волков Н. М., Гладков О. А., Карасева В. В., Сакаева Д. Д., Фадеева Н. В., Федянин М. Ю. 1. ВВЕДЕНИЕ Противоопухолевая лекарственная терапия является самостоятельным разделом онкологии, объединяющим химиотерапию, гормонотерапию, антиангиогенную, таргет- ную, иммуно- и поддерживающую терапию, и играет важную роль в лечении пациентов со злокачественными новообразованиями. Противоопухолевые препараты назначаются в соответствии с показаниями, указанными в инструкции к препарату. Допустимо назна- чение препарата по незарегистрированным показаниям или в незарегистрированных в инструкции дозировках при наличии информированного согласия пациента и соблю- дении следующих условий: a) данная опция указана в клинических рекомендациях; a) данная опция указана в клинических рекомендациях; б) данная опция не указана в клинических рекомендациях: • по жизненным показаниям по решению врачебной комиссии или онкологического консилиума; • по жизненным показаниям по решению врачебной комиссии или онкологического консилиума; • наличие научных обоснований эффективности данной терапии (научные пуб- ликации или регистрационные показания в других странах); • наличие научных обоснований эффективности данной терапии (научные пуб- ликации или регистрационные показания в других странах); • отсутствие более эффективных и (или) менее токсичных методов лечения. • отсутствие более эффективных и (или) менее токсичных методов лечени DOI: 10.18027 / 2224-5057-2022-12-3s2-27-40 DOI: 10.18027 / 2224-5057-2022-12-3s2-27-40 Цитирование: Трякин А. А., Бесова Н. С., Волков Н. М., Гладков О. А., Карасева В. В., Сакаева Д. Д. и соавт. Практические рекомендации по общим принципам проведения противоопухолевой лекар- ственной терапии. Злокачественные опухоли : Практические рекомендации RUSSCO #3s2, 2022 (том 12). 27–40. ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 27 Версия 2022 1.2. Виды противоопухолевой лекарственной терапии Адъювантная: назначается после радикального хирургического лечения или радикальной ЛТ с целью профилактики рецидива заболевания. Неоадъювантная: назначается при исходно операбельных опухолях до радикального хирургического лечения или до радикальной ЛТ с целью уменьшения объема опухоли и достижения патоморфологического ответа. Лечебная / индукционная: лекарственная терапия при метастатическом или местно- распространенном опухолевом процессе, направлена на увеличение продолжительности жизни пациента, в отдельных случаях может быть достигнуто выздоровление. Лечебная / индукционная: лекарственная терапия при метастатическом или местно- распространенном опухолевом процессе, направлена на увеличение продолжительности жизни пациента, в отдельных случаях может быть достигнуто выздоровление. Паллиативная: преследует цель устранения симптомов заболевания за счет уменьшения опухолевой массы, не преследует цель увеличения продолжительности жизни. Паллиативная: преследует цель устранения симптомов заболевания за счет уменьшения опухолевой массы, не преследует цель увеличения продолжительности жизни. Поддерживающая лекарственная терапия: направлена на  поддержание эффекта, достигнутого при лечебной / индукционной лекарственной терапии. Противоопухолевые лекарственные препараты могут быть использованы отдельно в виде монотерапии или в составе режимов комбинированной лекарственной терапии, а также могут сочетаться с другими видами терапии (лучевая, операция и др.). Противоопухолевые лекарственные препараты могут быть использованы отдельно в виде монотерапии или в составе режимов комбинированной лекарственной терапии, а также могут сочетаться с другими видами терапии (лучевая, операция и др.). Химиолучевая терапия — одновременное или  последовательное комбинированное применение лекарственной терапии (возможна ХТ, таргетная терапия, иммунотерапия) и ЛТ. Химиолучевая терапия — одновременное или  последовательное комбинированное применение лекарственной терапии (возможна ХТ, таргетная терапия, иммунотерапия) и ЛТ. Химиоиммунотерапия — одновременное комбинированное применение химиопрепаратов и иммунопрепаратов — моноклональных антител, интерферонов. Химиогормонотерапия — одновременное комбинированное применение химиопрепаратов и гормональных препаратов. Химиогормонотерапия — одновременное комбинированное применение химиопрепаратов и гормональных препаратов. 1.1. Цели лекарственного лечения злокачественных опухолей 1.1. Цели лекарственного лечения злокачественных опухолей • Излечение пациента • Излечение пациента • Контроль заболевания и увеличение продолжительности жизни • Устранение симптомов болезни и улучшение качества жизни • Контроль заболевания и увеличение продолжительности жизни • Устранение симптомов болезни и улучшение качества жизни том  /  vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS Russian Society of Clinical Oncology MALIGNANT TUMOURS Russian Society of Clinical Oncology Практические рекомендации 28 Практические рекомендации 1.3. Способы применения противоопухолевых лекарственных препаратов Системный: рассчитан на  резорбтивный противоопухолевый эффект при  приеме противоопухолевого препарата внутрь, введении п / к, в / в, в / м, ректально. Регионарный: лекарственный препарат вводится в сосуды, питающие новообразование, при этом системное воздействие, т. е. поступление цитостатика в другие органы минимально. Локальный метод: препараты наносятся на поверхностные очаги в виде мазей или растворов, вводятся непосредственно в опухолевый очаг, в серозные полости (брюшную — с целью купирования асцита, грудную — с целью купирования плеврита), спинномозговой канал (интратекально), применяются внутрипузырно. MALIGNANT TUMOURS Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology 29 ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ Версия 2022 2. ОБЩИЕ ПРИНЦИПЫ ЛЕКАРСТВЕННОЙ ТЕРАПИИ ЗЛОКАЧЕСТВЕННЫХ НОВООБРАЗОВАНИЙ Выбор терапии основывается на локализации и морфологической форме опухоли, ее молекулярном профиле, распространенности опухолевого процесса, эффективности ранее проводимой терапии, состоянии пациента, цели лечения в конкретной клинической ситуации, соотношении потенциальной пользы и токсичности лечения. Если целью является излечение, то даже значительные побочные эффекты являются приемлемыми. Когда целью лечения является продление жизни, то следует тщательно рассмотреть соотношение ожидаемой пользы и выраженности побочных эффектов терапии. При паллиативной терапии лечение следует выбирать так, чтобы вероятность выраженной токсичности была небольшой. В этом случае побочные эффекты не должны быть тяжелее, чем симптомы заболевания. При лечении метастатических форм опухолей может быть использовано несколько линий терапии. Переход от одной линии к другой производится при прогрессировании заболевания или при непереносимости терапии из‑за выраженных нежелательных явле- ний. При большинстве опухолей в случае непрерывного прогрессирования заболевания на фоне трех последовательных линий терапии (при раке молочной железы — до 5) дальнейшее лекарственное лечение не имеет смысла. том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии Российское общество клинической онкологии 2.1. Характеристики опухоли, учитываемые при планировании лекарственной терапии При постановке первичного диагноза предпочтительна его гистологическая вери- фикация. При тяжелом общем состоянии пациента, технических трудностях или невозможности проведения биопсии опухоли, можно основываться на результатах цитологического исследования. В ряде ситуаций, например, при герминогенных опухолях, хориокарциноме матки, гепатоцеллюлярном раке допускается начало терапии при наличии характерной клинической картины, данных инструментальных методов обследования и повышенного уровня специфических маркеров. При развитии рецидива заболевания (местный рецидив опухоли или появление метастазов) необходимо стремиться к его гистологической или цитологической вери- фикации, если она технически выполнима без существенного риска серьезного вреда для пациента, с целью исключения метахронных, в том числе, индуцированных зло- качественных новообразований, изменения морфологической формы (трансформации) и / или молекулярного профиля опухоли, особенно в следующих ситуациях: • при поздних рецидивах; • в ситуациях, когда возможное изменение молекулярного профиля опухоли влечет за собой коррекцию терапевтических подходов; • в ситуациях, когда возможное изменение молекулярного профиля опухоли влечет за собой коррекцию терапевтических подходов; рр р • в случае нетипичного варианта метастазирования опухоли; рр р • в случае нетипичного варианта метастазирования опухоли; • при наличии солитарного метастаза как единственного проявления заболевания. MALIGNANT TUMOURS Russian Society of Clinical Oncology том  /  vol. 12 № 3s2 • 2022 Практические рекомендации 30 Практические рекомендации При невозможности верификации рецидива заболевания, тактика лечения опре- деляется исходя из данных анамнеза, клинической картины, данных дополнительных инструментальных методов исследований, определения опухолевых маркеров. 2.3. Противопоказания к проведению лекарственной терапии Противопоказания к проведению лекарственной терапии могут носить абсолютный и относительный характер. 1. Мнение пациента и его выбор. 1. Мнение пациента и его выбор. 2. Возраст. 3. Общее состояние (оценивается в баллах по шкале ECOG). При статусе ECOG ≥ 3 баллов ХТ, как правило, не показана (за исключением больных, не получавших ранее терапии, с высокочувствительными к данному виду лечения опухолями, при которых высока вероятность достижения выраженного противоопухолевого эффекта); решение о назначении таргетной терапии, гормональной и иммунотерапии принимается индивидуально. 3. Общее состояние (оценивается в баллах по шкале ECOG). При статусе ECOG ≥ 3 баллов ХТ, как правило, не показана (за исключением больных, не получавших ранее терапии, с высокочувствительными к данному виду лечения опухолями, при которых высока вероятность достижения выраженного противоопухолевого эффекта); решение о назначении таргетной терапии, гормональной и иммунотерапии принимается индивидуально. 4. Сопутствующие заболевания. Необходима оценка спектра сопутствующей пато- логии, тяжести и прогноза для каждого заболевания. Выраженные сопутствующие заболевания и их лечение могут являться противопоказанием для проведения противоопухолевой лекарственной терапии. В случаях, когда продолжительность жизни пациента определяется не опухолевым, а сопутствующим заболеванием, проведение противоопухолевой лекарственной терапии нецелесообразно. 4. Сопутствующие заболевания. Необходима оценка спектра сопутствующей пато- логии, тяжести и прогноза для каждого заболевания. Выраженные сопутствующие заболевания и их лечение могут являться противопоказанием для проведения противоопухолевой лекарственной терапии. В случаях, когда продолжительность жизни пациента определяется не опухолевым, а сопутствующим заболеванием, проведение противоопухолевой лекарственной терапии нецелесообразно. 5. Предшествующее противоопухолевое лечение (количество курсов лечения, ис- пользованные лекарственные препараты, в случае применения антрациклинов — их суммарные дозы, достигнутый ранее ответ опухоли на терапию, переносимость терапии), остаточные проявления токсичности. 6. Психосоциальный статус пациента. Важной составляющей успешной терапии явля- ется комплаентность пациента — способность и готовность пациента соблюдать все предписания лечащего врача, график лечения и обследования. 6. Психосоциальный статус пациента. Важной составляющей успешной терапии явля- ется комплаентность пациента — способность и готовность пациента соблюдать все предписания лечащего врача, график лечения и обследования. 2.2. Характеристики пациента, учитываемые при планировании лекарственной терапии 1. Мнение пациента и его выбор. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии том  /  vol. 12 № 3s2 • 2022 2.3.1. Абсолютные противопоказания Абсолютным противопоказанием является крайне тяжелое (статус по  шкале ECOG 4 балла) общее состояние пациента. В каждой конкретной ситуации должно оцениваться соотношение потенциальных пользы и вреда противоопухолевой лекарственной терапии. Например, при излечимых опухолях (герминогенные опухоли, хориокарцинома матки, лимфомы) или при исполь- зовании малотоксичных высокоэффективных препаратов (ингибиторы EGFR при раке легкого, ГТ) терапия первой линии может проводиться пациентам и в крайне тяжелом том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology MALIGNANT TUMOURS Russian Society of Clinical Oncology ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 31 Версия 2022 состоянии. Однако при этих же опухолях проведение терапии поздних линий, когда шанс на излечение или значимое продление жизни минимален, задачей терапии ста- новится контроль симптомов заболевания при минимизации токсичности и риска тяжелых осложнений. том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии 2.3.2. Относительные противопоказания Относительными противопоказаниями к проведению терапии является активная инфекция, ранее перенесенный инфаркт миокарда, острое нарушение мозгового кровообращения давностью менее 6 мес., острый (в срок до 5–7 дней) тромбоз глубоких вен. Для многих препаратов существуют специфические относительные противо- показания. Для цисплатина: состояние по шкале ECOG > 1 балла; клиренс креати- нина < 50–60 мл / мин.; сенсоневральная тугоухость > 1ст. и периферическая нейро- патия > 1 ст.; сердечная недостаточность III–IV класса по NYHA. Для антиангиогенных препаратов — кровотечение, наличие свища, перфорации полых органов ЖКТ в ана- мнезе, артериальные тромбоэмболические события в анамнезе, неконтролируемая артериальная гипертензия. Острые инфузионные реакции 1–2 степени тяжести, купирующиеся введением антигистаминных препаратов и системных ГКС, не являются абсолютным противопо- казанием к повторному применению вызвавшего их препарата, но требуют усиления премедикации: 20 мг дексаметазона (или его эквивалента) внутрь приблизительно за 12 и 6 часов до введения препарата и / или 20 мг дексаметазона внутривенно примерно за 30–60 мин. до введения препарата, 50 мг дифенгидрамина (или его эквивалента) внутривенно и 50 мг ранитидина (или 20 мг фамотидина) внутривенно за 30–60 ми- нут до введения препарата, удлинения времени инфузии или применения того же препарата другой фирмы-производителя, проведения протокола десенсибилизации (например, при лечении карбоплатином, оксалиплатином) (см. возможный вариант десенсибилизации: Тюляндина А. С., Коломиец Л. А., Морхов К. Ю., и соавт. Практические рекомендации по лекарственному лечению рака яичников, первичного рака брюшины и рака маточных труб. Злокачественные опухоли: Практические рекомендации RUSSCO #3s2, 2021 (том 11). 10). В случае развития клинически значимых аллергических реакций, а также при отсут- ствии в медицинской организации необходимого противоопухолевого лекарственного препарата может быть произведена замена его на другой, схожий по эффективности препарат, в рамках одной группы: например, при меланоме пембролизумаб может быть заменен на ниволумаб в монотерапии; паклитаксел на доцетаксел и, наобо- рот, — при большинстве опухолей; карбоплатина на цисплатин — при раке яич- ников, раке молочной железы, раке легкого; оксалиплатина на цисплатин — при раке желудка, капецитабин на инфузионный фторурацил — при большинстве опухолей. Равноэффективными и взаимозаменяемыми также являются ингибиторы ароматазы, цетуксимаб и панитумумаб при колоректальном раке, пембролизумаб и ниволумаб при меланоме и т. д. том  /  vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии Практические рекомендации 32 2.4. Поддержание дозовой интенсивности противоопухолевой терапии Интенсивность цитотоксической ХТ определяется как доза, вводимая в единицу времени. Она может быть повышена за счет увеличения разовой дозы (высокодоз- ные режимы) или уменьшения интервалов между введениями (уплотненные режимы). При производственной необходимости или при клинических показаниях допускается раздельное применение цитостатических препаратов и противоопухолевых моно- клональных антител, интервал между которыми может достигать 7 дней. Например, комбинация доцетаксел + трастузумаб + пертузумаб (все препараты в день 1‑й, курсы каждые 3 недели) может быть применена как доцетаксел (день 1‑й), пертузумаб + трасту- зумаб (день 4‑й, курсы каждые 3 недели). С целью поддержания дозовой интенсивности терапии, а также профилактики развития фебрильной нейтропении после завершения цитостатической терапии возможно применение Г-КСФ, наиболее распространенным представителем которого является филграстим и его пролонгированные формы. Доза и продолжительность терапии филграстимом индивидуальны и определяются его клинической эффективностью, оцениваемой по динамике уровня нейтрофилов. Вме- сто дозы 5 мкг / кг допустимо применение филграстима в фиксированных суточных дозах 300 мкг или 480 мкг. Возможно досрочное прекращение терапии филграстимом в ситуациях, когда после первоначального падения уровня нейтрофилов отмечается их рост до ≥ 10 × 109 / л либо, если их снижение после ХТ не наблюдалось, при достижении уровня нейтрофилов ≥ 40–50 × 109 / л. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии Режимы лечения в зависимости от дозы цитотоксического препарата В зависимости от интенсивности дозы цитотоксического препарата выделяют: 1) низкодозную (низкоинтенсивную) ХТ: часто рекомендуется больным пожилого и стар- ческого возраста, пациентам с серьезной сопутствующей патологией, при тяжелом и среднетяжелом общем состоянии, при проведении терапии с паллиативной целью; 2) ХТ стандартной дозовой интенсивности; 1) низкодозную (низкоинтенсивную) ХТ: часто рекомендуется больным пожилого и стар- ческого возраста, пациентам с серьезной сопутствующей патологией, при тяжелом и среднетяжелом общем состоянии, при проведении терапии с паллиативной целью; 2) ХТ стандартной дозовой интенсивности; 3) высокодозную (высокоинтенсивную) ХТ, как правило, характеризующуюся высокой миело-и иммуносупрессивностью, вплоть до полной миелоаблативности; 3) высокодозную (высокоинтенсивную) ХТ, как правило, характеризующуюся высокой миело-и иммуносупрессивностью, вплоть до полной миелоаблативности; 4) сверхвысокодозную ХТ или ХТ сверхвысокой интенсивности, которая, как правило, представляет собой модификации уже существующих режимов высокодозной ХТ с добавлением дополнительных химиотерапевтических агентов или с дополнитель- ной эскалацией доз одного или нескольких химиотерапевтических агентов; 5) метрономную ХТ — регулярное назначение цитостатических препаратов в дозах, значительно меньших максимально переносимых (в низких дозах), с небольшими (значительно меньшими, чем в стандартных режимах) интервалами времени; 5) метрономную ХТ — регулярное назначение цитостатических препаратов в дозах, значительно меньших максимально переносимых (в низких дозах), с небольшими (значительно меньшими, чем в стандартных режимах) интервалами времени; 6) режим с уплотненным введением химиопрепаратов (доз-денсный режим): рас- сматривается при быстро пролиферирующих опухолях; характеризуется меньшим интервалом (2 недели вместо 3‑недельного интервала) между введениями цито- статических препаратов и требует профилактического использования Г-КСФ. 6) режим с уплотненным введением химиопрепаратов (доз-денсный режим): рас- сматривается при быстро пролиферирующих опухолях; характеризуется меньшим интервалом (2 недели вместо 3‑недельного интервала) между введениями цито- статических препаратов и требует профилактического использования Г-КСФ. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 33 Многие виды противоопухолевой лекарственной терапии (прежде всего, цитотокси- ческой) обладают узким терапевтическим окном, в связи с чем, важно придерживаться рекомендованных доз препаратов, а также интервалов между их введениями. Расчет дозы цитотоксического средства для большинства препаратов выполняется на площадь поверхности тела, определить которую можно по формуле Мостеллера, Дюбуа и Дюбуа в модификации, а также при помощи номограмм и калькуляторов. Рекомендуется округление расчетной площади поверхности тела до первого знака после запятой. Формула Мостеллера (Mosteller, 1987): Формула Дюбуа и Дюбуа (модификация): Формула Дюбуа и Дюбуа (модификация): том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии Практические рекомендации 34 Практические рекомендации переносимости редуцированных доз допускается их эскалация, вплоть до полных доз, на последующих курсах. Клинически значимая токсичность ранее проведенного курса терапии также является основанием для редукции разовых доз препаратов, реэскалация дозы на последующих курсах, как правило, не производится. ХТ часто носит цикловой характер. Цикл ХТ — период времени, исчисляемый с пер- вого дня первого введения цитостатиков до планируемого первого дня следующего курса, включает в себя дни введения препаратов и плановый перерыв между курсами. Примером 21‑дневного цикла является режим XELOX: продолжительность курса 14 дней, перерыв между курсами 7 дней; примером 14‑дневного цикла являются режимы типа FOLFOX или FLOT, повторное введение препаратов в которых запланировано на 15 день, считая с 1‑го дня введения препаратов предыдущего курса. Примером 6‑недельного цикла являются режимы, содержащие некоторые производные нитрозометилмочевины или митомицина С. Допускается смещение даты начала следующего лечебного цикла на 3 дня в мень- шую и большую стороны от запланированной даты без причин медицинского характера (производственная необходимость, государственные праздники, семейные обстоятель- ства и т. п.). Начало очередного цикла может быть отложено по медицинским показаниям в следующих ситуациях: • побочные эффекты лечения, не разрешившиеся до степени 0–1 к началу следующего цикла (за исключением алопеции); • побочные эффекты лечения, не разрешившиеся до степени 0–1 к началу следующего цикла (за исключением алопеции); • обострение сопутствующих заболеваний; • любое заболевание в острой фазе. Для большинства режимов ХТ стандартными требованиями к показателям гемо- граммы, позволяющим начать очередной курс ХТ, являются АЧН ≥ 1,5 × 109 / л и количе- ство тромбоцитов ≥ 100 × 109 / л. Для отдельных комбинаций и клинических ситуаций (лечение онкогематологических заболеваний, еженедельное введение паклитак- села, гемцитабина, комбинация гемцитабина с наб-паклитакселом, режим ВЕР или ЕР при герминогенных опухолях) применяются особые критерии гемограммы, достаточ- ные для начала курса / введения химиопрепарата и учитываемые при редукции доз препаратов на 8‑й и 15‑й дни. Уровень гемоглобина не должен служить поводом к отсрочке цикла терапии; в слу- чаях выраженной анемии требуется проведение гемотрансфузии с последующим про- должением лечения. ППТ (м2) = вес [кг] 0,425 × рост [см] 0,725 / 139,2 У больных с ожирением при отсутствии сопутствующей патологии расчет дозы препарата производится исходя из фактической массы тела, особенно если целью терапии является излечение. При наличии сопутствующей патологии, а также в случае паллиативных целей терапии при площади поверхности тела свыше 2 м 2 возможен расчет дозы на площадь 2 м2 на первом курсе терапии. При хорошей переносимости лечения на последующих курсах рекомендуется одномоментная или последовательная (в течение двух-трех курсов терапии) эскалация доз препаратов с расчетом на акту- альные вес и рост. Для некоторых химиотерапевтических препаратов установлены предельно допу- стимые разовые дозы (например, для винкристина — 2 мг, для блеомицина — 30 мг, для карбоплатина — 1000 мг в комбинациях и 1200 мг — в монотерапии). Для препаратов, обладающих дозолимитирующей кумулятивной токсичностью, установлены предельно допустимые кумулятивные дозы: например, с учетом кумулятивной кардиотоксичности антрациклинов, кумулятивная доза для доксорубицина составляет 550 мг / м2, для эпи- рубицина — 1000 мг / м2; для блеомицина с учетом потенциальной возможности развития фиброза легких кумулятивная доза составляет 360 мг. В отдельных ситуациях, когда отсутствуют другие более эффективные опции лечения и при условии тщательного мониторинга кардиотоксичности на фоне профилактической кардиотропной терапии возможно превышение кумулятивных доз антрациклинов. Допустимо округление реально вводимой дозы противоопухолевого лекарственного препарата в диапазоне до 10 % от расчетной. Перерасчет разовой дозы на последующих курсах производится по актуальным показателям роста и веса в том случае, если изме- нение массы тела пациента превысило 10 %. Допускается исходная редукция разовой дозы препарата в случаях, когда при- менение рекомендуемых доз представляет для пациента повышенный риск развития осложнений. К подобным ситуациям относятся общий статус по шкале ЕСОG 2–3, стар- ческий биологический возраст, большой объем облучения плоских костей в анамнезе, неудовлетворительные показатели функции костного мозга, почек, печени. При хорошей ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology том  /  vol. 12 № 3s2 • 2022 2.4.1. Обеспечение венозного доступа Так как большинство противоопухолевых лекарственных препаратов вводится в / в, необходимо обеспечение максимального удобства и комфорта пациентам. При хорошем состоянии периферических вен и коротких (менее 12 часов) в / в инфузиях возможно про- ведение лечения через периферические венозные катетеры. Для проведения режимов при затрудненном периферическом венозном доступе, а также при длительных (> 12 часов) инфузиях препарата необходимо обеспечение центрального венозного доступа, что может быть достигнуто различными путями: том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 35 Версия 2022 • установкой периферически имплантируемого центрального венозного катетера (ПИК-катетер), который подходит для краткосрочного и среднего по длительности (от 6 дней до 6 мес.) использования; • установкой периферически имплантируемого центрального венозного катетера (ПИК-катетер), который подходит для краткосрочного и среднего по длительности (от 6 дней до 6 мес.) использования; • установкой полностью имплантируемой венозной порт системы, для последующей работы с которой используется специальная игла Губера; является оптимальным вариантом при длительных (от 3 мес.) курсах лечения; • катетеризацией центральной вены на каждом курсе лечения (менее предпочтитель- ный подход). • катетеризацией центральной вены на каждом курсе лечения (менее предпочтитель- ный подход). Проведение суточных и многосуточных инфузий требует специального оборудова- ния, наиболее популярным является эластомерное инфузионное устройство одноразо- вого применения, специально разработанное для планового введения лекарственных препаратов с заданной скоростью и в определенный промежуток времени. Для ряда препаратов (например, фторурацила) необходимо защищать от разрушения светом инфузионные системы и линии. 2.5. Оценка эффективности лечения Плановая оценка эффективности ХТ выполняется каждые 6–12 недель (в зависимости от вида опухоли и задач терапии), эндокринной и иммунотерапии — каждые 8–16 недель, внеплановая оценка проводится по показаниям, в частности при подозрении на про- грессирование болезни. Интервал в оценке эффективности лечения при проведении адъювантной терапии раннего рака молочной железы может достигать 6 месяцев в связи с низким риском развития рецидива. Ориентиром для оценки эффекта лечения могут служить международные крите- рии оценки ответа солидных опухолей на терапию (Response Evaluation Criteria in Solid Tumors (RECIST) последняя версия 1.1 2016 или iRECIST при использовании иммуно- терапевтических препаратов), разработанные исходно для клинических исследований и основанные на определении размера опухоли методами лучевой диагностики (КТ, МРТ). На практике для оценки эффективности лечения допустимо применение методов ультразвуковой диагностики, планарной рентгенографии, эндоскопии и объективного физикального обследования. Для получения достоверных данных о динамике процесса необходимо использовать один и тот же диагностический метод, выбранный до начала терапии и позволяющий качественно визуализировать опухолевые очаги, с подробным описанием выявленных изменений в процессе лечения. том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии 2.5.1. Критерии RECIST 1.1 Современными критериями ответа опухоли на  терапию являются критерии RECIST 1.1 от 2009 г. Основу оценки составляет определение размера опухоли с помощью методов лучевой диагностики (рентгенографии, КТ, МРТ) (см. «Методические рекомен- дации по применению критериев ответа солидных опухолей на химиотерапевтическое лечение (RECIST 1.1). Правительство Москвы, Департамент здравоохранения г. Москвы, ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology том  /  vol. 12 № 3s2 • 2022 том  /  vol. 12 № 3s2 • 2022 том  /  vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology Практические рекомендации 36 Практические рекомендации ГБУЗ «Научно-практический центр медицинской радиологии Департамента здраво- охранения г. Москвы», Москва 2018 г.). Опухолевые очаги В соответствии с критериями RECIST опухолевые очаги подразделяются на два типа: 1. Измеряемые очаги — очаги, выявляемые и составляющие по наибольшему диаметру: • ≥ 10 мм при спиральной КТ при реконструкции срезов толщиной в 5 мм; • ≥ 20 мм при шаговой КТ с толщиной среза 10 мм; • ≥ 20 мм при рентгенографии грудной клетки; • лимфатические узлы > 15 мм в измерении, перпендикулярном максимальному диаметру (т. е. по короткой оси). 2. Неизмеряемые очаги — очаги, выявляемые и составляющие по наибольшему диа- метру при шаговом КТ < 20 мм, а при спиральной КТ < 10 мм. К неизмеряемым очагам относятся: мелкие очаги (< 10 мм), лептоменингеальные метастазы, плеврит, асцит, перикардит, кисты, лимфогенный канцероматоз кожи или легких, метастазы в костях, воспалительные изменения молочных желез, очаги с кистозной и некротической трансформацией, увеличение размеров органов (гепатоспленомегалия), перитоне- альные импланты. Алгоритм оценки 1. В первую очередь измеряемые очаги оцениваются количественно. 1. В первую очередь измеряемые очаги оцениваются количественно. 2. Далее нужно выбрать таргетные очаги, которые в процессе терапии будут исполь- зоваться для контроля; таргетный очаг сохраняется на протяжении всех повтор- ных исследований таргетным, даже если размеры очага уменьшатся до предела погрешности метода исследования. В качестве таргетного рекомендуется выбирать не наибольший, а наиболее четко визуализируемый и оптимальный для измерения очаг. В качестве таргетного рекомендуется выбирать не наибольший, а наиболее четко визуализируемый и оптимальный для измерения очаг. MALIGNANT TUMOURS Russian Society of Clinical Oncology ТА РГЕ ТНЫЕ ОЧ А ГИ • Измеряемые очаги, максимально до 2 очагов на орган, в сумме — до 5 очагов; очаги, отражающие пораженный орган, должны соответствовать критериям таргетного очага; • Измеряемые очаги, максимально до 2 очагов на орган, в сумме — до 5 очагов; очаги, отражающие пораженный орган, должны соответствовать критериям таргетного очага; • выбираются, исходя из их размеров (с наибольшим диаметром) и пригодности для точного повторного измерения; • выбираются, исходя из их размеров (с наибольшим диаметром) и пригодности для точного повторного измерения; • очаги в подвижных органах (например, в органах ЖКТ, яичниках) могут быть непод- ходящими для оценки; • очаги в подвижных органах (например, в органах ЖКТ, яичниках) могут быть непод- ходящими для оценки; • указываются только аксиальные размеры. • указываются только аксиальные размеры. том  /  vol. 12 № 3s2 • 2022 ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 37 Версия 2022 НЕ ТА РГЕ ТНЫЕ ОЧ А ГИ • Могут включать как измеряемые, так и неизмеряемые очаги; у р , р ; • к измеряемым нетаргетным очагам относят все измеряемые очаги в данном органе, которые не вошли в число таргетных в связи с превышением максимально допусти- мого количества (до 2 очагов на орган, всего — не более 5); • нетаргетные очаги могут не соответствовать критериям измеряемых, т. е. быть не измеряемыми; • нетаргетные очаги могут не соответствовать критериям измеряемых, т. е. быть не измеряемыми; р • в идеале должны включать все очаги, не отнесенные к категории таргетны ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии Критерии оценки рии оценки тичный ответ — уменьшение суммы диаметров таргетных очагов на ≥ 30 %. й ( ) б й ый ответ — уменьшение суммы диаметров таргетных очагов на ≥ 30 %. • Частичный ответ — уменьшение суммы диаметров таргетных очагов на ≥ 3 • Полный ответ — исчезновение всех (таргетных и нетаргетных) очагов; любой из ранее увеличенных лимфатических узлов должен иметь размер < 10 мм по короткой оси. • Прогрессирование заболевания — увеличение на ≥ 20 % суммы диаметров таргетных очагов; появление одного или нескольких новых очагов; безусловная прогрессия нетаргетных очагов. • Прогрессирование заболевания — увеличение на ≥ 20 % суммы диаметров таргетных очагов; появление одного или нескольких новых очагов; безусловная прогрессия нетаргетных очагов. • Стабилизация заболевания — признаки, не соответствующие критериям полного или частичного ответа, а также прогрессирования заболевания. Общий ответ Таргетные очаги Нетаргетные очаги Новые очаги Полный ответ Полный ответ Полный ответ Нет Частичный ответ Полный ответ Частичный ответ / ста- билизация заболева- ния Нет Частичный ответ Частичный ответ Отсутствие прогрессирования заболевания Нет Стабилизация заболевания Стабилизация заболевания Отсутствие прогрессирования заболевания Нет Прогрессирование заболевания Прогрессирование заболевания Любой ответ Есть или нет Прогрессирование заболевания Любой ответ Прогрессирование заболевания Есть или нет Прогрессирование заболевания Любой ответ Любой ответ Нет ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology 38 Практические рекомендации 2.6. Обеспечение безопасности лекарственной противоопухолевой терапии том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии 2.6.1. Минимальные лабораторные и инструментальные обследования при проведении противоопухолевой лекарственной терапии крови ранее, чем через 7 дней после начала курса цитостатической терапии в отсутствии клинических показаний. Рекомендуемая периодичность выполнения ЭКГ в процессе химио-и / или таргетной терапии составляет 3–4 мес. 2.6.2. Мониторинг безопасности противоопухолевой лекарственной терапии Оценка побочных эффектов терапии выполняется на каждом цикле. Общеприня- той шкалой являются «Общие терминологические критерии нежелательных явлений» (Common Terminology Criteria for Adverse Events (CTCAE), версия 5, 2017 г.). При развитии клинически значимых побочных явлений (3–4 степени, за исключением гематологиче- ской токсичности) выполняется редукция дозы на 20–40 %, если другое не предусмо- трено в инструкции по применению препарата. При купировании клинически значимых побочных явлений в отдельных ситуациях возможно возобновление лечения в полных дозах, если другой подход не предлагается в инструкции по применению препарата. Для некоторых видов кумулятивной токсичности, например, легочной, нейротоксич- ности клинически значимой может являться ≥ 2 степень выраженности. При проведении паллиативной химиотерапии назначение схемы каждой из линий и изменения доз пре- паратов определяются врачом-онкологом (специалистом по лекарственной терапии) без привлечения консилиума. Следует иметь ввиду, что для дозолимитирующих кумулятивных видов токсичности, таких как нейротоксичность, ототоксичность, легочная токсичность, кардиотоксич- ность отсутствуют профилактические меры с доказанной клинической активностью. Поскольку кумулятивная токсичность развивается в процессе цикловой ХТ по мере увеличения числа курсов, необходим целенаправленный и подробный сбор жалоб пациента перед началом каждого цикла лечения. При выявлении клинических сим- птомов, позволяющих заподозрить развитие кумулятивной токсичности, возможно привлечение (при наличии необходимости) профильных специалистов и назначение необходимых методов обследования и лечения с последующим мониторингом выра- женности клинических симптомов побочного эффекта и повторными обследованиями при нарастании их тяжести. 2.6.1. Минимальные лабораторные и инструментальные обследования при проведении противоопухолевой лекарственной терапии До начала первого курса лекарственной терапии рекомендуется выполнение про- цедур минимального уровня обследования, перечень которых может быть расширен по клиническим показаниям: • клинический анализ крови (с определением уровня гемоглобина, эритроцитов, лейкоцитов с подсчетом лейкоцитарной формулы, нейтрофилов, тромбоцитов); выполняется не более чем за 14 дней до начала лечения; • клинический анализ крови (с определением уровня гемоглобина, эритроцитов, лейкоцитов с подсчетом лейкоцитарной формулы, нейтрофилов, тромбоцитов); выполняется не более чем за 14 дней до начала лечения; • биохимический анализ крови (с определением уровня глюкозы, общего билирубина, АЛТ, АСТ, общего белка, креатинина); выполняется не более чем за 14 дней до начала лечения; • коагулограмма (фибриноген, АЧТВ, протромбиновое время); выполняется не более чем за 30 дней до начала лечения; • общеклинический анализ мочи (выполняется не более чем за 14 дней до начала лечения); • ЭКГ (выполняется не более чем за 30 дней до начала лечения); • по показаниям: – эхоКГ с определением ФВлж (при планировании кардиотоксичной терапии трастузумабом, пертузумабом, трастузумабом эмтанзином, антрациклинами); – аудиометрия (при наличии / усугублении клинических симптомов снижения слуха при планировании / проведении терапии цисплатином, но не карбоплатином или оксалиплатином); – консультации специалистов — по показаниям, определяемым лечащим врачом. Например, консультация эндокринолога или кардиолога — при необходимости назначения или коррекции проводимой терапии в случае ее недостаточной эффективности. – по показаниям (выраженная общая слабость, удлинение интервала QT) — опре- деление уровня магния у пациентов, получающих цетуксимаб или панитумумаб. – по показаниям (выраженная общая слабость, удлинение интервала QT) — опре- деление уровня магния у пациентов, получающих цетуксимаб или панитумумаб. В дальнейшем в процессе химио- или таргетной терапии перед началом каждого курса должен быть выполнен общеклинический (выполняется не более чем за 5 дней до проведения очередного курса) и биохимический анализы крови в вышеуказанном объеме (выполняется не более чем за 7 дней до проведения очередного курса), при еже- недельных режимах биохимический анализ крови может выполняться каждые 3–4 недели, остальные анализы — по клиническим показаниям. При проведении ГТ, обладающей минимальной токсичностью (антиэстрогены, ингибиторы ароматазы, агонисты ГРГ) вышеуказанный лабораторный мониторинг допускается проводить реже (до 1 раза в два месяца). Не рекомендуется рутинно выполнять контрольный общеклинический анализ том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 39 Версия 2022 крови ранее, чем через 7 дней после начала курса цитостатической терапии в отсутствии клинических показаний. Рекомендуемая периодичность выполнения ЭКГ в процессе химио-и / или таргетной терапии составляет 3–4 мес. том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологи MALIGNANT TUMOURS Russian Society of Clinical Oncology 2.6.3. Длительность лекарственной терапии Длительность неоадъювантной и адъювантной терапии устанавливается клиниче- скими рекомендациями. При паллиативной и лечебной ХТ в некоторых случаях также существует четко опре- деленное рекомендациями число циклов, превышение которого не улучшает отдаленные результаты лечения (например, 4–6 циклов ХТ при немелкоклеточном раке легкого, 4 цикла химиотерапии по схеме BEP при герминогенных опухолях, 16–18 недель индукционной ХТ первой линии колоректального рака). В других случаях лечение может быть продол- жено до прогрессирования заболевания, непереносимой токсичности или достижения максимально допустимой кумулятивной дозы используемых препаратов, обладающих кумулятивной токсичностью. том  /  vol. 12 № 3s2 • 2022 том  /  vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии Практические рекомендации 40 Практические рекомендации При паллиативной ХТ важно оценивать соотношение пользы (эффекта) и вреда (токсичности) от продолжения терапии. При достижении стабилизации заболевания или максимального эффекта без дальнейшего уменьшения опухолевых очагов при ряде опухолей рекомендуется провести еще 2–3 цикла ХТ с последующим наблюдением или поддерживающей терапией. Таргетная терапия, ГТ и иммунотерапия обычно при пал- лиативном лечении проводятся до прогрессирования заболевания или непереносимой токсичности. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии том  /  vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ Российское общество клинической онкологии MALIGNANT TUMOURS Russian Society of Clinical Oncology
https://openalex.org/W2995302914
https://hal.inrae.fr/hal-03326088/document
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Edible Films and Coatings Functionalization by Probiotic Incorporation: A Review
Polymers
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10,921
Edible Films and Coatings Functionalization by Probiotic Incorporation: A Review Oana L Pop, Carmen R Pop, Marie Dufrechou, Dan C Vodnar, Sonia A Socaci, Francisc V Dulf, Fabio Minervini, Ramona Suharoschi To cite this version: Oana L Pop, Carmen R Pop, Marie Dufrechou, Dan C Vodnar, Sonia A Socaci, et al.. Edible Films and Coatings Functionalization by Probiotic Incorporation: A Review. Polymers, 2020, 12 (1), pp.1-15. ￿10.3390/polym12010012￿. ￿hal-03326088￿ Distributed under a Creative Commons Attribution 4.0 International License Edible Films and Coatings Functionalization by Probiotic Incorporation: A Review Oana L. Pop 1, Carmen R. Pop 1 , Marie Dufrechou 2, Dan C. Vodnar 1 , Sonia A. Socaci 1 , Francisc V. Dulf 3 , Fabio Minervini 4,* and Ramona Suharoschi 1,* Oana L. Pop 1, Carmen R. Pop 1 , Marie Dufrechou 2, Dan C. Vodnar 1 , Sonia A. Socaci 1 , Francisc V. Dulf 3 , Fabio Minervini 4,* and Ramona Suharoschi 1,* 1 Department of Food Science, University of Agricultural Sciences and Veterinary Medicine, Calea Mănă¸stur 3-5, 400372 Cluj-Napoca, Romania; oanalelia.pop@gmail.com (O.L.C.); carmen-rodica.pop@usamvcluj.ro (C.R.P.); dan.vodnar@usamvcluj.ro (D.C.V.); sonia.socaci@usamvcluj.ro (S.A.S.) 1 Department of Food Science, University of Agricultural Sciences and Veterinary Medicine, Calea Mănă¸stur 3-5, 400372 Cluj-Napoca, Romania; oanalelia.pop@gmail.com (O.L.C.); carmen-rodica.pop@usamvcluj.ro (C.R.P.); dan.vodnar@usamvcluj.ro (D.C.V.); sonia.socaci@usamvcluj.ro (S.A.S.) j 2 USC 1422 GRAPPE, INRA, Ecole Supérieur d’Agriculture, SFR 4207 QUASAV, 55 rue Rabelais, BP 30 4900 Agnes Cedex 01, France; m.dufrechou@groupe-esa.com 3 Department of Biochemistry, University of Agricultural Sciences and Veterinary Medicine, Calea Mănă¸stur 3-5, 400372 Cluj-Napoca, Romania; francisc_dulf@yahoo.com 4 Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70121 Bari, Italy * Correspondence: fabio.minervini@uniba.it (F.M.); ramona.suharoschi@usamvcluj.ro (R.S.); Tel.: +39-0805442946 (F.M.); +40-771636781 (R.S.) Received: 1 December 2019; Accepted: 17 December 2019; Published: 19 December 2019 Abstract: Edible coatings and films represent an alternative packaging system characterized by being more environment- and customer-friendly than conventional systems of food protection. Research on edible coatings requires multidisciplinary efforts by food engineers, biopolymer specialists and biotechnologists. Entrapment of probiotic cells in edible films or coatings is a favorable approach that may overcome the limitations linked with the use of bioactive compounds in or on food products. The recognition of several health advantages associated with probiotics ingestion is worldwide accepted and well documented. Nevertheless, due to the low stability of probiotics in the food processing steps, in the food matrices and in the gastrointestinal tract, this kind of encapsulation is of high relevance. The development of new and functional edible packaging may lead to new functional foods. This review will focus on edible coatings and films containing probiotic cells (obtaining techniques, materials, characteristics, and applications) and the innovative entrapment techniques use to obtained such packaging. Keywords: edible films; edible coatings; probiotics; functional food; antibacterial activity HAL Id: hal-03326088 https://hal.inrae.fr/hal-03326088v1 Submitted on 25 Aug 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 Polymers 2020, 12, 12; doi:10.3390/polym12010012 1. Introduction Edible films or coatings (edible packaging or EP) are defined by any material meant to be applied (wrapping or coating) to food in order to extend the shelf life and may be consumed together with the food. Due to the many disadvantages related to plastic films and packaging, edible films have gained popularity in the scientific world, and drawn the attention of authorities and consumers [1] concerned about environmental protection. Indeed, conventional synthetic packages have a very damaging effect on the environment [2]. EP, especially those containing microorganisms, can be considered as a living ecosystem that selectively allows for the exchanges of respiration gases (e.g., oxygen, carbon dioxide and ethylene) between food and the atmosphere, diminishes or prevents loss of moisture and aromas and/or protects against undesired microorganisms [3]. Depending on the exact purpose, the film/coating can totally coat the food or can be applied between food constituents [4]. The materials that are utilized for the edible films/coatings production Polymers 2020, 12, 12; doi:10.3390/polym12010012 www.mdpi.com/journal/polymers 2 of 15 Polymers 2020, 12, 12 are biopolymers, proteins, lipids or composites. Thus, even if they are not consumed with food, they can be more rapidly and easily degraded with respect to plastic materials [5]. are biopolymers, proteins, lipids or composites. Thus, even if they are not consumed with food, they can be more rapidly and easily degraded with respect to plastic materials [5]. The main difference between coating and film is in their preparation and application process. Indeed, edible films are usually obtained in parallel to food and then applied to the surface, whereas coatings are directly prepared on food surface [6]. Both coatings and films can entrap live probiotic microorganisms. g Due to handling and hygienic limitations, EP can be combined with ecofriendly non-EP [6–8 The utilization of films for food preservation dates back to the 12th century in China, where wax was utilized to delay moisture loss from fruits. Sixteen centuries ago, the first edible films made from soymilk were used in Japan for fruits preservation and in order to obtain a shiny surface [9,10]. Due to the narrow variety of materials used to protect fruits and vegetables at that time, no big interest was shown to this type of package. Refrigeration, controlled/modified atmosphere, heat or radiation sterilization, smoking have ever received stronger attention than edible packaging. 1. Introduction Of course, food conservation methods have considerably increased and have offered unlimited opportunities to prepare, store and consume any type of food in any season. However, EP can currently be applied to a large variety of food products, with unique, tailored and innovative ways of action than conventional food preservation techniques [1]. Among various roles played by EP, physical protection [11] amplification and protection of food properties, carriers of food additives and prolongation of shelf life are the most important ones. EP may be categorized according to the class of their constituent material. Hydrocolloids (polysaccharides and proteins) and lipids are the most used materials. Among these, polysaccharides are the easiest to purchase and more suitable to form films or coatings. The presence of a large number of hydroxyl groups and hydrogen bonds favor the formation of film. Different properties can be observed between films and coatings made of negatively charged gums (i.e., alginate, pectin, or carboxymethyl cellulose) [7]. Proteins used for EP have mostly animal origin (gelatin, casein, whey proteins, collagen or egg albumin). However, plant-derived proteins (e.g., corn, soybean, wheat, cottonseed, peanut, and rice) are also appreciated and compatible with the vegetarian diet. The film/coating forming process is started, in most of the cases, with protein denaturation using heat or pH adjustment, followed by a conglomeration of peptide chains through new intermolecular interactions [12]. These type of films are suitable mainly for meat products, due to their affinity to hydrophilic surfaces [13–15]. Lipids do not form cohesive films, unlike hydrocolloids. For this reason, they are used especially for coatings or in mixture with polysaccharides in order to obtain an optimized water vapor barrier [16]. Lipids do not form cohesive films, unlike hydrocolloids. For this reason, they are used especially for coatings or in mixture with polysaccharides in order to obtain an optimized water vapor barrier [16]. The integration of different additives (i.e., probiotic microorganisms but also organic acids [17], essential oils [18], plant extracts [19], and antibacterial compounds [20]) into the EP has the benefit The integration of different additives (i.e., probiotic microorganisms but also organic acids [17], essential oils [18], plant extracts [19], and antibacterial compounds [20]) into the EP has the benefit of ensuring slower release of these compounds to food [21]. The aim of this paper is to review the application of various types of natural EP that incorporates live probiotic microorganisms. 3. Probiotics in Food and Human Health Nowadays, probiotics are associated with a world that lives on and inside humans and animals, modulating the host’s health [28,29]. The amount of bacteria that can be found in the human body exceeds the number of human cells by more than ten times. Due to the great impact of the gut microbiota on the human body and its health modulation, the human gastrointestinal network is also called “the other brain” [30]. The human gut hosts about 1500 bacterial species, of which about 500 species have pathogenic or probiotic traits. The gut’s influence on health, exerted by microorganisms inhabiting human body (especially some sections of the gut), starts in the womb of mother, depends on the child’s delivery (C-section or vaginal) modality, milk (breast-fed or artificial milk) ingested by newborn, and afterwards, is mostly modulated by diet. Other genetic and epigenetic factors, as well as environmental drivers (geographic location, stress, physical activity, and drug intake), further modulate the balance in the gut microbiota. While being relatively stable in adulthood, during aging, the gut microbiota composition continuously changes [31]. In elderly individuals, the frequently observed decrease in the Bacteroidetes/Firmicutes ratio is correlated with functionality decline of the immune system. Any modification in the diversity of the gut microbiota (dysbiosis) may result in the onset of certain illnesses and dysfunctions. The use of probiotic supplements is a possible, cost-effective and easy-to-use solution to counteract dysbiosis and face the pressing issue of microorganisms capable of resisting multiple antibiotics [32]. The current definition of probiotic microorganism underlines it as a viable, single or mixed, culture of bacteria or yeast which beneficially impact animal or human health when ingested in the adequate amount [33]. Members of the genera Bifidobacterium and Lactobacillus are consistently used for their probiotic effect, whereas members of the genera Streptococcus and Enterococcus contain several opportunistic pathogens [34,35]. Some yeasts, mostly Saccharomyces boulardii, are accepted for use as probiotics. Probiotics help to prevent or, in some cases, treat diarrhea, ulcerative colitis, irritable bowel syndrome, allergies, obesity, and diabetes [36,37]. Several modes of action are well-known for probiotics; for instance, they are able to modulate nutrients absorption [38], act as a barrier against pathogenic bacteria at the level of intestinal mucosa [39], have an impact on the immune system [40], and influence the gut–brain axis [41]. 3. Probiotics in Food and Human Health Some mechanisms of action exerted by probiotic microorganisms are mediated by their metabolites, such as molecules with antimicrobial activity (e.g., organic acids, ethanol, hydrogen peroxide, and bacteriocins) and short chain fatty acids that are used by enterocytes as nutrients [42]. 2. Bioactive Molecules in EP and Perspectives in Food Industry EP may be used not only for their protective effect but as carriers of bioactive substances too. Some examples of bioactive molecules are: antimicrobial compounds, probiotics, anti-browning compounds, omega-3 fatty acids, and other nutraceuticals [22]. Active food packaging that incorporates bioactive molecules not only acts as a traditional protective system for the food product, but also promotes the health of consumers. Even more, the utilization of byproducts in order to obtain the edible package or to extract a bioactive molecule that will be further incorporated into the package will sustain the economical approach [23]. Obviously, when compared with fresh fruits and vegetables, the utilization of agricultural byproducts, like fruit peels to prepare edible films, seems much more profitable from the perspective of resource recycling and environmental protection, and needs further study [24]. 3 of 15 Polymers 2020, 12, 12 The utilization of EP that contain bioactive molecules have multiple advantages [25]. One example of EP that can sustain human health and influence the final product are fried food products. A decrease of oil intake in deep-fried food products represents a significant application of EP, namely coatings. Obesity and heart diseases have been linked to an excess of fat in food. The use of film made of methylcellulose and hydroxypropyl/methylcellulose allows for a decrease in oil absorption by food, thus helping to reduce fat intake [26]. The processing techniques used to obtain EP vary depending on the material and bioactive compounds added in the EP [27]. Legislation, polymer types, active molecules, destination and are all factors that influence the matrix choice. 4. Entrapped Probiotics in EP In order to benefit from the consumption of probiotics, a dose of 108–9 viable cells per day is recommended. In many products, to reach this dose is challenging due to high sensibility of probiotics to environmental conditions. Survival of probiotics depends on strain, food characteristics (e.g., pH), processing technologies, storage conditions and time [43]. Biological activities of probiotic bacteria and yeasts can be negatively affected by their loss of viability during food processing and storage. The use 4 of 15 Polymers 2020, 12, 12 of encapsulated probiotics in edible films or coatings could favor the optimal survival of beneficial microorganisms in food. EP that incorporate probiotics display, besides those characteristics that are peculiar to all the EP, features specifically addressed to maintain the host in good health (Figure 1). In addition, since probiotic microorganisms often showed inhibitory activity towards spoilage or pathogenic bacteria, their incorporation in EP can increase food stability and safety. Food packaged in coatings or films containing probiotics may be regarded as functional food, a special group of food items that, if regularly introduced in diet, benefit health, beyond their nutritional value [44]. Polymers 2019, 11, x FOR PEER REVIEW a special group of food items that, if regularly introduced in diet, benefit health, beyond nutritional value [44]. Figure 1 Characteristics of edible packaging (EP) containing probiotics and some of the most st Figure 1. Characteristics of edible packaging (EP) containing probiotics and some of the most studied healthy effects exerted by probiotics. Figure 1 Characteristics of edible packaging (EP) containing probiotics and some of the most s Figure 1. Characteristics of edible packaging (EP) containing probiotics and some of the most studied healthy effects exerted by probiotics. healthy effects exerted by probiotics. E l ti f bi ti i EP b bt i d i d i [45] ith Encapsulation of probiotics in EP may be obtained using spray drying [45] with or without protectants, spray freeze-drying or electrospray, and cross-linking gelation. healthy effects exerted by probiotics. E l i f bi i i EP b b i d i d i [45] i h Encapsulation of probiotics in EP may be obtained using spray drying [45] with or without protectants, spray freeze-drying or electrospray, and cross-linking gelation. Encapsulation of probiotics in EP may be obtained using spray drying [45] with or w protectants, spray freeze-drying or electrospray, and cross-linking gelation. 4. Entrapped Probiotics in EP The addition of probiotics to EP is much less frequent than the addition of plant ex Nowadays, this technology allows widening the range of probiotic-carrier food products that v probiotics, satisfying the demand for nondairy foods, fostered by vegan consumers and la intolerant people [40]. In one of the first research studies about the encapsulation of probiotics Bifidobacterium animalis subsp. lactis BB-12 was entrapped in alginate and gellan-based edible co of apple and papaya slices. The addition of BB-12 seemed to cause an increase in the space be the polymer chains. During 10 days of storage at 2 °C, the cell density of the strain was i i d d (106 C l F i U it / CFU/ ) [46 47] H th The addition of probiotics to EP is much less frequent than the addition of plant extracts. Nowadays, this technology allows widening the range of probiotic-carrier food products that vehicle probiotics, satisfying the demand for nondairy foods, fostered by vegan consumers and lactose-intolerant people [40]. In one of the first research studies about the encapsulation of probiotics in EP, Bifidobacterium animalis subsp. lactis BB-12 was entrapped in alginate and gellan-based edible coatings of apple and papaya slices. The addition of BB-12 seemed to cause an increase in the space between the polymer chains. During 10 days of storage at 2 ◦C, the cell density of the strain was above minimum recommended (106 Colony-Forming Units/g or CFU/g) [46,47]. However, the coating containing the probiotic strain showed higher (50%) water vapor permeability than the control coating [46]. minimum recommended (106 Colony-Forming Units/g or CFU/g) [46, 47]. However, the c containing the probiotic strain showed higher (50%) water vapor permeability than the c coating [46]. EP containing probiotics could be exploited to overcome the otherwise unavoidable l viability of beneficial microorganisms during food processes carried out at high tempera Microcapsules containing a probiotic strain of L. acidophilus were dispersed (1% or 2%) in a (5%) solution which covered the surface of bread dough [48] This technology allowed L acido EP containing probiotics could be exploited to overcome the otherwise unavoidable loss of viability of beneficial microorganisms during food processes carried out at high temperatures. Microcapsules containing a probiotic strain of L. acidophilus were dispersed (1% or 2%) in a starch (5%) solution, which covered the surface of bread dough [48]. This technology allowed L. 4. Entrapped Probiotics in EP acidophilus to keep its viability after baking, without any negative impact on the taste of bread and texture properties of the crumb. In addition, the edible coating reduced bread crust crispness [48]. ( ) , to keep its viability after baking, without an 5. Materials and Techniques Used for Probiotic EP Due to the materials due to the incorporated active molecules, the EP are meant to protect the food or just to act as er for the active compounds, to reduce contamination, to improve/maintain the food product l appearance, to enhance the mechanical properties of fragile food products or to boost the ance and flavor (Figure 2). Figure 2. Edible films and coatings components and roles. Figure 2. Edible films and coatings components and roles. 2. Edible films and coatings components and roles. Figure 2. Edible films and coatings components and roles. iverse biocompatible components, such as hydrocolloids, lipids and composites, are used in paration [50]. According to their specific purpose, miscellaneous compounds may be exploited trapment of probiotics in EP are miscellaneous In these cases the package is defined as Diverse biocompatible components, such as hydrocolloids, lipids and composites, are used in EP preparation [50]. According to their specific purpose, miscellaneous compounds may be exploited for entrapment of probiotics in EP are miscellaneous. In these cases, the package is defined as composite [5,16]. trapment of probiotics in EP are miscellaneous. In these cases, the package is defined as site [5, 16]. ydrocolloids include polysaccharides and proteins. Among polysaccharides, cellulose and its tives, dextran, inulin, alginate, carrageenan, starch derivatives, pectin derivatives, chitosan, ed extracts, and galactomannan are the most utilized for edible films and packages [14, 24, 51]. lysaccharides successfully protect food from oxygen, odor, and oil absorption; on the other they show high water permeability [49] In subsequent paragraphs, a concise presentation of Hydrocolloids include polysaccharides and proteins. Among polysaccharides, cellulose and its derivatives, dextran, inulin, alginate, carrageenan, starch derivatives, pectin derivatives, chitosan, seaweed extracts, and galactomannan are the most utilized for edible films and packages [14,24,51]. All polysaccharides successfully protect food from oxygen, odor, and oil absorption; on the other hand, they show high water permeability [49]. In subsequent paragraphs, a concise presentation of the most utilized materials is made: they show high water permeability [49]. In subsequent paragraphs, a concise presentation of st utilized materials is made: ellulose and cellulose derivate (e.g., methylcellulose and hydroxypropyl methylcellulose) nt oil absorption from fried food items [52] and have been successfully used for EP-containing tics [53-55]. Alginic acid, also known as alginate, may be conveniently applied to meat cts, where it considerably delays lipid oxidation [56, 57] [58]. ( ) , to keep its viability after baking, without an 5. Materials and Techniques Used for Probiotic EP properties of the crumb. In addition, the edible coating reduced bread crust crispness [48]. 5. Materials and Techniques Used for Probiotic EP Commonly, EP are expected to be transparent, flavorless and unable of modifying the se properties of food products. However, some applications (e.g., sushi wraps) may require sp e o y o e tie a la k of e olutio of e ati e o a ole ti ha a te i ti EP a e u Commonly, EP are expected to be transparent, flavorless and unable of modifying the sensory properties of food products. However, some applications (e.g., sushi wraps) may require specific sensory properties as lack of evolution of negative organoleptic characteristics. EP are usually composed of two major components: (i) a macromolecule-based substance, biopolymers, (ii) additives as plasticizers, cross-linkers, nanoreinforcements and (iii) precursors as proteins, polysaccharides, lipids or resins 5 of 15 Polymers 2020, 12, 12 (Figure 2). The macromolecule-based substance represents the base that, dissolved in a solvent (usually water), forms a cohesive assembly. The plasticizer is added in order to improve mechanical properties of package (e.g., elasticity, toughness, resistance to tearing), so that the package gains flexibility and higher stability [14]. Plasticizers, such as sorbitol, polyethylene glycol, glycerol and sucrose, are commonly needed when the package is composed of proteins and polysaccharides. In some cases, emulsifiers are used, instead of plasticizers, in order to increase the stability of film/coatings, made of lipids and polysaccharides [49]. Due to the materials and/or due to the incorporated active molecules, the EP are meant to protect the food or just to act as a carrier for the active compounds, to reduce contamination, to improve/maintain the food product natural appearance, to enhance the mechanical properties of fragile food products or to boost the appearance and flavor (Figure 2). 2019, 11, x FOR PEER REVIEW 5 of 18 hylene glycol, glycerol and sucrose, are commonly needed when the package is composed of ns and polysaccharides. In some cases, emulsifiers are used, instead of plasticizers, in order to se the stability of film/coatings, made of lipids and polysaccharides [49]. ( ) , to keep its viability after baking, without an 5. Materials and Techniques Used for Probiotic EP Chitosan is obtained from chitin ylation and is usually obtained from the exoskeleton of crustaceans and fungal cell walls [59]. acetylation process influences the chitosan molecular weight and, in turns its properties (i.e., linity, hydrophobicity, degradation, tensile strength and moisture content) [60, 61]. Chitosan antimicrobial properties [62, 63]. Starch and its derivatives are cost-effective and easy to . In addition, they are typically clear, inodorous and insipid [64, 65]. The starch films and g characteristics are strongly influenced by the amylose/amilopectin ratio. A strong and flexible obtained from a starch rich in amylose content [66]. Pectin, frequently utilized in jams and Cellulose and cellulose derivate (e.g., methylcellulose and hydroxypropyl methylcellulose) prevent oil absorption from fried food items [52] and have been successfully used for EP-containing probiotics [53–55]. Alginic acid, also known as alginate, may be conveniently applied to meat products, where it considerably delays lipid oxidation [56–58]. Chitosan is obtained from chitin deacetylation and is usually obtained from the exoskeleton of crustaceans and fungal cell walls [59]. The deacetylation process influences the chitosan molecular weight and, in turns its properties (i.e., crystallinity, hydrophobicity, degradation, tensile strength and moisture content) [60,61]. Chitosan shows antimicrobial properties [62,63]. Starch and its derivatives are cost-effective and easy to handle. In addition, they are typically clear, inodorous and insipid [64,65]. The starch films and coating characteristics are strongly influenced by the amylose/amilopectin ratio. A strong and flexible film is obtained from a starch rich in amylose content [66]. Pectin, frequently utilized in jams and jellies, was used to produce films and coatings containing probiotics [67,68]. Polymers 2020, 12, 12 6 of 15 Proteins are dissolved or dispersed in solvents (i.e., water or ethanol) that are further evaporated in order to obtain the package. The protein-based structure forming process is favored by heat or acid conditions [69,70]. Compared to polysaccharides, proteins have lower vapor permeability. Lipids may be commonly utilized in the form of waxes, oils, fats, and resins for building EP-entrapping probiotics [71]. Since they are characterized by a high resistance to water penetration, in most cases, the lipids are combined with polysaccharides or proteins [72]. Table 1 summarizes some of the types of composite used in order to obtain EP, the materials and the designated food products with some generic and specific effects. ( ) , to keep its viability after baking, without an 5. Materials and Techniques Used for Probiotic EP The materials used for EP may be derived from food industry byproducts, such as whey, corn zein (source of proteins), mung bean or fruit pomace (source of pectin). This represents an environmentally friendly solution and assists in cost reduction. Nevertheless, the utilization of food byproducts in EP could signal consumer mistrust due to confusion between byproducts and wastes. Extensive applications of mentioned materials has been literally obstructed by some difficulties in the material preparation process [73,74]. Most of these difficulties are related to the solubility of the materials in solvents that are accepted in food industry. However, scientists innovate in order to obtain best properties of the EP. An edible biocomposite film was proposed to be obtained directly from psyllium seed, but it was proven that the utilization of seeds husk and husk flour was more suitable [75]. In general, lipids are difficult to apply on the surface of some foods due to their poor adhesion to food products with hydrophilic surfaces [76]. Chitosan can ensure many benefits, such as excellent hydrophilicity, high porosity, big adhesion area, and can be cross-linked to avoid dissolution in acidic solutions (pH < 2). The use of chitosan as material for the entrapment of probiotics has been widely studied, but the too-soft texture and similarities between the density of the EP and that of water (leading to easy float) limits its industrial function. Therefore, efforts have been made to support the structure through the addition of activated clay and crosslinking with glutaraldehyde, which has been demonstrated to permit superior operational stability. However, these alternatives are not suitable for the food industry. Nevertheless, more studies regarding the challenges in the materials preparation process need to be conducted in order to smooth the processes and sustain this environmentally friendly method [77]. In order to sustain the applicability of probiotic EP in the food industry at an industrial scale, new and innovative techniques need to be developed. Nanotechnology and the utilization of nanomaterials is a promising area that can broaden the use of probiotic EP. Formulation of non-nanomaterials in nanosized structures can bring enormous benefits due to the new and unique obtained bioactive properties [78]. The utilization of electrospinning in the preparation of EP materials can be a suitable technique for the restructuration of biopolymers in nanoscale. 7 of 15 Polymers 2020, 12, 12 Table 1. ( ) , to keep its viability after baking, without an 5. Materials and Techniques Used for Probiotic EP Some polysaccharides, proteins, lipids, and composites-based EP for different food products with their generic and specific functions. Materials/Methods Generic Effects Specific Composition Type of Food Specific Effects Reference Polysaccharides Starch + colorless + oil-free appearance + reduced caloric content + prolong shelf life + suitable for fruits, vegetables, meat + control oxygen transmission + reduce darkening of the surface - no moisture barrier - hydrophilic nature Starch-based coatings with D-glucose, silver nitrate. Chicken Sausages Antimicrobial activity. [79] Cellulose and derivatives Hydroxypropyl methylcellulose (HPMC) and beeswax coatings. Cherry tomatoes Prevent weight loss, sustain fruit firmness, improved sensory attributes. [80] Pectin Pectin and sodium alginate coatings with citral and eugenol essential oils. Raspberries Maintain the color, prevent weight loss, trolox equivalent antioxidant capacity, prevent microbial growth. [81] Pullulan Pullulan-based coatings with sweet basil extract. Apples Sustain color, appearance and sensory attributes during hypothermia storage. [82] Alginates Alginate-chitosan and ZnO nanoparticle Guavas Increase the shelf-life of the fruit. [83] Chitosan Chitosan-based coatings with vacuum packaging. Beef Effects on color preservation and lipid oxidation during retail presentation. [84] Proteins Vegetable-based proteins + provide mechanical stability + good transparency - not suitable for some diets (vegan) Whey proteins coatings with lysozyme. Salmon Overall quality of salmon. [85] Gluten and zein coatings with potassium caseinate, rennet casein, xanthan gum, locust bean additives. Trout Fillets Sensorial attributes and the physical biochemical qualities. [86] Animal-based proteins Caseinate-based coatings with ascorbic acid additives. Beef Effect of gamma irradiation on microbiological characteristics of ground beef. [87] Furcellaran-gelatin-based edible coating. Salmon sushi Exhibit good transparency, mechanical and barrier properties and can be manufactured by extrusion or casting processes. [88] Fats Oils + reduce water transmission Lipid-based (sunflower oil and chocolate) coating with stearic acid, polyglycerol. Apple slices Moisture barrier. [89] Waxes Candelilla wax coating with ellagic acid. Avocado Antifungal characteristics to enhance shelf life. [90] Carnauba wax coating. Eggplant Increase in the water vapor resistance and reduction in weight loss. [91] Candelilla wax coatings with mineral oil. Guava fruit Weight loss ethylene emission, gloss, retention of the color, firmness. [92] Chitosan-Beeswax coating. Strawberries Reduction in weight loss. [93] Multicomponents/Composites + special tailored for specific characteristics + enhance the permeability or mechanical properties - may get expensive Composites of carrageenan and whey protein coatings with CMC sodium salt, polyethylene glycol, calcium chloride, glycerol and oxalic acid additives. Apples Reduce brownness. [94] Composite of chitosan and gelatin coatings. 6. Probiotics Viability in EP Probiotics may be used in pharmaceutical or food-based products [99,100]. The edible coating or films may be regarded as a carrier of probiotics. The major challenge to be faced by probiotic microorganisms is their resistance to entrapment, an essential prerequisite for their viability in the final product. Only viable probiotics at adequate cell numbers can successfully colonize the colon. Some studies were specifically devoted to investigating the viability of probiotics entrapped in edible coatings/films. Composition and storage temperature affect viability of probiotics in edible coatings/films. Pullulan is a polysaccharide that can be used as a base for EP. A pullulan-based film embedding probiotic lactobacilli (L. reuteri ATCC (American Type Culture Collection) 55730, L. rhamnosus GG ATCC 53103, and L. acidophilus DSM 20079) proved to sustain the viability of probiotics, during 10 and 20 days’ storage at room temperature at levels of 10.3 and 4.5 log CFU/mL, respectively. A similar film, but containing a mixture of pullulan and potato starch, was less effective in maintaining the viability of probiotic lactobacilli. In detail, the higher the starch content, the lower the probiotic viability. However, when lower storage temperature (4 ◦C) was applied, no differences were found in terms of viability loss between the pullulan- and pullulan/starch-based film. The viability loss did not exceed 10% even after 30 days of storage [101]. Entrapment of L. rhamnosus GG in a film based on a mixture of starch (from rice and corn) and proteins (bovine gelatin, sodium caseinate, and soy protein) resulted in higher viability of the probiotic strains at refrigeration than at room temperature. The presence of proteins increased viability of L. rhamnosus GG during the film formation process [102]. L. plantarum and Kluyveromyces marxianus incorporated in a film composed of kefiran (a polysaccharide secreted by lactic acid bacteria) and glycerol did not negatively affect the film optical and physical properties. Compared to a suspension, both microorganisms showed better tolerance to acid conditions in the film and maintained their viability through storage at room temperature. In addition, the yeast showed higher resistance to the film-forming procedure than the lactic acid bacterium [103]. B. animalis subsp. lactis BB-12 was incorporated in alginate and gellan (2% solutions) edible coatings and applied on fresh-cut apple and papaya. Although a viability decrease of the probiotic higher than 85% was observed, BB-12 was maintained above the minimum recommended (106 CFU/g) [46]. ( ) , to keep its viability after baking, without an 5. Materials and Techniques Used for Probiotic EP Red bell peppers Improve firmness, diminish weight loss, and ethanol concentration. [95] Composite of hydroxypropyl methyl cellulose (HPMC) and lipid coating with potassium sorbate, sodium benzoate, sodium propionate, stearic acid, glycerol additives. Oranges Antifungal properties improved during long-term cold storage. [96] Shellac, gelatin and Persian gum. Orange Improve permeability characteristics. [97] Hydroxypropyl methylcellulose-lipid composite edible coatings. Citrus fruits Maintain postharvest quality. [98] es-based EP for different food products with their generic and specific functions. 8 of 15 Polymers 2020, 12, 12 8. Antimicrobial Effects of Probiotics Incorporated in EP Besides their positive effects on human health, probiotic microorganisms incorporated in EP could protect food from pathogenic bacteria, leading to increased food safety. They could also inhibit spoilage microorganisms, thus extend the shelf-life of food. A probiotic strain belonging to Lactobacillus sakei was embedded in a sodium caseinate-based film through either direct incorporation in the film-forming suspension or by spraying on an already-formed film. The film, and its counterpart not containing probiotic lactobacilli, were applied on plates of tryptic soy agar on fresh beef slices, which were inoculated with Listeria monocytogenes. During four days of incubation on plates, the probiotic strain increased of one log cycle its cell density. L. monocytogenes decreased (3.0–3.6 log cycles) during the 12 days of storage. In the beef slices stored at 4 ◦C for 21 days, L. sakei cell density was higher than 6 log CFU/cm2. In addition, the cell density of the pathogenic bacterium was two log cycles lower than in the probiotic-free film [106]. In the presented study, it can be observed that the presence pf probiotics from lactobacillus species negatively influenced the multiplication of L. monocytogenes on the beef slices by producing bacteriocin-like substance. Thus, the production of this substance was nonexistent after a long period of time. This fact can be explained by the death of lactobacillus as an effect of the environmental conditions and lack of nutrients. A similar study that echoes the above-presented results is an alginate-based film containing Carnobacterium maltaromaticum, a potential probiotic bacterium normally found as commensal of various fish species [107,108], was applied on smoked salmon, inoculated with L. monocytogenes at 4 log CFU/cm2. This film had a bacteriostatic effect towards L. monocytogenes during 28 days of storage at 4 ◦C [109]. The authors of the study declare that the antibacterial effect can be explained due to the neutralized supernatant and therefore was not due to acidity or pH. A gelatin-based coating containing probiotic strains of L. acidophilus and Bifidobacterium bifidum was applied to hake (Merluccius merluccius). Both probiotic strains maintained their initial level (109 CFU/mL) of viability for 6 days of storage at 2 ◦C. The spoilage agent Shewanella putrefaciens, typically producer of H2S, was found in coated hake at significantly lower counts than the uncoated hake. However, the antibacterial effect had no relevant link to the presence of probiotics in the edible package. 7. Synbiotics in EP Probiotics may be combined with prebiotic compounds, i.e., substances capable of favoring beneficial microbes in the human gut. The term “synbiotics” is used for indicating products containing at least one probiotic microorganism and one prebiotic substance. Such products may help to maintain the cell viability of probiotics and have been experimented inside edible films. The presence of fructooligosaccharides (FOS) as prebiotic compounds in a methylcellulose-based film containing two probiotic strains (L. delbrueckii subsp. bulgaricus CIDCA 333 and L. plantarum CIDCA 83114) was effective in the protection of both probiotics. However, it had a negative effect in the film forming-process, by reducing the glass transition temperature of the film [66]. Inulin, galacto-oligosaccharide and FOS in chitosan-based film favored viability of probiotic Bifidobacterium infantis ATCC 15697 and Lactobacillus fermentum ATCC 9398. Besides the prebiotic effect, the oligosaccharides increased the extensibility of the film, compared to a prebiotic-free film [104]. Viability of L. rhamnosus GG was monitored during time in a gelatin-based film added with inulin, polydextrose, gluco-oligosaccharides and wheat dextrin. The presence of prebiotic compounds did not impair the film structure. Viability loss was found regardless of the type of prebiotic compound, but especially with film containing gluco-oligosaccharides (about 40%) or polydextrose (almost 85%). Among the tested prebiotics, inulin allowed to maintain viability of the probiotic strain at acceptable level over 100 days of storage, whereas in the film containing the other compounds an acceptable viability was maintained for a shorter time (63–83 days) [102]. Thus, the limitations and difficulties in the utilization of pro- and prebiotics in EP formulations need to be addresses, despite the fact that very few scientific papers discuss this aspect. The utilization of prebiotics, together with the probiotics may lead to serious changes in the final properties of EP. 9 of 15 Polymers 2020, 12, 12 Ensuring low production costs is the main challenge for future EP process and formulation technologies. The exploitation of food-grade raw materials such as native, and physically or enzymatically processed biopolymers, is one example of a method that has the potential to meet the challenge of widening the range of EP types into which probiotic and prebiotic can be favorably incorporated [105]. Novel developments for control release systems from the EP will also provide new possibilities. 7. Synbiotics in EP Negative changes in the EP formulation, that are not affecting the characteristics of food products and ensure the extension of shelf life (i.e., transparency, brightness, etc.) will be accepted by the consumers only if they realize that the benefits related to the presence of prebiotics in the probiotic EP are greater than the characteristic related to the food appearance. 8. Antimicrobial Effects of Probiotics Incorporated in EP Treatment of coated hake through high hydrostatic pressure (200 MPa for 10 min at 20 ◦C) proved to be effective in decreasing the spoiling agent, but had no effect on the viability of probiotics [98]. The ability of an agar-based film, incorporating green tea extract and two probiotic strains (Lactobacillus paracasei L26 and B. animalis subsp. lactis B94), to inhibit two spoiling bacteria was investigated in hake fillets. The spoiling agents, S. putrefaciens and Photobacterium phosphoreum were deliberately added (103–104 CFU/g) to hake fillets, prior to film application. The results showed that probiotic bacteria migrated from the film to fish and that fish wrapped in the film displayed lower values of spoilage indicators compared to untreated fish (e.g., pH, count of H2S-producing bacteria, concentration of trimethylamine). Overall, the use of this probiotic film extended the shelf life of hake fillets for at least one week [110]. The type of material constituting the edible package affects probiotics viability and their antimicrobial activity. A probiotic strain of L. plantarum was embedded in an edible film based on sodium caseinate, pea protein, methylcellulose or hydroxymethylcellulose [54]. The probiotic strain 10 of 15 Polymers 2020, 12, 12 showed higher viability in protein than in cellulose-based film. Interestingly, when applied in the cellulose-based film, L. plantarum produced higher levels of bacteriocin, resulting in the total inactivation of Listeria innocua during 8 days of storage at refrigerated temperature [54]. When incorporated in sodium caseinate- or methylcellulose-based film, L. acidophilus displayed higher viability than L. reuteri. After three days of storage, higher antilisterial activity was found for the methylcellulose-based film than for the one made of sodium caseinate. Compared to similar films without probiotic lactobacilli, Listeria sp. decreased by about 1.5 log cycle after 12 days of storage [111]. Alginate, whey proteins, or a mixture thereof were used for forming an edible coating containing L. rhamnosus GG and was applied to bread [43]. During the two drying processes considered (60 ◦C for 10 min, 180 ◦C for 2 min), the composite-based coating provided L. rhamnosus GG with higher protection, with respect to alginate- or whey proteins-based coating. However, following simulated gastrointestinal digestion, the highest cell density of L. rhamnosus GG (106 CFU/g) was found in the bread coated with alginate [43]. g The antibacterial activity of probiotics embedded in EP is limited due to the specific activity of the probiotic metabolites. 8. Antimicrobial Effects of Probiotics Incorporated in EP This fact can explain why same probiotic strain act as antimicrobials against certain pathogens and some have no influence. Nevertheless, as seen [54], the material used for the incorporation of probiotics has a great impact regarding the antimicrobial activity of the probiotic strain. This activity modulation can be correlated to the permeability of the EP for the antimicrobials metabolites produced by the probiotic cells and by the material capacity to protect the active cells. 9. Concluding Remarks Nowadays, the increasing consciousness of consumers about the link between dietary habits and health fosters the market of food containing probiotic microorganisms. EP technologies allow us to broaden the fields of application of probiotics to unexplored food items (e.g., baked goods). Overall, at an industrial scale, the number of applications of edible coatings/films containing probiotics is much lower than that of research studies carried out in the laboratory. One of the major challenges to be faced in order to achieve a wider industrial application is to obtain the perfect combination of materials, technologies and probiotic strains, tailored to specific foods and consumers’ needs, and at an acceptable cost. Another challenge is in the need to maintain a high cell density of probiotics during the formation process of EP and, especially, after ingestion. This is a prerequisite to impact human health positively. Future research efforts should be dedicated to these two challenges. In addition, a higher number of studies about the health benefits of EP are essential. Author Contributions: O.L.P. researched studies and wrote the manuscript; C.R.P. researched studies and collected data, M.D., D.C.V., S.A.S., F.V.D., provided ideas, discussion, corrected the manuscript, F.M. and R.S. revised and corrected the manuscript and supervised this project. All authors have read and agreed to the published version of the manuscript. Funding: The publication was supported by funds from the National Research Development Projects to fina xcellence (PFE)-37/2018–2020 granted by the Romanian Ministry of Research and Innovation. Acknowledgments: We kindly thank Marian Rodion Pop for image processing. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. uilbert, S.; Gontard, N.; Cuq, B. 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Tomás, S.A.; Bosquez-Molina, E.; Stolik, S.; Sánchez, F. Effects of mesquite gum-candelilla wax based edible coatings on the quality of guava fruit (Psidium guajava L.). J. Phys. IV Fr. 2005, 125, 889–892. [CrossRef] 93 V li k E Wi k lh E K S Al VD M ldã M ti M I t f hit b 92. Tomás, S.A.; Bosquez-Molina, E.; Stolik, S.; Sánchez, F. Effects of mesquite gum-candelilla wax based edible coatings on the quality of guava fruit (Psidium guajava L.). J. Phys. IV Fr. 2005, 125, 889–892. [CrossRef] 93. Velickova, E.; Winkelhausen, E.; Kuzmanova, S.; Alves, V.D.; Moldão-Martins, M. Impact of chitosan-beeswax edible coatings on the quality of fresh strawberries (Fragaria ananassa cv Camarosa) under commercial storage conditions. LWT 2013, 52, 80–92. [CrossRef] 15 of 15 Polymers 2020, 12, 12 94. Lee, J.Y.; Park, H.J.; Lee, C.Y.; Choi, W.Y. Extending shelf-life of minimally processed apples with edible coatings and antibrowning agents. LWT 2003, 36, 323–329. [CrossRef] 95. Poverenov, E.; Zaitsev, Y.; Arnon, H.; Granit, R.; Alkalai-Tuvia, S.; Perzelan, Y.; Weinberg, T.; Fallik, E. Effects of a composite chitosan–gelatin edible coating on postharvest quality and storability of red bell peppers. Postharvest Biol. Technol. 2014, 96, 106–109. [CrossRef] 96. Valencia-Chamorro, S.A.; Pérez-Gago, M.B.; del Río, M.Á.; Palou, L. Effect of antifungal hydroxypropyl methylcellulose (HPMC)–lipid edible composite coatings on postharvest decay development and quality attributes of cold-stored ‘Valencia’ oranges. Postharvest Biol. Technol. 2009, 54, 72–79. [CrossRef] 97. Khorram, F.; Ramezanian, A.; Hosseini, S.M.H. Shellac, gelatin and Persian gum as alternative coating for orange fruit. Sci. Hortic. 2017, 225, 22–28. [CrossRef] 98. Guimarães, J.E.R.; de la Fuente, B.; Pérez-Gago, M.B.; Andradas, C.; Carbó, R.; Mattiuz, B.-H.; Palou, L. References Antifungal activity of GRAS salts against Lasiodiplodia theobromae in vitro and as ingredients of hydroxypropyl methylcellulose-lipid composite edible coatings to control Diplodia stem-end rot and maintain postharvest quality of citrus fruit. Int. J. Food Microbiol. 2019, 301, 9–18. [CrossRef] [PubMed] 99. Lin, D.C. Probiotics as functional foods. Nutr. Clin. Pract. 2003, 18, 497–506. [CrossRef] 100. Vieira da Silva, B.; Barreira, J.C.M.; Oliveira, M.B.P.P. Natural phytochemicals and probiotics as bioactive ingredients for functional foods: Extraction, biochemistry and protected-delivery technologies. Trends Food Sci. Technol. 2016, 50, 144–158. [CrossRef] 101. Kanmani, P.; Lim, S.T. Development and characterization of novel probiotic-residing pullulan/starch edible films. Food Chem. 2013, 141, 1041–1049. [CrossRef] 102. Soukoulis, C.; Singh, P.; Macnaughtan, W.; Parmenter, C.; Fisk, I.D. Compositional and physicochemical factors governing the viability of Lactobacillus rhamnosus GG embedded in starch-protein based edible films. Food Hydrocoll. 2016, 52, 876–887. [CrossRef] 103. Piermaria, J.; Diosma, G.; Aquino, C.; Garrote, G.; Abraham, A. Edible kefiran films as vehicle for probiotic microorganisms. Innov. Food Sci. Emerg. 2015, 32, 193–199. [CrossRef] 104. Tang, Y.; Xie, F.; Zhang, D.; Zhu, M.; Liu, L.; Liu, P.; Gu, C. Physical properties and prebiotic activity of maize starch-based functional films. Starch Stärke 2015, 67, 124–131. [CrossRef] 104. Tang, Y.; Xie, F.; Zhang, D.; Zhu, M.; Liu, L.; Liu, P.; Gu, C. Physical properties and prebiotic activity of maize starch-based functional films. Starch Stärke 2015, 67, 124–131. [CrossRef] 105. Mattila-Sandholm, T.; Myllärinen, P.; Crittenden, R.; Mogensen, G.; Fondén, R.; Saarela, M. Technological 05. Mattila-Sandholm, T.; Myllärinen, P.; Crittenden, R.; Mogensen, G.; Fondén, R.; Saarela, M. Technolog challenges for future probiotic foods. Int. Dairy J. 2002, 12, 173–182. [CrossRef] 106. Gialamas, H.; Zinoviadou, K.G.; Biliaderis, C.G.; Koutsoumanis, K.P. Development of a novel bioactive packaging based on the incorporation of Lactobacillus sakei into sodium-caseinate films for controlling Listeria monocytogenes in foods. Food Res. Int. 2010, 43, 2402–2408. [CrossRef] 107. Socaciu, M.-I.; Semeniuc, C.A.; Vodnar, D.C. Edible Films and coatings for fresh fish packaging: focus on quality changes and shelf-life extension. Coatings 2018, 8, 366. [CrossRef] 108. Lauzon, H.L.; Pérez-Sánchez, T.; Merrifield, D.L.; Ringø, E.; Balcázar, J.L. Probiotic Applications in Cold Water Fish. Species. In Aquaculture Nutrition; John and Wiley and Sons: Hoboken, NJ, USA, 2014; pp. 223–252. 109. Concha-Meyer, A.; Schöbitz, R.; Brito, C.; Fuentes, R. Lactic acid bacteria in an alginate film inhibit Listeria monocytogenes growth on smoked salmon. Food Control 2011, 22, 485–489. [CrossRef] 110. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References López de Lacey, A.M.; López-Caballero, M.E.; Montero, P. Agar films containing green tea extract and probiotic bacteria for extending fish shelf-life. LWT 2014, 55, 559–564. [CrossRef] 110. López de Lacey, A.M.; López-Caballero, M.E.; Montero, P. Agar films containing green tea extract and probiotic bacteria for extending fish shelf-life. LWT 2014, 55, 559–564. [CrossRef] 111. Sánchez-González, L.; Quintero Saavedra, J.I.; Chiralt, A. Antilisterial and physical properties of biopolymer 111. Sánchez-González, L.; Quintero Saavedra, J.I.; Chiralt, A. Antilisterial and physical properties of biopolymer films containing lactic acid bacteria. Food Control 2014, 35, 200–206. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient
Journal of the Belgian Society of Radiology
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RAMAZAN ORKUN ONDER ALPTEKIN TOSUN TÜMAY BEKCI *Author affiliations can be found in the back matter of this article CORRESPONDING AUTHOR: Ramazan Orkun Onder CORRESPONDING AUTHOR: Ramazan Orkun Onder This article describes the case of a 56-year-old male patient who presented with a headache and swelling on the left side of his head. Medical examinations, including non-contrast computed tomography (CT) and contrast-enhanced magnetic resonance imaging (MRI), revealed the presence of an expansile soft tissue lesion in the frontoparietal bone structures, causing compression of the brain tissue. Further immunohistochemical analysis confirmed the diagnosis of extramedullary hematopoiesis (EMH) in the bone lesion, with predominant involvement of the myeloid series. EMH is a compensatory mechanism of the body to meet the demand for erythropoiesis. While EMH is commonly associated with thalassemia in children and myelofibrosis in adults, it can occur in various tissues throughout the body, including the intracranial region, although it is rare. The imaging findings of EMH on MRI typically show iso-hypointense signals on T1-weighted images and hypointense signals on T2-weighted images due to the presence of hemosiderin. However, in this case, the lesions appeared hyperintense on T2-weighted images, which posed a challenge in the differential diagnosis. The article highlights the importance of clinical evaluation and imaging findings in diagnosing EMH, as these lesions can resemble other conditions such as metastases, angiomatous meningioma, chloroma, or epidural hematoma. Giresun University, Faculty of Medicine, Turkey Giresun University, Faculty of Medicine, Turkey orkunonder535@gmail.com IMAGES IN CLINICAL RADIOLOGY Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient RAMAZAN ORKUN ONDER ALPTEKIN TOSUN TÜMAY BEKCI *Author affiliations can be found in the back matter of this article IMAGES IN CLINICAL RADIOLOGY Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient RAMAZAN ORKUN ONDER ALPTEKIN TOSUN TÜMAY BEKCI *Author affiliations can be found in the back matter of this article KEYWORDS: intracranial extramedullary hematopoiesis; metastases; meningioma; chloroma; epidural hematoma Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient IMAGES IN CLINICAL RADIOLOGY TO CITE THIS ARTICLE: TO CITE THIS ARTICLE: Orkun Onder R, Tosun A, Bekci T. Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient. Journal of the Belgian Society of Radiology. 2023; 107(1): 63, 1–3. DOI: https://doi. org/10.5334/jbsr.3268 Teaching Point: Isolated cases of intracranial EMH should be considered as a differential diagnosis in adult patients as they may be radiologically confused with malignant conditions such as metastases, angiomatous meningioma, chloroma or epidural hematoma. Orkun Onder et al. Journal of the Belgian Society of Radiology DOI: 10.5334/jbsr.3268 2 CASE HISTORY dominance of the myeloid series. The patient underwent a whole body CT scan and no other foci were found. A 56-year-old male patient presented with headache and swelling on the left side of the head. His medical history was unremarkable. Hemoglobin was 11.7 g/dL. Non- contrast computed tomography (CT) of the brain showed an expansile soft tissue lesion eroding the frontoparietal bone structures extending subcutaneously from the epidural space, and compressing the brain parenchyma on the left (Figure 1). Magnetic resonance imaging (MRI) showed that the lesion was hyperintense on T2- weighted images (WI) (Figure 2A), iso- to hypointense on T1WI (Figure 2B) and homogenously enhancing on contrast imges (Figure 3; red arrow). Smaller similar lesions were also observed in the ethmoid bone, orbit and other calvarial bone structures. (Figure 3; green arrows). Immunohistochemical examination confirmed the diagnosis of extramedullary hematopoiesis (EMH) in the frontoparietal bone lesion, characterized by diffuse staining with myeloperoxidase and CD33, indicating Figure 3. in our case. These lesions are defined as extra-axial masses that can potentially compress the underlying neuroparenchyma and resemble conditions such as metastases, angiomatous meningioma, chloroma or epidural hematoma [1]. Alptekin Tosun orcid.org/0000-0003-1783-9171 Giresun University, Faculty of Medicine, Department of Radiology, Giresun, Turkey Tümay Bekci orcid.org/0000-0002-3147-2786 Giresun University, Faculty of Medicine, Department of Radiology, Giresun, Turkey Alptekin Tosun orcid.org/0000-0003-1783-9171 Giresun University, Faculty of Medicine, Department of Radiology, Giresun, Turkey Alptekin Tosun orcid.org/0000-0003-1783-9171 Giresun University, Faculty of Medicine, Department of Radiology, Giresun, Turkey Tümay Bekci orcid.org/0000-0002-3147-2786 Giresun University, Faculty of Medicine, Department of Radiology, Giresun, Turkey AUTHOR AFFILIATIONS Ramazan Orkun Onder orcid.org/0000-0003-0616-2564 Giresun University, Faculty of Medicine, Turkey Ramazan Orkun Onder orcid.org/0000-0003-0616-2564 Giresun University, Faculty of Medicine, Turkey s12098-018-2618-z The authors have no competing interests to declare. 1. Merchant R, Choudhari AJ, Verma M, Patkar DP, Doctor P. Intracranial Hematopoiesis in Beta Thalassemia: A Case Series. Indian J Pediatr. 2018; 85(8): 679–681. Epub 2018 Jan 25. PMID: 29368108. DOI: https://doi.org/10.1007/ 1. Merchant R, Choudhari AJ, Verma M, Patkar DP, Doctor P. Intracranial Hematopoiesis in Beta Thalassemia: A Case Series. Indian J Pediatr. 2018; 85(8): 679–681. Epub 2018 Jan 25. PMID: 29368108. DOI: https://doi.org/10.1007/ TO CITE THIS ARTICLE: Orkun Onder R, Tosun A, Bekci T. Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient. Journal of the Belgian Society of Radiology. 2023; 107(1): 63, 1–3. DOI: https://doi.org/10.5334/jbsr.3268 Submitted: 11 July 2023 Accepted: 28 July 2023 Published: 25 August 2023 COMMENTS EMH acts as the body’s compensatory mechanism to meet the demand for erythropoiesis. Thalassaemia primarily causes EMH in children, while myelofibrosis is more common in adults. In addition to the bone marrow, EMH can occur in a wide variety of tissue and organss, including the liver, spleen, lymph nodes, adrenal glands, thymus, pleura, pulmonary interstitium, skin, gastrointestinal tract, paranasal sinuses, and even the dura mater. Intracranial EMH is particularly rare. Clinical evaluation with imaging findings is typically used for the diagnosis of EMH. On MRI, lesions exhibit iso-hypointense signal on T1WI and hypointense signals on T2WI due to the presence of hemosiderin. However, the lesions were slightly hyperintense on T2WI EMH acts as the body’s compensatory mechanism to meet the demand for erythropoiesis. Thalassaemia primarily causes EMH in children, while myelofibrosis is more common in adults. In addition to the bone marrow, EMH can occur in a wide variety of tissue and organss, including the liver, spleen, lymph nodes, adrenal glands, thymus, pleura, pulmonary interstitium, skin, gastrointestinal tract, paranasal sinuses, and even the dura mater. Intracranial EMH is particularly rare. Clinical evaluation with imaging findings is typically used for the diagnosis of EMH. On MRI, lesions exhibit iso-hypointense signal on T1WI and hypointense signals on T2WI due to the presence of hemosiderin. However, the lesions were slightly hyperintense on T2WI Figure 1. Figure 2. Figure 1. Figure 1. Figure 2. Figure 2. Figure 2. 3 Orkun Onder et al. Journal of the Belgian Society of Radiology DOI: 10.5334/jbsr.3268 3 Figure 3. REFERENCE The authors have no competing interests to declare. The authors have no competing interests to declare. COPYRIGHT: © 2023 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. Journal of the Belgian Society of Radiology is a peer-reviewed open access journal published by Ubiquity Press.
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https://link.springer.com/content/pdf/10.1007%2Fs00540-016-2241-y.pdf
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A comparative analysis of the effects of sevoflurane and propofol on cerebral oxygenation during steep Trendelenburg position and pneumoperitoneum for robotic-assisted laparoscopic prostatectomy
Journal of anesthesia
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J Anesth (2016) 30:949–955 DOI 10.1007/s00540-016-2241-y ORIGINAL ARTICLE ORIGINAL ARTICLE Keywords  Robotic-assisted laparoscopic prostatectomy (RALP) · Steep Trendelenburg position · Cerebral oxygenation · Sevoflurane · Propofol Keywords  Robotic-assisted laparoscopic prostatectomy (RALP) · Steep Trendelenburg position · Cerebral oxygenation · Sevoflurane · Propofol Keywords  Robotic-assisted laparoscopic prostatectomy (RALP) · Steep Trendelenburg position · Cerebral oxygenation · Sevoflurane · Propofol 1 Department of Anesthesiology, School of Medicine, Iwate Medical University, 19‑1 Uchimaru, Morioka 020‑0023, Japan A comparative analysis of the effects of sevoflurane and propofol on cerebral oxygenation during steep Trendelenburg position and pneumoperitoneum for robotic‑assisted laparoscopic prostatectomy Aya Doe1 · Motoi Kumagai1   · Yuichiro Tamura1 · Akira Sakai1 · Kenji Suzuki1 Received: 14 April 2016 / Accepted: 13 August 2016 / Published online: 26 August 2016 © The Author(s) 2016. This article is published with open access at Springerlink.com r2 = 0.065; p = 0.004) shows a weak relationship betw SjO2 and SctO2. Conclusions  Sevoflurane maintains higher SjO2 levels propofol during RALP. SctO2 does not accurately re SjO2. r2 = 0.065; p = 0.004) shows a weak relationship between SjO2 and SctO2.l * Motoi Kumagai bear@kxd.biglobe.ne.jp Abstract Purpose  Steep Trendelenburg position and pneumoperito- neum during robotic-assisted laparoscopic prostatectomy (RALP) increase intracranial pressure (ICP) and may alter cerebral blood flow (CBF) and oxygenation. Volatile anes- thetics and propofol have different effects on ICP, CBF, and cerebral metabolic rate and may have different impact on cerebral oxygenation during RALP. In this study, we meas- ured jugular venous bulb oxygenation (SjO2) and regional oxygen saturation (SctO2) in patients undergoing RALP to evaluate cerebral oxygenation and compared the effects of sevoflurane and propofol. We also verified whether SctO2 may be an alternative to SjO2. Conclusions  Sevoflurane maintains higher SjO2 levels than propofol during RALP. SctO2 does not accurately reflect SjO2. Introduction Robotic-assisted laparoscopic prostatectomy (RALP) requires a steep Trendelenburg position and pneumoperi- toneum, both of which can increase intracranial pressure (ICP) [1, 2]. Increased ICP, in turn, may decrease cerebral perfusion pressure and cerebral oxygenation [3, 4]. Methods  Fifty patients scheduled for RALP were ran- domly assigned to undergo sevoflurane (group S) or propo- fol (group P) anesthesia. SjO2, SctO2, mean arterial pres- sure (MAP), heart rate (HR), cardiac index (CI), central venous pressure (CVP), partial pressures of arterial oxygen (PaO2) and carbon dioxide (PaCO2), hemoglobin concen- tration (Hb), Bispectral Index (BIS) and nasopharyngeal temperature (BT) were recorded 5 min before surgery com- mencement, 5 min after pneumoperitoneum, 5, 30, 60, 90, and 120 min after pneumoperitoneum in a Trendelenburg position, and after desufflation in a supine position. Although sevoflurane and propofol are commonly used anesthetics, they have different effects on cerebral blood flow (CBF) and the cerebral metabolic rate for oxygen (CMRO2). Volatile anesthetics primarily increase CBF via local vasodilatation and reduce CMRO2. At low concentra- tions, where the effect of CMRO2 is dominant, these anes- thetics constrict cerebral vessels by suppressing CMRO2 and prevent an increase in the CBF and CBF/CMRO2 ratio. At the medium-to-high concentrations commonly used in clinical settings, the direct vasodilatory effect becomes dominant, causing CBF and the CBF/CMRO2 ratio to increase. With sevoflurane, this effect is accompanied by an increase in cerebral blood volume (CBV) and an elevated ICP [5]. In RALP, where ICP is already increased, it is con- ceivable that the CBV increase caused by volatile anesthet- ics could further increase ICP and in turn lower the CBF/ CMRO2 ratio and cerebral oxygenation. Supporting this Results  SjO2 was significantly higher in group S than in group P at all measurement points [group S vs. group P: 77 % (11) vs. 65 % (13), mean of all measurement points (1SD); p < 0.01]. Linear regression analysis (β = 0.106; 1 J Anesth (2016) 30:949–955 950 kg) with an oxygen fraction (FiO2) of 0.45. The respira- tory rate was adjusted to maintain an end-tidal carbon diox- ide tension (ETCO2) of 35–40 mmHg. In both groups, the concentrations of sedative drugs were adjusted to achieve a Bispectral Index (BIS) of 40–60. Sevoflurane was initi- ated at concentrations of 2.0 % and adjusted above 1.7 % (one minimum alveolar concentration; MAC), and propo- fol was maintained by TCI. Introduction Also, remifentanil was adjusted to maintain a mean arterial pressure (MAP) within 20 % of the preinduction value in both groups. When the MAP decreased to 80 % of the preinduction value, it was treated with an 8-mg bolus of ephedrine. Patients not anesthe- tized under these conditions were excluded from the final analysis. hypothesis, one study showed cerebral oxygen desatura- tion in a steep Trendelenburg position and pneumoperito- neum with isoflurane anesthesia [4]. Propofol reduces CBF by cerebral vasoconstriction and suppresses CMRO2. The relationship between the vasoconstriction and CMRO2 sup- pression effects of propofol is unclear; some studies have reported approximate equivalency [6], and others have reported that vasoconstriction is slightly dominant [7]. Propofol at least does not increase the CBF/CMRO2 ratio. Volatile anesthetics and propofol each have the potential to decrease cerebral oxygenation during RALP. Patients undergoing RALP are typically elderly and often have cerebrovascular disease that is associated with impaired cerebral oxygenation. These are not only asso- ciated with cerebral vasoconstriction or vasospasm dur- ing surgery but also with postoperative cognitive decline (POCD) [8]. Some studies suggest that the choice of anes- thetic affects cerebral oxygenation and POCD [9, 10]. Thus, it is important to monitor cerebral oxygenation and choose anesthetics that best preserve cerebral oxygenation. Routine monitoring including electrocardiography, pulse oximetry, and non-invasive automated blood pres- sure measurement, started on arrival in the operating room. After the induction of anesthesia, a radial artery catheter was inserted for direct arterial blood pressure measurement and blood sampling. The arterial line was also connected to a FloTrac sensor and a third-generation Vigileo moni- toring system (Edwards Lifesciences, Irvine, CA, USA) to monitor the cardiac index (CI) data. The ETCO2 and end- tidal concentration of sevoflurane were measured using a Vamos anesthetic gas monitor (Dräger Japan, Tokyo, Japan). Central venous pressure (CVP) was measured by a single-lumen central venous catheter that was inserted into the right internal jugular vein. A BIS electrode was applied to the forehead and was measured continuously using an Aspect BIS A2000 monitor v3.31 (Aspect Medical Sys- tems, Natick, MA, USA). Nasopharyngeal temperature (BT) was monitored continuously. Jugular venous bulb oxygen saturation (SjO2) accurately reflects the CBF/CMRO2 ratio but is invasive and complex [11]. By contrast, near-infrared spectroscopy (NIRS) can non-invasively assess SctO2, which can reflect the CBF/ CMRO2 ratio [12]. However, it has also been suggested that SctO2 and SjO2 are poorly correlated [12, 13]. Introduction To date, the difference in SjO2 between common anes- thetic agents has not been investigated during RALP. In this study, we compared the effects of sevoflurane and propofol on SjO2, and verified whether SctO2 could be used as an alternative to SjO2 during RALP. For SctO2 measurement, the cerebral oximeter probes were placed bilaterally ≥1 cm above each eyebrow. SctO2 was monitored with an INVOS5100B cerebral oximeter (Somanetics, Troy, MI, USA), and the values from each side were averaged. For the continuous monitoring of SjO2, a Pre-Sep Oximetry Catheter (Edwards Lifesciences, Irvine, CA, USA) was connected to the Vigileo system and placed in the left jugular venous bulb. The catheter position was verified radiographically. 1 3 Methods The correlation between SjO2 and SctO2 was evaluated by simple linear regression. Significance was determined at p  <  0.05, and we used XLSTAT 2015 for Windows (Addinsoft, New York, USA) for all analyses. Data are expressed as mean (standard deviation). Fig. 1   The time courses of jugular venous oxygen saturations (SjO2) in groups that received sevoflurane (filled circle) and propo- fol (opened circle). Values are expressed as mean (SD). Multi symbol p < 0.05 vs. group P Fig. 2   The time courses for regional oxygen saturations (SctO2) in groups receiving sevoflurane (filled circle) and propofol (opened cir- cle). Values are expressed as mean (SD). *p < 0.05 vs. T0 Methods There were no statistically significant differences between the two groups Fig. 1   The time courses of jugular venous oxygen saturations (SjO2) in groups that received sevoflurane (filled circle) and propo- fol (opened circle). Values are expressed as mean (SD). Multi symbol p < 0.05 vs. group P that we needed a minimum sample size of 17 patients per group to detect an effect size of 0.89 using independent Student’s t tests with an α of 0.05 and a power of 0.80. Thus, we aimed to include 25 patients per each group to allow for a 30 % drop-out rate. The Shapiro–Wilk test was used to test the normality of the data. Demographic vari- ables, duration of surgery and anesthesia, ephedrine and remifentanil doses, blood loss, urine output, and intrave- nous fluid volume between groups were compared using unpaired Student’s t tests. A two-way repeated-measures analysis of variance (ANOVA) with post hoc unpaired t test and Bonferroni correction was used to compare SjO2, SctO2, MAP, HR, CI, CVP, PaO2, PaCO2, Hb, BT, and BIS between the groups. A one-way repeated-measures ANOVA with Bonferroni post hoc tests was used to ana- lyze these variables across time within the groups. All statistical tests were two-tailed. The correlation between SjO2 and SctO2 was evaluated by simple linear regression. Significance was determined at p  <  0.05, and we used XLSTAT 2015 for Windows (Addinsoft, New York, USA) for all analyses. Data are expressed as mean (standard deviation). that we needed a minimum sample size of 17 patients per group to detect an effect size of 0.89 using independent Student’s t tests with an α of 0.05 and a power of 0.80. Thus, we aimed to include 25 patients per each group to allow for a 30 % drop-out rate. The Shapiro–Wilk test was used to test the normality of the data. Demographic vari- ables, duration of surgery and anesthesia, ephedrine and remifentanil doses, blood loss, urine output, and intrave- nous fluid volume between groups were compared using unpaired Student’s t tests. A two-way repeated-measures analysis of variance (ANOVA) with post hoc unpaired t test and Bonferroni correction was used to compare SjO2, SctO2, MAP, HR, CI, CVP, PaO2, PaCO2, Hb, BT, and BIS between the groups. A one-way repeated-measures ANOVA with Bonferroni post hoc tests was used to ana- lyze these variables across time within the groups. All statistical tests were two-tailed. Methods The institutional ethics committee of Iwate Medical Uni- versity Hospital, Japan, approved this study, and it has been registered in the UMIN Clinical Trials Registry (UMIN000016685). After obtaining written informed con- sent, we enrolled adult male patients who were undergo- ing RALP with the requirement that they were American Society of Anesthesiologists physical status class 1 or 2. Patients with known histories of cerebral ischemia or hem- orrhage were excluded. Participants were randomized by computer generation into groups that received either sevo- flurane (group S) or propofol (group P). We recorded the SjO2, SctO2, MAP, heart rate (HR), CI, CVP, PaO2, PaCO2, hemoglobin concentration (Hb), BIS, and BT at the following points: 5 min before surgery (T0); 5 min after inducing a 15 mmHg pneumoperitoneum (T1); 5, 30, 60, 90, and 120 min after inducing the pneumoperi- toneum in a 30° Trendelenburg position (T2, T3, T4, T5, and T6, respectively); and after desufflation in a supine position (T7). Anesthesia was induced in group S by a bolus injec- tion of thiopental (5 mg/kg) and in group P by an effect- site target-controlled infusion (TCI) of propofol of 5  µg/ ml; remifentanil (0.2–0.4  µg/kg/min) was used in both groups. After administering rocuronium (0.8  mg/kg), the trachea was intubated and the lungs were mechanically ventilated in volume control mode (tidal volume 8–10 ml/ Based on a pilot study, the sample size was calculated to detect a 9 % difference in the mean SjO2 value at T7 using G power: 68 % (±9 %) for group P compared with 77 % (±11 %) for group S. The power analysis indicated 1 3 1 3 J Anesth (2016) 30:949–955 951 Data are presented as the mean (SD). There were no statistically significant differences between the two groups Group S (n = 22) Group P (n = 19) p value Age (years) 67 (4) 66 (4) 0.21 Weight (kg) 67 (8) 68 (6) 0.65 Height (cm) 165 (6) 166 (5) 0.22 Anesthetic time (min) 284 (39) 282 (37) 0.69 Operation time (min) 201 (40) 201 (32) 0.73 Fluid administered (ml) 1132 (362) 1103 (287) 0.78 Blood loss (ml) 94 (119) 108 (81) 0.65 Urine output (ml) 163 (124) 121 (82) 0.15 Ephedrine administered (mg) 47 (20) 40 (16) 0.11 Total dose of remifentanil administered (mg) 4.2 (1.2) 4.7 (0.8) 0.19 Table 1   Demographic and anesthetic data Data are presented as the mean (SD). Results Of the 50 patients who were scheduled to enroll in the study, seven (three in group S and four in group P) were excluded. One patient in group P was found to have a his- tory of cerebral infarction after consenting to the study and six withdrew their consent before the operation. In group P, an additional two patients were excluded, one because nica- rdipine was administered to treat intraoperative hyperten- sion, and another because the FiO2 needed to be increased beyond the protocol limit to maintain the SaO2 >94 %. All of the measured values were normally distributed. There were no major complications on normal postoperative Fig. 2   The time courses for regional oxygen saturations (SctO2) in groups receiving sevoflurane (filled circle) and propofol (opened cir- cle). Values are expressed as mean (SD). *p < 0.05 vs. T0 rounds in either group. As shown in Table  1, there were no significant differences in the demographic or anesthetic data between the groups. 1 3 J Anesth (2016) 30:949–955 952 952 J Anesth (2016) 30:949 955 Fig. 3   Changes in a mean arterial pressure (MAP), b heart rate (HR), c cardiac index (CI), d central venous pressure (CVP), e naso- pharyngeal temperature (BT), f Bispectral Index (BIS), g partial pres- sures of arterial oxygen (PaO2), h partial pressures of carbon dioxide (PaCO2), and i hemoglobin concentration (Hb). Measurements were performed: 5 min before the commencement of surgery (T0); 5 min after a 15 mmHg pneumoperitoneum (T1); 5, 30, 60, 90 and 120 min after the Trendelenburg position (T2, T3, T4, T5, and T6, respec- tively); and after exsufflation in the supine position (T7). Plus symbol p < 0.05 vs. T0 in group S. Hash symbol p < 0.05 vs. T0 in group P. Multi symbol p < 0.05 vs. group P Fig. 3   Changes in a mean arterial pressure (MAP), b heart rate (HR), c cardiac index (CI), d central venous pressure (CVP), e naso- pharyngeal temperature (BT), f Bispectral Index (BIS), g partial pres- sures of arterial oxygen (PaO2), h partial pressures of carbon dioxide (PaCO2), and i hemoglobin concentration (Hb). Measurements were performed: 5 min before the commencement of surgery (T0); 5 min after a 15 mmHg pneumoperitoneum (T1); 5, 30, 60, 90 and 120 min after the Trendelenburg position (T2, T3, T4, T5, and T6, respec- tively); and after exsufflation in the supine position (T7). Results Plus symbol p < 0.05 vs. T0 in group S. Hash symbol p < 0.05 vs. T0 in group P. Multi symbol p < 0.05 vs. group P Figure 1 shows that the SjO2 was significantly higher in group S at all measurement points (p < 0.05) with no sig- nificant changes throughout the procedure when compared with T0. By contrast, Fig. 2 shows that there was no sig- nificant difference in the SctO2 values between the groups at each point, but that SctO2 was significantly higher at T2 than at T0 within each group. pneumoperitoneum in the Trendelenburg position (T2–T6) compared with T0. In group P, Hb was significantly lower at T7 than at T0. Compared with T0, there were no differ- ences within each group for PaO2, PaCO2, CI, Hb, BT, and BIS at each time point. Figure  4 shows the linear regression analysis for SjO2 and SctO2 during anesthesia, with evidence of only a weak relationship (β = 0.106; r2 = 0.065; p = 0.004). Figure  3 shows that there were no differences across the measured time points between the groups in most of the key parameters that could affect SjO2 and SctO2. How- ever, HR in group S was lower than that in group P at T0 and T1. Compared with T0, the MAP was significantly higher at T1–T6 in group S, and at T1, T2, and T6 in group P. In both groups, CVP was significantly higher during Discussion Although volatile anesthet- ics and propofol presumably have the potential to decrease cerebral oxygenation during RALP, we showed that neither did so. Considering the lack of significant changes in SjO2 compared with the baseline (T0), it is conceivable that pneu- moperitoneum and a 30° Trendelenburg position may not affect cerebral oxygenation within 2 h. This may be because the predicted raise in ICP was within the physiological com- pensation, such as cerebrospinal fluid (CSF) and blood mov- ing into the spinal canal and extracranial vasculature not to suppress cerebral oxygenation, and also may be due to the effect of cerebrovascular autoregulation although that might be partly impaired in group S. Fig. 4   Linear regression showing a weak relationship between regional oxygen saturation (SctO2) and jugular venous oxygen satura- tion (SjO2). Data are for 328 measurement pairs from 41 patients and showed that sevoflurane had significantly higher lev- els of SjO2 than propofol at all measurement points. We also observed no intergroup differences in MAP, CI, CVP, BT, BIS, PaO2, PaCO2, Hb at any time point and in the total ephedrine and remifentanil dose, even though HR was lower in group S at T0 and T1. The difference in HR may be explained by the higher doses of ephedrine administered in group P to treat hypotension during this period. Although not statistically significant, the PaO2 of group S was higher by about 15 mmHg at all measurement points, but this is likely too small to affect SjO2 physiologically and also from a lit- erature review [14]. BT and BIS have been strongly related to cerebral metabolism [15, 16], but there were no signifi- cant differences in these values between the groups suggest- ing that there was no difference in CMRO2. Because we used sevoflurane at a mid-range concentration (above 1 MAC), we presumed that the cerebrovascular dilation effect was domi- nant in group S and that the intergroup differences in SjO2 were due to the differences in CBF. Cerebral oxygen desaturation during surgery can predict early POCD and has been associated with delirium and pro- longed hospitalization among the elderly [17, 18]. Current study might show that sevoflurane is better than propofol for RALP, but the result of our study does not directly indicate that sevoflurane is a superior choice for RALP. Discussion The present study is the first to compare cerebral oxygena- tion by SjO2 between anesthetic regimens during RALP 3 1 3 953 J Anesth (2016) 30:949–955 Fig. 4   Linear regression showing a weak relationship between regional oxygen saturation (SctO2) and jugular venous oxygen satura- tion (SjO2). Data are for 328 measurement pairs from 41 patients SjO2 due to cerebrovascular stenosis during pneumoperito- neum and a steep Trendelenburg position. A study compar- ing sevoflurane and propofol anesthesia among patients with baseline SjO2 values <50 % showed no differences in post- operative cognitive function [20], but the study did not show the SjO2 difference between the groups and did not control for BP, CI, or PaCO2, so we cannot assess the true relation- ship between the anesthetics, SjO2, and postoperative cogni- tive function. Several studies have shown the differences in cerebral oxygenation depending on the anesthetic used in a variety of clinical settings, but only a few have done so for pneumoperitoneum in the Trendelenburg position. One study tested whether SctO2 differed between propofol and sevoflu- rane anesthesia during laparoscopic surgery in a 20° Tren- delenburg position [21], and concluded that propofol was associated with significantly lower SctO2 than sevoflurane. In situations of raised ICP (e.g., brain tumors), research with volatile anesthetics have shown that they may maintain a significantly higher SjO2 compared with propofol [22]. Fur- ther, in the supine position for normal surgery [23], in the sitting position for arthroscopy [13], and during one-lung ventilation for lung surgery [24], volatile anesthetics can maintain higher cerebral oxygenation than propofol. One pilot study has reported that, compared with sevoflurane, propofol increased cerebral SctO2 only when the stimulus of surgery was intense [25]. Although the authors specu- lated that an increased propofol dose due to the stimulation caused the CMRO2 to decrease, the mechanism remains unclear because the concentrations of sevoflurane were not disclosed. Most of the studies discussed above, including the current study, indicate that volatile anesthetics cause higher cerebral oxygenation levels with no differences in neurologi- cal complications or POCD when compared with propofol anesthesia. We also showed that SjO2 was well preserved in both groups (>50 %; Fig. 1). Although volatile anesthet- ics and propofol presumably have the potential to decrease cerebral oxygenation during RALP, we showed that neither did so. Discussion Considering the lack of significant changes in SjO2 compared with the baseline (T0), it is conceivable that pneu- moperitoneum and a 30° Trendelenburg position may not affect cerebral oxygenation within 2 h. This may be because the predicted raise in ICP was within the physiological com- pensation, such as cerebrospinal fluid (CSF) and blood mov- ing into the spinal canal and extracranial vasculature not to suppress cerebral oxygenation, and also may be due to the effect of cerebrovascular autoregulation although that might be partly impaired in group S. SjO2 due to cerebrovascular stenosis during pneumoperito- neum and a steep Trendelenburg position. A study compar- ing sevoflurane and propofol anesthesia among patients with baseline SjO2 values <50 % showed no differences in post- operative cognitive function [20], but the study did not show the SjO2 difference between the groups and did not control for BP, CI, or PaCO2, so we cannot assess the true relation- ship between the anesthetics, SjO2, and postoperative cogni- tive function. Several studies have shown the differences in cerebral oxygenation depending on the anesthetic used in a variety of clinical settings, but only a few have done so for pneumoperitoneum in the Trendelenburg position. One study tested whether SctO2 differed between propofol and sevoflu- rane anesthesia during laparoscopic surgery in a 20° Tren- delenburg position [21], and concluded that propofol was associated with significantly lower SctO2 than sevoflurane. In situations of raised ICP (e.g., brain tumors), research with volatile anesthetics have shown that they may maintain a significantly higher SjO2 compared with propofol [22]. Fur- ther, in the supine position for normal surgery [23], in the sitting position for arthroscopy [13], and during one-lung ventilation for lung surgery [24], volatile anesthetics can maintain higher cerebral oxygenation than propofol. One pilot study has reported that, compared with sevoflurane, propofol increased cerebral SctO2 only when the stimulus of surgery was intense [25]. Although the authors specu- lated that an increased propofol dose due to the stimulation caused the CMRO2 to decrease, the mechanism remains unclear because the concentrations of sevoflurane were not disclosed. Most of the studies discussed above, including the current study, indicate that volatile anesthetics cause higher cerebral oxygenation levels with no differences in neurologi- cal complications or POCD when compared with propofol anesthesia. We also showed that SjO2 was well preserved in both groups (>50 %; Fig. 1). References veins, and capillaries combined; and is affected by extrac- erebral blood flow, cerebrospinal fluid, and systemic blood pressure. By contrast, SjO2 measures only the intracerebral mixed venous saturation [27, 28]. Interestingly, in this study, SctO2 showed similar changes to BP across time, whereas SjO2 did not. Third, NIRS calculates SctO2 based on a fixed artery-to-vein ratio, regardless of changes induced by the Trendelenburg position and anesthetic agents [9, 28]. 1. Mavrocordatos P, Bissonnette B, Ravussin P. Effects of neck position and head elevation on intracranial pressure in anesthetized neurosurgical patients. J Neurosurg Anesth. 2000;12:10–4. 2. Halverson A, Buchanan R, Jacobs L, Shayani V, Hunt T, Riedel C, Sackier J. Evaluation of mechanism of increased intracranial pressure with insufflation. Surg Endosc. 1998;12:266–9. 3. Dunham CM, Sosnowski C, Porter JM, Siegal J, Kohli C. Cor- relation of noninvasive cerebral oximetry with cerebral perfu- sion in the severe head injured patient: a pilot study. J Trauma. 2002;52:40–6. The present study has several limitations. First, we did not routinely perform cognitive tests. Thus, it remains unclear whether postoperative cognitive outcomes correlate with differences in cerebral oxygenation by different anes- thetic agents; further research is needed. Second, we meas- ured SjO2 unilaterally, and although no research has shown a clear difference between left and right SjO2, this may have affected the results. Third, we did not include patients with cerebrovascular disease and complications, so we cannot state how the SjO2 or SctO2 would have responded to RALP in patients with a cerebral pathology. Fourth, the values for ICP, CBF, and CMRO2 were not measured directly, which should be rectified in future research. Fifth, we limited the time spent with a pneumoperitoneum in the Trendelenburg position to 120 min, yet there have been reports of neuro- logical complications due to brain edema when this proce- dure exceeds 8 h [29]. Actually, cerebrovascular autoregula- tion gradually changes with prolonged pneumoperitoneum in the Trendelenburg position; one study showed that it is decreased from 170  min [19]. Considering the inhibitory effect of volatile anesthetics on cerebrovascular autoregula- tion, it may be worth comparing the effects of longer sur- gery and steeper Trendelenburg positions in the future.l 4. Lee JR, Lee PB, Do SH, Jeon YT, Lee JM, Hwang JY, Han SH. The effect of gynaecological laparoscopic surgery on cerebral oxygenation. J Int Med Res. 2006;34:531–6. 5. Archer DP, Labrecque P, Tyler JL, Meyer E, Trop D. References Cerebral blood volume is increased in dogs during administration of nitrous oxide or isoflurane. Anesthesiology. 1987;67:642–8. 6. Oshima T, Karasawa F, Satoh T. Effects of propofol on cerebral blood flow and the metabolic rate of oxygen in humans. Acta Anaesthesiol Scand. 2002;46:831–5. 7. Stephan H, Sonntag H, Schenk HD, Kohlhausen S. Effect of Dis- oprivan (propofol) on the circulation and oxygen consumption of the brain and CO2 reactivity of brain vessels in the human. Anaesthesist. 1987;36(2):60–5. 8. Monk TG, Weldon BC, Garvan CW, Dede DE, van der Aa MT, Heilman KM, Gravenstein JS. Predictors of cognitive dysfunction after major noncardiac surgery. Anesthesiology. 2008;108:18–30. 9. Liu J, Wang P, Zhang X, Zhang W, Gu G. Effects of different concentration and duration time of isoflurane on acute and long- term neurocognitive function of young adult C57BL/6 mouse. Int J Clin Exp Pathol. 2014;7:5828–36. 10. Ballard C, Jones E, Gauge N, Aarsland D, Nilsen OB, Saxby BK, Lowery D, Corbett A, Wesnes K, Katsaiti E, Arden J, Amoako D, Prophet N, Purushothaman B, Green D. Optimised anaesthe- sia to reduce post-operative cognitive decline (POCD) in older patients undergoing elective surgery, a randomised controlled trial. PLoS One. 2012;7:e37410. We conclude that sevoflurane allows for a higher SjO2 than propofol during RALP. We also conclude that SctO2 does not accurately reflect SjO2 during this procedure. Ulti- mately, further research is needed to determine the effect of anesthetics on hyperemia and cerebral edema during longer pneumoperitoneums and in steeper Trendelenburg positions, as well as on the potential correlation between postoperative cognitive outcomes and differences in cerebral oxygenation between anesthetic agents. 11. Schell RM, Cole DJ. Cerebral monitoring: jugular venous oxi- metry. Anesth Anal. 2000;90:559–66.i 12. Casati A, Spreafico E, Putzu M, Fanelli G. New technology for noninvasive brain monitoring: continuous cerebral oximetry. Minerva Anesthesiol. 2006;72:605–25. 13. Jeong H, Jeong S, Lim HJ, Lee JU, Yoo KY. Cerebral oxygen saturation measured by Near-infrared spectroscopy and jugu- lar venous bulb oxygen saturation during arthroscopic shoul- der surgery in beach chair position under sevoflurane-nitrous oxide or propofol-remifentanil anesthesia. Anesthesiology. 2012;116:1047–56.l 14. Legrand M, Vallée F, Mateo J, Payen D. Influence of arterial dis- solved oxygen level on venous oxygen saturation: don’t forget the PaO2! Shock. 2014;41:510–3. Discussion Cerebrovas- cular dilation effect of sevoflurane also implies the cerebro- vascular dysregulation, which can be amplified during RALP [19] and could result in hyperemia and cerebral edema. These aspects must be clarified if we are to demonstrate the superiority of sevoflurane over propofol in a clinical setting because they might occur prior to a decrease in SjO2 or cere- bral perfusion pressure. Although we performed no cognitive tests, our study might suggest the appropriateness of sevoflu- rane over propofol for patients with lower baseline values for We also found only a weak relationship between SjO2 and SctO2. There are several factors that could contribute to the inconsistency between the two values. First, SjO2 is an index of oxygenation for the entire brain, whereas SctO2 is an index of localized oxygenation in the frontal lobe [9–11, 26]. Second, NIRS measures blood oxygenation in arteries, 1 J Anesth (2016) 30:949–955 954 References Compliance with ethical standards 15. Michenfelder JD. Anesthesia and brain: clinical, functional, metabolic, and vascular correlates. New York: Churchill Living- stone; 1988. Conflict of interest  The authors declare no conflicts of interest asso- ciated with this manuscript. 16. Alkire MT. Quantitative EEG correlations with brain glucose metabolic rate during anesthesia in volunteers. Anesthesiology. 1998;89:323–33. Open Access  This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://crea- tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 17. Tang L, Kazan R, Taddei R, Zaouter C, Cyr S, Hemmerling TM. Reduced cerebral oxygen saturation during thoracic surgery pre- dicts early postoperative cognitive dysfunction. Br J Anaesth. 2012;108:623–9. 18. Kadoi Y, Saito S, Goto F, Fujita N. Decrease in jugular venous oxygen saturation during normothermic cardiopulmonary bypass 1 3 J Anesth (2016) 30:949–955 955 predicts short-term postoperative neurologic dysfunction in elderly patients. J Am Coll Cardiol. 2001;38:1450–5. during one-lung ventilation under sevoflurane- or propofol- based anesthesia for lung surgery. J Cardiothorac Vasc Anesth. 2008;22:71–6. y p 19. Schramm P, Treiber AH, Berres M, Pestel G, Engelhard K, Wer- ner C, Closhen D. Time course of cerebrovascular autoregulation during extreme Trendelenburg position for robotic-assisted pros- tatic surgery. Anaesthesia. 2014;69:58–63. 25. Sen P, Izdes S, But A. Effects of sevoflurane and propofol anaes- thesia on cerebral oxygenation during normocapnia and mild hypercapnia: a pilot study. Br J Anaesth. 2013;110:318–9. g y 20. Guo JY, Fang JY, Xu SR, Wei M, Huang WQ. Effects of propo- fol versus sevoflurane on cerebral oxygenation and cognitive outcome in patients with impaired cerebral oxygenation. Ther Clin Risk Manag. 2016;12:81–5. 26. Yoshitani K, Kawaguchi M, Iwata M, Sasaoka N, Inoue S, Kuru- matani N, Furuya H. Comparison of changes in jugular venous bulb oxygen saturation and cerebral oxygen saturation during variations of haemoglobin concentration under propofol and sevoflurane anaesthesia. Br J Anaesth. 2005;94:341–6. g 21. Kim SJ, Kwon JY, Cho AR, Kim HK, Kim TK. The effects of sevoflurane and propofol anesthesia on cerebral oxygena- tion in gynecological laparoscopic surgery. Korean J Anesth. 2011;61:225–32. 27. Yoshitani K, Kawaguchi M, Miura N, Okuno T, Kanoda T, Ohni- shi Y, Kuro M. Effects of hemoglobin concentration, skull thick- ness, and the area of the cerebrospinal fluid layer on near-infrared spectroscopy measurements. Compliance with ethical standards Anesthesiology. 2007;106:458–62. ; 22. Muñoz HR, Núñez GE, de la Fuente JE, Campos MG. The effect of nitrous oxide on jugular bulb oxygen saturation during remifentanil plus target controlled infusion propofol or sevoflu- rane in patients with brain tumors. Anesth Anal. 2002;94:389–92. 28. Pollard V, Prough DS, DeMelo AE, Deyo DJ, Uchida T, Widman R. The influence of carbon dioxide and body position on near- infrared spectroscopic assessment of cerebral hemoglobin oxy- gen saturation. Anesth Anal. 1996;82:278–87. 23. Valencia L, Rodríguez-Pérez A, Kühlmorgen B, Santana RY. Does sevoflurane preserve regional cerebral oxygen saturation measured by near-infrared spectroscopy better than propofol? Ann Fr Anesth Reanim. 2014;33:e59–65. 29. Pandey R, Garg R, Darlong V, Punj J, Chandralekha, Kmar A. Unpredicted neurological complications after robotic laparo- scopic radical cystectomy and ileal conduit formation in steep Trendelenburg position: two case reports. Acta Anaesthesiol Belg. 2010;61:163–6. 24. Iwata M, Inoue S, Kawaguchi M, Takahama M, Tojo T, Tani- guchi S, Furuya H. Jugular bulb venous oxygen saturation 1 3 1 3 3
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toe, liet algemeene verschooningsrecht, waarop de openbare accountant aanspraak kan maken. toe, liet algemeene verschooningsrecht, waarop de openbare accountant aanspraak kan maken. p Op den beambte-accountant, de collega met de specifieke accoun- tantskennis, maar in dienstbetrekking bij een particuliere onderneming, rust, dit spreekt welhaast vanzelf, de zwijgplicht. Hij kan echter geen aanspraak op verschooningsrecht maken. p p g Hoe is bij het Nederlandsch Instituut van Accountants het beroeps­ geheim geregeld? Het antwoord kan kort zijn. Het woord beroepsgeheim komt nog niet in Statuten en Reglementen voor. Evenwel is thans een wijziging van het Reglement van Arbeid in behandeling; art. 2 van het ontwerp der herziening schrijft o.a. voor dat de leden gehouden zijn hun arbeid te verrichten met eerlijkheid, nauwkeurigheid en onzijdigheid en met inachtneming van de meest mogelijke geheimhouding. Onder ,,de meest mogelijke geheimhouding” moet niet worden verstaan: „absolute geheimhouding”. Vervolgens enkele opmerkingen over enkele Koninklijke Besluiten, wel­ ke na de bevrijding zijn gepubliceerd en waarin het verschooningsrecht wordt 'aangetast. Dit zijn het Besluit herstel rechtsverkeer (E 100) en het Besluit vijandelijk vermogen (E 133). In deze besluiten wordt uitdrukkelijk bepaald, dat advocaten, procureurs, notarissen, accountants, bankiers en ambtenaren, als getuigen of deskundigen opgeroepen, zich niet kunnen beroepen op eenig beroeps- of ambtsgeheim. Onder erkenning van het groote gemeenschapsbelang, dat is gelegen in rechtsherstel en in het achterhalen van vijandelijk en on-Vaderlandsch vermogen, aarzelt de inleider toch deze aantasting van het verschooningsrecht goed te keuren, want wordt niet aan een ander groot gemeenschapsbelang, de onbe­ schroomde raadpleging van vertrouwenslieden, groote schade toege­ bracht? Voorts vestigt de inleider nog de aandacht op een brief, welken het bestuur van het Nederlandsch Instituut van Accountants schreef aan den Raad van Ministers in verband met de opheffing van het beroepsgeheim, zooals bepaald in het Buitengewoon Navorderingsbesluit. Tenslotte gaat de inleider nog summier na, welke opvattingen in het buitenland heerschen met betrekking tot het beroepsgeheim van den open­ baren accountant en van de wijzigingen, welke daarin gedurende of na den oorlog zijn gekomen. BOEKBESPREKING Sociale spanningen, rede uitgesproken bij de aanvaarding van het ambt van hoogleraar aan de Rijksuniversiteit te Gronin­ gen, door Dr. P. J. Bouman. Uitgeverij H. ]. Paris, Amster­ dam, 30 blz. d D F H k Sociale spanningen, rede uitgesproken bij de aanvaarding van het ambt van hoogleraar aan de Rijksuniversiteit te Gronin­ gen, door Dr. P. J. Bouman. Uitgeverij H. ]. Paris, Amster­ dam, 30 blz. d D F H k door Dr. F. van Heek In vrijwel alle wetenschappen hebben sterk sprekende feiten en duide­ lijk aan den dag komende verschijnselen eerder de aandacht getrokken dan langzame veranderingen of verborgen, min of meer gelijkmatig verloo- pende processen. Een soortgelijke ontwikkeling valt bij de geschiedenis en de sociologie waar te nemen. De voorkeur der oudere historiografie voor gewelddadige gebeurtenissen — met veronachtzaming der geschie- m a b blz. 27 a b blz. m a b b m a b denis van het alledaagsche leven —■ is bekend. In de sociologie vinden wij een rijke literatuur over revoluties, terwijl de sociale spanningen vrijwel buiten beschouwing blijven. Dat deze verwaarloozing zeer ten onrechte geschiedt wordt door den schrijver van de te bespreken publicatie duide­ lijk aangetoond. j g De sociale spanning kenmerkt zich volgens Bouman door een massaal karakter. Daarbij komt een zekere mate van duurzaamheid; vluchtige emoties mogen er niet toe worden gerekend. Een derde kenmerk van de sociale spanning is haar latenten aard. De spanning is een phychische gesteldheid die zich ieder oogenblik in daden kan omzetten. Noodzakelijk is dit echter niet. In vele gevallen zullen spanningen fluctueeren en menig­ maal zelfs zoodanig verzwakken, dat zij hun naam ternauwernood meer verdienen. In dit verband gezien is het methodisch gewenscht de psy­ chische gesteldheid van de spanning te onderscheiden van het handelen dat uit de spanning resulteert. Zoo gaat de Marxistische klassenstrijd- theorie uit van de veronderstelling dat sociale tegenstellingen in ieder geval tot revoluties leiden. Marx verwart echter de symptomen die een aanwezigheid van sociale spanningen verraden, met klassenstrijd als zoodanig. g In Bouman’s rede wordt bijzondere aandacht geschonken aan de sociale spanningen, die hun oorsprong vinden in het ressentiment van een massa, die zich als rechteloos beschouwt en de bestaande sociale orde veroor­ deelt Deze spanningen zijn, volgens Mannheim, in de tegenwoordige maatschappij bijzonder gevaarlijk omdat onze samenleving in steeds min­ dere mate beheerscht wordt door de cultureel leiding gevende groepen. BOEKBESPREKING Groote massa’s laten zich gemakkelijk mobiliseeren door psychopaten die de gevoelens van hun individueel ressentiment op de door hen gelei­ den weten over te dragen. Zoo kan onze fragmentarisch over-gerationa- liseerde maatschappij een plotselinge omslag vertoonen naar wilde en on- beheerschte sociale bewegingen, waarin de maatschappelijke spanningen zich een uitweg zoeken. Bouman stelt de vraag, welke middelen ons ten dienste staan om dit gevaar op te heffen. Hij wijst op de noodzakelijk­ heid van redelijk overleg bij tegenover elkaar staande groepen en op de wenschelijkheid van deugdelijke voorlichting. Het is evenwel bekend, dat een beroep op het verstand of het redelijk inzicht in zijn effect niet mag worden overschat. Somtijds kunnen echter ongemotiveerde gevoe­ lens van haat of verachting gecompenseerd worden door een gemeen­ schappelijk élan b.v. in een emotioneel nationaal saamhoorigheidsgevoel. Het is beschamend te constateeren, dat een dergelijk élan gewoonlijk slechts in oorlogstijd wordt waargenomen. Is het mogelijk onder de huidige maatschappelijke omstandigheden een samenvattend ideaal te vinden, even krachtig als de oorlog zelf? Kan het ideaal der sociale rechtvaardigheid daartoe dienen of moet alle heil ver­ wacht worden van een herleving der christelijke moraal? Schrijver stelt deze vragen zonder ze te beantwoorden. Een derde weg om de sociale spanningen te doen verminderen, is het tegengaan van de massale opeen- hoopingen van menschen, die door sociaal-cultureele omstandigheden aan geestelijke en moreele nivelleering zijn prijsgegeven. Oordeelskracht en persoonlijk verantwoordelijkheidsgevoel gedijen slechts in kleiner groeps­ verband, waarin nog gemeenschapsgedachten leven. In verband met het bovenstaande noemt Bouman als sociaal en cultu­ reel hoopgevende tendenties van dezen tijd, de bereidwilligheid der ker­ ken om geestelijk leiding te geven bij groepsvorming van velerlei aard, de toenemende belangstelling voor bedrijfssolidariteit, de opkomst van de m ab blz. 28 m ab blz. 28 z.g. „wijkgedachte” in de steden (waarin stadswijken en buurten als orga­ nische eenheden tot hun recht komen) en ten slotte ook symptomen van sociale, cultureele herleving op het platteland, o.a. het werk van de volks- hoogescholen. g Het door Bouman gekozen onderwerp kan wegens de veelzijdigheid van het object moeilijk in het kader van een inaugurale rede worden samengevat. Niettemin is hij er in geslaagd een van de belangrijkste socio­ logische vraagstukken van dezen tijd op treffende wijze aan de orde te stellen. MEDEDEELING VAN HET NEDERLANDSCH INSTITUUT VAN ACCOUNTANTS Leden van verdienste; Bestuurssamenstelling; Nieuwe leden In de laatstgehouden Algemeene Vergadering van het Nederlandsch Instituut van Accountants zijn tot leden van verdienste dier vereeniging benoemd de heeren: Prof. R. A. Dijker te Wassenaar; H. R. Reder te Amsterdam. Prof. R. A. Dijker te Wassenaar; Tot bestuursleden werden gekozen de heeren: Tot bestuursleden werden gekozen de heeren: T. Keuzenkamp te 's-Gravenhage; G. Hartog te Amsterdam; G. Hartog te Amsterdam; K C Sl t t A t d K. C. Sluyter te Amsterdam. De bestuurssamenstelling is voor 1947 als volgt: De bestuurssamenstelling is voor 1947 als volgt: De bestuurssamenstelling is voor 1947 als volgt: J. Kraayenhof te Amsterdam, Voorzitter; T K k ’ G h Pl T. Keuzenkamp te ’s-Gravenhage, Plaatsvervangend-voorzitter: G A d S i G. Hartog te Amsterdam, Secretaris; A Th d L d Pl A. Th. de Lange, ec. drs., Plaatsvervangend-secretaris; K. C. Sluyter te Amsterdam, Penningmeester: J P d k d ' G h B J. Paardekooper, ec. drs. te 's-Gravenhage, Bibliothecaris; W . C. Koppenberg te Borne; Mr. B. Moret, ec. drs. te Rotterdam; A. Nierhoff te Amsterdam. In bedoelde Algemeene Vergadering werden de volgende heeren tot Ik! benoemd: J. G. Baak Aerdenhout Amsterdam A d S. C. Bakkenist, ec. drs. E B k Amsterdam A f E. Beekman J H d J. H. van der Burg A C Amersfoort A. M. Cats Den Haag d E. Driessen Amsterdam Den Haag d J. Ferdinandusse H. Fros Amsterdam Den Haag R d A. C. ). de Jong k g Rotterdam d R. Jonker C. F. Kamer Amsterdam J. G. Klimbie Haarlem Amsterdam A. J. König m a b blz. 29
https://openalex.org/W3091088548
https://link.springer.com/content/pdf/10.1007/s40572-020-00292-3.pdf
English
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Nutrition Transition and Climate Risks in Nigeria: Moving Towards Food Systems Policy Coherence
Current environmental health reports
2,020
cc-by
8,997
ERROR: type should be string, got "https://doi.org/10.1007/s40572-020-00292-3\nCurrent Environmental Health Reports (2020) 7:392–403 https://doi.org/10.1007/s40572-020-00292-3\nCurrent Environmental Health Reports (2020) 7:392–403 CLIMATE CHANGE AND HEALTH (C GOLDEN, SECTION EDITOR) Abstract Purpose of Review The purpose of this review is to describe the combined impacts of the nutrition transition and climate change\nin Nigeria and analyze the country’s national food-related policy options that could support human and planetary health\noutcomes. Recent Findings This paper uses a food systems framework to analyze how the nutrition transition and climate change interact in\nNigeria affecting both diets and the double burden of malnutrition, resulting in what has been termed the syndemic. Interactions\nbetween climate change and the nutrition transition in Nigeria are exacerbating diet-related inequities and will continue to do so if\nfood systems continue on their current trajectory and without significant transformation. Siloed policy actions that attempt to\nmitigate one aspect of food system risk can create a negative feedback loop in another aspect of the food system. Our analysis\nfinds that Nigeria has five national policies that include actionable steps to address food system insufficiencies; however, each of\nthese policies is constrained by the boundaries of singular nutrition, climate change, and agricultural objectives. The country\nshould consider a coherent policy environment that explicitly identifies and links underlying systemic and institutional drivers\nbetween climate change and malnutrition that simultaneously and comprehensively address both human and planetary health\noutcomes of food systems. Summary The systemic and institutional outcomes of this emerging syndemic—undernutrition, obesity, and climate change—\nare inexorably linked. Nigeria lacks a coherent policy environment taking on this challenging syndemic landscape. The analysis\nin this paper highlights the need for Nigeria to prioritize their national nutrition and agricultural and climate policies that uncouple\nfeedback loops within food systems to address climate change and malnutrition in all its forms. Keywords Nigeria . Food systems . Policy coherence . Nutrition transition . Climate change . Planetary health Nutrition Transition and Climate Risks in Nigeria: Moving Towards\nFood Systems Policy Coherence Alexandra E. Morgan1 & Jessica Fanzo2 Accepted: 16 September 2020\n# The Author(s) 2020\n/ Published online: 1 October 2020 Accepted: 16 September 2020\n# The Author(s) 2020\n/ Published online: 1 October 2020 2\nThe Nitze School of Advanced International Studies, Bloomberg\nSchool of Public Health, Berman Institute of Bioethics, Johns\nHopkins University, Washington, DC, USA 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA This article is part of the Topical Collection on Climate Change and\nHealth Introduction staple grains and salt, and sanitation and hygiene improve-\nments are partially responsible for reductions in the worldwide\nburden of undernourishment from 14.8 to 10.8% [3]. However, urbanization, income shifts, and work and lifestyle\npatterns have simultaneously produced an increase in over-\nweight and obesity across all regions [3]. The past two decades have produced a dynamic shift in world\ndiets for low and lower middle-income countries [1•, 2]. Increased income and some significant interventions such as\nthe promotion of exclusive breastfeeding, fortification of As populations become more affluent, dietary consumption\npatterns shift. While diets become more diverse, moving away\nfrom meals composed predominantly with staple grains and/\nor tubers, there is also higher intake of refined carbohydrates\nand highly processed foods (and drinks) with higher amounts\nof added sugars, unhealthy fats, and sodium, and animal\nsourced foods. This typical dietary shift is known as the nu-\ntrition transition [4, 5]. This pattern is associated with global-\nization and urbanization factors but also an increasingly sed-\nentary lifestyle [6], and the combination of these trends leads\nto shifts away from infectious diseases towards diet-related This article is part of the Topical Collection on Climate Change and\nHealth 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA Curr Envir Health Rpt (2020) 7:392–403 393 non-communicable diseases such as cardiovascular disease,\ndiabetes, and stroke [4, 5]. undernutrition, and unsustainable food systems are associated\nwith both burdens [8•, 15••, 18]. If no action is taken to ad-\ndress climate change and its impacts on food systems, the\nhistoric progress made to reduce undernutrition could be lost,\nand overweight and obesity trends will continue to increase\n[1•, 8•, 15••]. Human and planetary health outcomes cannot be\ndecoupled, and effective policies must target the feedback\nloops linking the health of both the planet and people [9••,\n15••]. What results from this transition is a population dealing\nwith both undernutrition and overweight and obesity—what\nhas been termed the double burden of malnutrition (also\ntermed the multiple burdens of malnutrition) [1•]. The inter-\naction between the nutrition transition and the epigenetic ef-\nfects of undernutrition has fueled this trend. Introduction Early nutritional\ndeficits put certain populations at risk for overweight and obe-\nsity later into adulthood [1, 7]. Both of these trends affect\nmorbidity and mortality, and poor diets remain a high-risk\nfactor for childhood death as well as adult non-\ncommunicable disease. Nigeria is the most populous country in Africa, and its\npopulation of 196 million people [19] is expected to double\nby 2050 [20]. Within the Economic Community of West\nAfrican States, Nigeria makes up more than two thirds of\nGDP [21]. Because of its economic growth and political status\nin the region, continent, and globally, guiding Nigeria towards\na trajectory towards sustainable development is critical\n[21–23]. Food systems connect and contribute to these dietary shifts,\npoor health outcomes, and environmental degradation [8•,\n9••]. Unsustainable food production systems allocate calories\nand nutrients in inefficient ways, while also contributing to\nclimate change [8•, 9••] and natural resource degradation\n[9••, 10••, 11]. Because of this circularity, climate change will\ncontinue to compound the health effects of malnutrition in all\nits forms [8•, 12–14]. The food systems framework (Fig. 1)\nhighlights the interactions between environment, food, and\nhealth. Nigeria is already experiencing the earlier described\n“syndemic.” Average maximum temperatures and average\nnumber of hot days have increased, while precipitation has\ndecreased in the country [24]. Temperature change has led\nto increasing desertification in the north, erratic and changing\nrainfall across the country, and increased flood risk in coastal\nregions and along the major river systems [25–28]. These\nchanges make food production, distribution, and utilization\nmore vulnerable, which in turn affect dietary and nutrition\noutcomes [16••, 29]. At the same time, Nigeria is experiencing\nthe double burden of malnutrition [30–32] with malnutrition\nranked as the number one risk factor for death and disability,\nand sub-optimal diets have risen to become the 7th highest\nrisk in the past decade [33]. Both climate change and the\nnutrition transition interact with the demographic shifts Climate change, undernutrition, and obesity share underly-\ning societal causes, and feedback loops between them magni-\nfy negative health and nutrition outcomes. This “syndemic” is\na synergy of epidemics that occur at the same time and place,\ninteract, and share common drivers [15••]. These emerging\nsyndemic risks exist in most countries but are particularly\nacute for the rural poor in the global South [16••, 17]. Food Systems Framing Food systems are composed of the people and activities that\nplay a part in growing, transporting, supplying, and, ultimate-\nly, eating food. These processes also involve elements that\noften go unseen, such as food preferences and resource invest-\nments. Food systems influence diets by determining what\nkinds of foods are produced and available in markets. They\nalso influence what foods people want to eat and are able to\naccess. As shown in Fig. 1, the different parts of the food\nsystem include food supply chains, food environments, and\nconsumer behavior (not shown in this framework for simpli-\nfication purposes). Food supply chains consist of the activities\nand actors that take food from production, storage and distri-\nbution, process and packaging, retail, and waste [34, 35]. Food environments are the physical, economic, political and\nsocio-cultural surroundings, opportunities, and conditions that\ncreate everyday prompts, shaping people’s dietary preferences\nand choices as well as nutritional status [34, 36]. They can be\nmarkets, restaurants, or cafeterias. These different parts shape\nfood systems and can lead to both positive and negative out-\ncomes across a range of outcomes [34]. Both rural-urban and north-south divisions are character-\nized by different nutritional challenges in Nigeria. While the\npopulation is evenly split between rural and urban residents\n[49], currently, urban populations are more likely to experi-\nence obesity and overweight while rural populations are more\nlikely to experience undernutrition [45]. However, evidence\nsuggests that rural places are beginning to catch up with urban\nobesity trends [50]. At the same time, DHS data show that in\nthe last 20 years underweight and vitamin deficiencies have\nbeen higher in the northern arid zones of the country, while\noverweight and obesity have been higher in the southern\nzones. Although northern zones produce more food for the\ncountry, residents in the north are the most likely to be food\ninsecure [23]. Childhood stunting and undernutrition are par-\nticularly acute in northern Nigeria. Almost half of all children\nin the northeast and northwest are stunted [51]. Food systems are not static. They are in constant transition\nand are shaped and shifted by different internal and external\ndrivers that push or pull the system in different directions [37]. While Fig. Introduction An\nincreasing number of countries are experiencing a double bur-\nden of overweight and obesity alongside continued Food supply chains\nFood environments\nFood production \nsystems and \ninput supply\nStorage and distribution\nProcessing and \npackaging\nDiets\nNutrition and \nhealth \noutcomes\nOther impacts\nSocial\nEconomic\nEnvironmental\nClimate \nChange\nGlobalization \nand Trade\nIncome \nGrowth and \nDistribution\nFood availability\nFood affordability\nProduct properties\nVendor properties\nFood messaging\nUrbanization\nFig. 1 Food systems framework. Source: 35 Income \nGrowth and \nDistribution Urbanization Nutrition and \nhealth \noutcomes Processing and \npackaging Fig. 1 Food systems framework. Source: 35 Curr Envir Health Rpt (2020) 7:392–403 394 occurring in Nigeria, exacerbating dietary inequalities and af-\nfecting access to high quality, nutrient-dense, and safe food on\na reliable basis. environments. For the purposes of this paper, we are focusing\non the gray-shaded elements and relationships of Fig. 1. Climate change and natural resources shape food system\nfunctionality, efficiency, and resiliency [38]. In turn, food sys-\ntems are a cause of climate change and environmental degra-\ndation [39]. Food systems contribute to greenhouse gas emis-\nsions, depletion of freshwater resources, deforestation, and\nnutrient pollution on landscapes and waterways [12]. The di-\nversity, safety, and quality of diets very much depend on\nthriving food systems. However, evidence suggests that sub-\noptimal diets dominate across the world, contributing to both\nenvironmental and climate degradation and detrimental health\noutcomes [9••]. This paper uses an adapted food systems framework (Fig. 1) from the High-Level Panel of Experts Report on Nutrition\nand Food Systems [34] to analyze the combined effects of\nclimate change and the nutrition transition on Nigerian diets,\nexamining multiple ways climate change will affect food sup-\nply chains and environments and its dietary and nutritional\nimpacts on consumers. It also reviews the literature on two-\nway relationship of role climate change in accelerating the\nnutrition transition and vice versa in Nigeria and assesses to\nwhat degree current national food-related policies in Nigeria\naddress the syndemic. Siloed actions that attempt to mitigate\nthe effects of one issue can create potential negative feedback\nloops and impacts in another area [15••]. In order to address\nclimate change and malnutrition, Nigeria must prioritize pol-\nicies that address the syndemic—by uncoupling feedback\nloops between climate, undernutrition, and obesity by\nstrengthening food system actions. Nutrition Transition of Nigeria Malnutrition in all its forms is the number one risk factor for\ndeath and disease in Nigeria [33]. The combined effects of\nrising obesity and stagnating stunting in children indicate that\nNigeria is going through a nutrition transition. Demographic\nand Health Survey (DHS) data have shown slight declines in\nstunting for children and adult female thinness as well as in-\ncreasing adult female overweight and obesity over the last\n20 years [40–44]. Independent studies have found that urban\nobesity increased by 20% in Nigeria between 2002 and 2010\n[45]. While some populations have access to sufficient calo-\nries, stunting and thinness have decreased slowly, and under-\nnourishment overall has risen since 2000, indicating that ad-\ndressing malnutrition will require multiple strategies that span\nhealth, food, care, and the environment [46–48]. Food Systems Framing 1 does not show all the myriad drivers that shape\nfood systems, climate change, urbanization, and globalization\nare significant drivers that instigate feedback loops of food\nsystem outcomes—diets, nutrition and health, environment,\nand livelihoods—on food supply chains and food Currently, Nigeria cannot meet food requirements through\ndomestic production [52]. The major food crops produced in\nthe country include cassava, cowpea, and sorghum [31, 53]. The Government of Nigeria (GON) reports that demand ex-\nceeds supply by more than 50% for rice, wheat, fish, milk, and\ntomatoes. Nigeria also underproduces maize, chicken, yams, Curr Envir Health Rpt (2020) 7:392–403 395 to 2010, and sugar-sweetened beverages have increased. There are increases, although small, in the consumption of\nmilk and fruits. This data source does not capture fish and\nseafood consumption, but these food sources are important\ncomponents of the Nigerian diet. Overall, the health of the\ndiet has not changed much over the last 15 years. Across the\nentire country, only 56% of women meet the minimum criteria\nfor dietary diversity, a marker for dietary quality, in the pre-\nvious day [40]. and oil palm to a lesser extent [52] and has significant losses of\nfruits and vegetables as these perishable foods move along the\nsupply chain [54]. Because it cannot meet its population’s\ndemand with its own production, Nigeria imports between\n$3 and $5 billion worth of food annually [55]. Food imports both reduce Nigeria’s foreign exchange re-\nserves and make consumers vulnerable to international prices. Over half of the population lives on less than $2 per day,\nincluding the majority of rural residents [4]. While some\nsmallholder farmers produce enough food for family con-\nsumption, the average rural family only produces a quarter\nof food they consume [18, 56]. Across the country, the aver-\nage household spends 65% of its income on food expenditure\n[57]. The unaffordability of nutritious food can be a signifi-\ncant barrier to consuming a healthy diet. The cost of a nutri-\ntionally adequate diet (CoNA) measures the minimum daily\ncost of meeting nutrient and energy requirements for a refer-\nence healthy adult woman, as a percent of daily per capita\nhousehold spending on food and non-alcoholic beverages. In\nNigeria, the CoNA is 92%, whereas in South Africa it is 42%\nand in the USA it is 32% [53]. Climate Vulnerability for Nigeria and Nigeria’s\nFood System In Nigeria, an increase in the number of days\nwith extreme temperature will reduce the output of cassava,\ncocoyam, sweet potatoes, cowpeas, and maize [63] By 2050,\nhotter temperatures are expected to reduce output of rice, sor-\nghum, millet, maize, yam, and cassava [68]. Increased rainfall\nwill also compromise sweet potato and rice production [64]. In the short term, warmer temperatures are associated with\nincreased production of millet, onions, tomatoes, and melon\nin northern Nigeria [63]; however, erratic rainfall patterns may\nreduce any positive effects of temperature. Pricing feedback loops highlight the economic effects of\nclimate change. Lowered or shifted yields will impact liveli-\nhoods for 60% of Nigeria’s labor force involved in agricultur-\nal production, 90% of which are smallholder farmers [27]. The\nprice effects of lowered yields will also affect non-farming\nand urban populations reducing their access to high quality\nand nutritious diets. Additional challenges affecting the food\nsystem include certain push factors such as increasing move-\nment from rural to urban areas, putting more stress on food\nsupply chains to urban areas and encroachment of urbaniza-\ntion into adjacent rural land (hinterlands) that are near major\nurban centers [26]. Figure 3 summarizes some effects of cli-\nmate change on food system elements (focusing on food sup-\nply chains and food environments), intermediary diet out-\ncomes, and nutrition and health outcomes in Nigeria using\nthe food systems framework in Fig. 1. Nutrient content will also shift in major food crops and\nconsumers’ access to nutrients will be negatively affected by\nchanges to food storage. Increased concentrations of atmo-\nspheric carbon dioxide will reduce zinc, iron, and protein in\nrice [69] and cassava will have increased concentrations of\ncyanide [60]. Heat will also make storage of vegetables more\ndifficult [29]. Consumers will also have elevated exposure to\nmycotoxins, such as aflatoxin, and mold increases because\nonly 10% of maize traders across the country fumigate and\ndry their wares [64]. Increased surface water temperature, sea level rise, and\nocean acidification will negatively affect aquaculture and fish-\neries [70, 71]. This will affect nutrient access for much of\nNigeria since fish make up about ½ of protein intake [70]. Human control may make aquaculture more resilient to cli-\nmate change [70]; however, farmed fish may hold less nutri-\ntional value than wild caught fish [72]. Nigeria is one of the\nseveral countries identified by Golden et al. Climate Vulnerability for Nigeria and Nigeria’s\nFood System The Nigerian government predicts that the major impacts for\nagricultural production will include lower yields and poor\nlivestock performance in the Sahel and savanna agro-\necological zones, rapid farm wastage, decreased soil fertility,\nand leaching. Seawater incursion and desertification will re-\nduce arable land [29]. Flooding will also affect coastal and\nfreshwater fisheries production negatively through siltation\nand contamination [61]. Some climate models suggest that\nrainfed agriculture may decline up to 50% by 2080, while\noverall agricultural productivity could decline by 10–25%\n[65]. irrigation. Only 1% of agricultural land currently uses modern\nirrigation [26, 28, 73]. The north central region may have\nlower sensitivity to rainfall changes in the short run, if lake\nwater in the region is harnessed to provide irrigation [26]. However, irrigation is not a panacea given uncertain changes\nin rainfall. Extreme weather may also cause flooding and a\nwidespread lack of flood control will make erratic rain partic-\nularly damaging [29, 62]. The agricultural impact of severe flooding caused by heavy\nrains in 2012 provides an example of the multiple pathways in\nwhich climate change may affect household nutrition. In\n2012, heavy rainfall strained dam infrastructure and flooded\nsettlements along the Niger, Benue, and Gongola rivers, caus-\ning $16.9 billion dollars worth of damage [62]. The disaster\ndestroyed 30% of the year’s rice production. The majority of\nfamilies affected by floods that relied on agricultural produc-\ntion to meet their family’s nutritional needs were forced to turn\nto markets to buy the majority of their food [62]. Increased\ndemand created a subsequent negative feedback loop elevat-\ning the price index for food and making market consumption\nmore inaccessible. Scarcity coping strategies used by families\nincluded borrowing food, reducing or skipping meals, and\nrelying on less preferred foods [62]. Countrywide surveys\nshow that even absent crisis level shocks, many families resort\nto food coping strategies. In 2010 and 2011, countrywide\nsurveys showed that between 7 and 9% of those in the lowest\nincome quintile used severe coping strategies such as going to\nbed hungry or borrowing food from the community or local\nNGOs as food assistance in the past 7 days [56]. Increasing temperature [63], shifts in rainfall, [64, 66] and\natmospheric carbon dioxide concentration [60] will drive\nshifts in agricultural yield. Across West Africa, a hotter dry\nseason will cause rice yields to decline, even with increased\nirrigation [67]. Climate Vulnerability for Nigeria and Nigeria’s\nFood System Temperatures in Nigeria are estimated to rise between 1.1 and\n2.6 °C by 2060 [60] leading to losses of up to 30% of GDP\n[61]. Sea level rise is expected to reach 1 m by 2050 [27]\naffecting coastal regions and river systems. Temperature in-\ncreases will affect agriculture mainly through changes in rain-\nfall. Climate change is projected to make the northern zones\ndrier and the southern zones more wet [27, 28, 60] Increasing\ndesertification will harm the northern part of the country,\nwhile sea level rise, saltwater intrusion, and flooding will af-\nfect the southern part of the country [62–64]. While GON analysis focuses mainly on staple crops, low\nconsumption of fruits and vegetables are also associated with\nnegative health outcomes, and the combination of both do-\nmestically produced and imported fruits and vegetables in\nNigeria do not meet the dietary requirements for daily vege-\ntable consumption [58]. Climate change will impact Nigeria’s agro-ecological\nzones in different ways. Nigeria has two main types of\nvegetation—tropical rainforest in the south and savannah in\nthe north. Erratic rainfall and unpredictable severe weather\nevents may affect agricultural production across both areas. While specific climate risks will differ depending on the agro-\necological zone, most key challenges will be related to water Figure 2 shows how diets have changed in Nigeria com-\nparing data from 1990 to 2010 (the most recent available)\nfrom the Global Dietary Database. There are interesting\ntrends. Whole grains and legumes have decreased from 1990 27.8\n98.1\n1.8\n100.2\n37.9\n15.3\n5.8\n25.6\n25.4\n53.2\n113.9\n58.9\n28.1\n103.3\n2.8\n82.3\n41.3\n17.5\n5.7\n25.3\n26.2\n72.3\n117.6\n44.8\n0.0\n20.0\n40.0\n60.0\n80.0\n100.0\n120.0\n140.0\nfiber\nfruits\nfruit juices\nlegumes\nmilk\nnuts and seeds\nprocessed meats\nred meats\nseafood\nsugar-sweetened beverages\nvegetables\nwhole grains\ngrams/day\n2010\n1990\nFig. 2 Changes in the Nigerian diets, 1990–2010. Source: [59] 2010\n1990 Fig. 2 Changes in the Nigerian diets, 1990–2010. Source: [59] Curr Envir Health Rpt (2020) 7:392–403 396 resources. In the north, dryness and drought will affect both\nagricultural production and livestock, while in the south, soil\nporosity issues will heighten the effects of sudden increased\nrain and flooding [26]. Climate change will impact each zone\ndifferently but will also shift the boundaries between the zones\n[27], challenging traditional agricultural cultivation patterns. Climate Vulnerability for Nigeria and Nigeria’s\nFood System as most reliant on\nfish, which also has estimated marine catch reductions of over\n20% by 2050 [72]. Climate Change-Driven Food System Impacts Nutrition and Health Outcomes Intermediary Dietary Outcomes Intermediary Dietary Outcomes Climate Change-Driven Food System Impacts\nIntermediary Dietary Outcomes\nNutrition and Health Outcomes\nReduced caloric intake\nReduced dietary quality and diversity\nIncreased risk of foodborne illness\nIncreased consumption of less \nperishable staple grains and tubers\nWasting \nStunting \nMicronutrient Deficiencies \nOverweight and Obesity\nNCDs\nFood supply chains\nReduced fruit and vegetable production\nIncreased loss of perishable foods\nIncreased contamination of foods with aflatoxin and molds\nIncreased concentration of cyanide in cocoyams\nDisrupted transportation and distribution\nLowered livestock productivity\nReduced fisheries and aquaculture production\nLowered production of rice, cocoyam, sweet potatoes\nReduced micronutrient content in rice and cassava\nFood environments\nIncreased vulnerability to infrastructure of markets\nReduced ability to get to markets Less foods available \nin markets\nIncrease waste of perishable foods\nIncreased cost of food\nFig. 3 Impacts of climate change on food systems for Nigeria. Sources: [26, 27, 29, 60, 61, 63, 64, 67] Food supply chains Increased risk of foodborne illness Wasting \nStunting \nMicronutrient Deficiencies \nOverweight and Obesity\nNCDs Reduced caloric intake\nReduced dietary quality and diversity Increased consumption of less \nperishable staple grains and tubers Fig. 3 Impacts of climate change on food systems for Nigeria. Sources: [26, 27, 29, 60, 61, 63, 64, 67] CCN), the National Policy on Food and Nutrition (NPFN), the\nAgriculture Sector Food Security and Nutrition Policy\n(ASFS), and the National Strategic Plan of Action on\nNutrition (NSPAN), to determine whether they used a food\nsystems framing to develop interventions targeting the\nsyndemic—malnutrition, nutrition-sensitive agriculture, and\nclimate change. Nigeria committed to the creation of a health\nand environment strategic alliance in the Libreville declara-\ntion, acknowledging the important link between environmen-\ntal risks and health outcomes [74]. Overall, Nigeria has devel-\noped strong policy guiding documents and national strategies\non climate change, nutrition, and agriculture. However, policy\nimplementation has been limited due to budgetary constraints\nand stakeholder fragmentation [23, 75] with state budgets and\npolicies not matching federal planning [23, 76]. affordability of diets or the choice architecture of these built\nplaces to facilitate healthier eating. More significantly, only 3\nof the policies contain a cost analysis of implementing the\ngoals and actions set out in those policies. All of the policies\ncontained a monitoring and evaluation (M&E) plan. Assessment of Relevant Nigerian Food-\nand Climate-Related Policies Using the food systems framework as a guide, we assessed\nfive national policies in Nigeria, the Agriculture Promotion\nPolicy (APP), the National Adaptation Strategy (NASPA- Rainfall change will have particularly negative effects on\nagriculture because of Nigeria’s dependence on rainfed Curr Envir Health Rpt (2020) 7:392–403 397 Climate Change-Driven Food System Impacts The most\ncomprehensive set of interventions along the supply chain\ncomes from the Agriculture Sector Food Security and\nNutrition Policy; however, this plan does not explicitly inte-\ngrate the effects of climate change on the supply chain. Table 1 below summarizes the results of the analysis. Challenges to Implementation While Nigeria’s policy documents highlight actionable items\nto create nutrition-sensitive and climate-smart interventions,\nsignificant challenges remain. The NASPA-CCN was devel-\noped in 2011, but fragmented implementation of the strategy\nhas made farm-level adaptation to climate change difficult to\nachieve [78]. The agricultural section of the National Climate\nPolicy focuses on the diversification of livestock, increased\naccess to drought resistant crops, better soil management prac-\ntices, national early warning systems, and increased use of\nirrigation and crop cover [61]. The government also suggests\nthe increased provision of crop insurance at subsidized rates\nfor smallholder farmers, provided by non-profits, or through\npublic-private partnerships. However, adaptation results have\nbeen limited. While the climate policy has ostensibly been in\neffect for 9 years, a 2019 study in two northern communities\nfound that while farmers were using adaptation strategies, they\nwere not utilizing the strategies best suited for their dryland The Nigerian government has made inroads to combine\nagriculture and nutrition policies using a systems perspective. However, the explicit integration of climate change adaptation\nand mitigation actions into nutrition policy has been limited. Three of 5 policies recognize the double burden of undernu-\ntrition and overweight/obesity, while two policies focus on\nundernutrition exclusively. The three nutrition polices have a\nweak focus on climate change, while the climate change pol-\nicy has little focus on nutrition. All five policies make target\ninterventions at various points along the supply chain, but\nagriculture production focused interventions are the most\nprevalent in the climate change and agriculture policies, while\ndemand focused interventions to improve diets are stronger in\nthe nutrition policies. Food environment interventions overall\nwere limited with very little focus on how to change the Curr Envir Health Rpt (2020) 7:392–403 Table 1\nNational policy environment in Nigeria\nNational strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Challenges to Implementation Cost\nanalysis\nM&E plan\nNational Adaptation Strategy\nand Plan of Action\n(NASPA-CCN)\n2011–current No\nUndernutrition only\nNo\n•Focus on climate impacts on\nagricultural production\n•Focus on food access\n•No mention of nutritional\nquality of food\nNo\nFood supply chains\n•Improved agriculture systems for\ncrops and livestock\n•Increase irrigation\nFood environment/markets\n•Facilitate credit to smallholder\nfarmers\nNo\nYes*\n*Focused on building\nout framework, not\ninterventions\nAgriculture Promotion\nPolicy\n(APP)\n2016–2020\nYes\nRecognizes double burden of\nunderweight, overweight,\nand obesity\nNo\n•Focus on agribusiness supply chains\n•Integrating climate-smart agriculture*\n•Integrating nutrition-sensitive programming*\n*Mentioned as a goal to integrate throughout all\nobjectives, but many objectives lack integration of\nnutrition and climate\nYes\nFood supply chains\n•Fertilizer, increased soil nutrients\n•Irrigation\n•Pest and disease control services\n•National grain storage centers\nFood environment/markets\n•Infrastructure improvement\nDiets\n•Import substitution to reduce\neffects of exchange rate policy\non consumers\n•School feeding programs\n•Strategic reserves of food\nNo\nYes\nNational Policy on Food and\nNutrition\n(NPFN)\n2016–Current Yes\nRecognizes burden of\nmalnutrition in all its forms\nNo\n•Links agriculture and food security\n•Does not mention climate change\nYes\nFood supply chains\n•Promote priority value chain\ncrops\n•Improved on-farm storage\n•Myco-toxin prevention\n•Improve processing and\npreservation\nFood environments/markets\n•Effective food distribution\nsystems\n•Improved infrastructure\nDiets\n•Biofortification of staple foods,\nvitamin provision\n•Food quality standards\n•National buffer stock to reduce\nprice volatility\n•School feeding programs\nNo\nYes\n2016–2025\nYes\nNo\nYes\nYes\nYes\n8\nCurr Envir Health Rpt (2020) 7:392–403 398 p\ny\ng\nNational strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Challenges to Implementation Cost\nanalysis\nM&E plan\nNational Adaptation Strategy\nand Plan of Action\n(NASPA-CCN)\n2011–current No\nUndernutrition only\nNo\n•Focus on climate impacts on\nagricultural production\n•Focus on food access\n•No mention of nutritional\nquality of food\nNo\nFood supply chains\n•Improved agriculture systems for\ncrops and livestock\n•Increase irrigation\nFood environment/markets\n•Facilitate credit to smallholder\nfarmers\nNo\nYes*\n*Focused on building\nout framework, not\ninterventions\nAgriculture Promotion\nPolicy\n(APP)\n2016–2020\nYes\nRecognizes double burden of\nunderweight, overweight,\nand obesity\nNo\n•Focus on agribusiness supply chains\n•Integrating climate-smart agriculture*\n•Integrating nutrition-sensitive programming*\n*Mentioned as a goal to integrate throughout all\nobjectives, but many objectives lack integration of\nnutrition and climate\nYes\nFood supply chains\n•Fertilizer, increased soil nutrients\n•Irrigation\n•Pest and disease control services\n•National grain storage centers\nFood environment/markets\n•Infrastructure improvement\nDiets\n•Import substitution to reduce\neffects of exchange rate policy\non consumers\n•School feeding programs\n•Strategic reserves of food\nNo\nYes\nNational Policy on Food and\nNutrition\n(NPFN)\n2016–Current Yes\nRecognizes burden of\nmalnutrition in all its forms\nNo\n•Links agriculture and food security\n•Does not mention climate change\nYes\nFood supply chains\n•Promote priority value chain\ncrops\n•Improved on-farm storage\n•Myco-toxin prevention\n•Improve processing and\npreservation\nFood environments/markets\n•Effective food distribution\nsystems\n•Improved infrastructure\nDiets\n•Biofortification of staple foods,\nvitamin provision\n•Food quality standards\n•National buffer stock to reduce\nprice volatility\n•School feeding programs\nNo\nYes\n2016–2025\nYes\nNo\nYes\nYes\nYes\n8\nCurr Envir Health Rpt (2020) 7:392–403 No No Curr Envir Health Rpt (2020) 7:392–403 399 (continued)\nl strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Challenges to Implementation Cost\nanalysis\nM&E plan\nure Sector Food\nity and Nutrition\ny\nRecognizes burden of\nmalnutrition in all its forms\n•Strong focus on agriculture and nutrition\n•Climate change mentioned once\nFood supply chains\n•Expand production of\nbio-fortified foods\n•Scale up fruit and vegetable\nproduction\n•Reduce post-harvest loss through\nincreased cold storage and\nprocessing\n•National aflatoxin control\ninitiative\nFood environments/markets\n•Facilitate access to credit for\nsmallholder farmers\n•Improved nutrition labeling\nDiets\n•Develop improved food-based\ndietary guidelines\nSector Component of\nnal Food and\ntion Policy\nN)\n2014–2019\nNo\nFocus on undernutrition,\nspecifically for mothers and\nchildren\nDoes not address overweight\nand obesity\nNo\n•No mention of agriculture or climate change\nNo\nDiets\n•Vitamin supplementation\n•Feeding centers for severe acute\nmalnutrition\n•Breastfeeding promotion\nYes\nYes\n[30, 31, 52, 61, 77] National strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Cost\nanalysis\nM&E plan\nAgriculture Sector Food\nSecurity and Nutrition\nPolicy\n(ASFS)\nRecognizes burden of\nmalnutrition in all its forms\n•Strong focus on agriculture and nutrition\n•Climate change mentioned once\nFood supply chains\n•Expand production of\nbio-fortified foods\n•Scale up fruit and vegetable\nproduction\n•Reduce post-harvest loss through\nincreased cold storage and\nprocessing\n•National aflatoxin control\ninitiative\nFood environments/markets\n•Facilitate access to credit for\nsmallholder farmers\n•Improved nutrition labeling\nDiets\n•Develop improved food-based\ndietary guidelines\nHealth Sector Component of\nNational Food and\nNutrition Policy\n(NSPAN)\n2014–2019\nNo\nFocus on undernutrition,\nspecifically for mothers and\nchildren\nDoes not address overweight\nand obesity\nNo\n•No mention of agriculture or climate change\nNo\nDiets\n•Vitamin supplementation\n•Feeding centers for severe acute\nmalnutrition\n•Breastfeeding promotion\nYes\nYes 400 Curr Envir Health Rpt (2020) 7:392–403 environment [79]. Irrigation policies are also critical for cli-\nmate adaptation, but efforts to scale up irrigation have been\nfragmented and duplicative [73, 80]. large strides to improve its policy environment and coherence. Its 2017 agricultural and nutrition policy highlights a systemic\nunderstanding of the drivers of malnutrition. Integrating cli-\nmate change into a more coherent food systems framework\nwould provide nuance to future assessments of malnutrition\nand will allow Nigeria to address and mitigate climate risks for\nnutrition. Doing so would require some major revisions to the\npolicy. Ecker et al. Challenges to Implementation (2020) also recommended that Nigeria’s\npolicymakers consider adopting a food systems framework to\nreformulate national food and nutrition policies in order to\nimprove household diets and reduce the multiple burdens of\nmalnutrition [89]. Numerous surveys of Nigerian farmers have highlighted\nthe continued challenges to increase the use of adaptation\nstrategies. Studies of farmers’ perceptions of climate change\nhave shown that farmers accurately perceive changes in tem-\nperature and rainfall [81], but climate adaptation strategies\ndepend on farmer income, perception of risk, and environment\n[82–84]. Other surveys have found that adaptation strategies\nfor farmers differ between older and younger farmers [82] and\nthat in some communities, challenges remain regarding\nfarmers’ perception of the causes of climate change. Farmers\nwith larger farms, higher incomes, and more schooling are\nmore likely to practice adaptation strategies [82] while those\nthat link climate change to religious beliefs are less likely to\nproactively use adaptive strategies [81]. A significant infor-\nmation gap exists between farmers [83]. Without increasing\nexpenditure on agriculture and other related industries such as\naquaculture and agroforestry, smallholder farmers are likely to\nbe left out of mitigation and adaptation strategies. One revision would be to fulfill the Comprehensive Africa\nAgriculture Development Program (CAADP) pledge to invest\nin agriculture, and, within agriculture, nutrition-climate-\nsensitive agriculture. Nigeria is a signatory to CAADP, which\nhas set a goal of 10% of government budget going towards\nagriculture. However, Nigeria has consistently\nunderperformed on this metric. Average yearly agricultural\nspending between 2008 and 2012 only totaled 4.6% of the\nnational budget. [90]. The most recent CAADP performance\nassessment gave Nigeria 0% progress on its three goals related\nto modernized agriculture, well-nourished citizens, and envi-\nronmentally sustainable and climate resilient economies [91]\nNigeria must increase domestic funding for climate, agricul-\nture, and nutrition in order to address malnutrition. Nutrition policies also lack budget allocations. The Scaling\nup Nutrition (SUN) Movement reports that Nigeria has met\n70% of the criteria for an enabling policy environment for\nnutrition as of 2019 [85]. However, budget allocations have\nnot matched the goals outlined in the costed strategies. Surveys of government and NGO stakeholders in 2015 and\n2016 found concerns about appropriate resource allocation at\nthe federal and state level [86]. In 2019, Nigeria only spent\n0.2% of its budget on nutrition specific interventions [85]. Budget delays lead to limited implementation. Challenges to Implementation A review of\nfederal stakeholders with responsibility for the NSPAN\n(2014–2019) found that only 1/3 had begun implementation\nof the plan by 2018 [87]. Limited domestic resource mobili-\nzation means that nutrition programs rely heavily on interna-\ntional donors for funding [85] and those resources can vary\nwidely depending on donor motivations. A second revision would be to consider double- or triple-\nduty actions. Double- and triple-duty actions simultaneously\nact on two or three of the epidemics (undernutrition, over-\nweight, and/or climate). One example could be reducing red\nmeat consumption to prevent cancer/obesity (obesity/NCDs),\nincrease land efficiency to grow food for human consumption\n(undernutrition), and lower greenhouse gas emissions (climate\nchange). These actions are carefully calibrated so that a focus\non undernutrition does not lead to an increase in overweight\nand obesity for certain populations [92••]. Swinburn and col-\nleagues articulated areas in which double- or even triple-duty\nactions could mitigate the sydemic, many with direct actions\nwith food environments, the place where consumers engage\nwith food systems [15••]. However, if Nigeria’s national pol-\nicies that touch on food systems do not address the impacts of\nclimate change, today’s investments may have little impact in\nthe next three decades. An integrated, holistic food systems\nframing linking climate-smart and nutrition-sensitive agricul-\nture on both land and water could identify priority areas for\ntriple-duty actions to make a more significant impact on the\nsyndemic in Nigeria [9••]. Some examples include scaling up\nnutrition-sensitive agriculture programs with climate adapted\nseeds and irrigation technologies, adapting food-based dietary\nguidelines to promote nutrient-dense and climate-conscious\ndiets, taxing highly processed foods and devoting revenue\nstreams to climate-smart agricultural policies, and developing\nmore urban agriculture. While some of these elements are Strong policy guidance with a lack of implementation\nfunding has been a continued problem for the agriculture,\nclimate, and nutrition sectors in Nigeria. Increased govern-\nment spending on agriculture has been linked to economic\ngrowth [88]. However, Nigerian budgets are heavily depen-\ndent on world oil prices [23, 26], and current price slumps\njeopardize programming for agriculture and nutrition\n(Table 1). • Of importance • Of importance •• Of major importance 1.• Popkin BM, Corvalan C, Grummer-Strawn LM. Dynamics of the\ndouble burden of malnutrition and the changing nutrition reality. Lancet. 2020;395(10217):65–74 This study describes the chang-\ning nutrition dynamics across the world as malnutrition stag-\nnates and overweight/obesity rises. They argue the double bur-\nden of malnutrition is rising in low- and middle-income\ncountries. 16.•• Fanzo J, Mclaren R, Davis C, Choufani J. Climate change and\nvariability: What are the risks for nutrition, diets and food systems? IFPRI Discussion Paper 01645. 2017. This report provides a\nthorough analysis of the multiple ways climate change will im-\npact nutrition along the supply chain. The report stresses the\nneed to create solutions for vulnerable populations that will be\nparticularly affected by climate-related food insecurity. 2. Dietz WH. Double-duty solutions for the double burden of malnu-\ntrition. Lancet. 2017;390(10113):2607–8. 2. Dietz WH. Double-duty solutions for the double burden of malnu-\ntrition. Lancet. 2017;390(10113):2607–8. 17. IPCC. Global Warming of 1.5°C. An IPCC Special Report on the\nimpacts of global warming of 1.5°C above pre-industrial levels and\nrelated global greenhouse gas emission pathways, in the context of\nstrengthening the global response to the threat of climate change,\nsustainable development, and efforts to eradicate poverty [Masson-\nDelmotte V, Zhai P, Pörtner HO, Roberts D, Skea J, Shukla JR\net al]. In Press. 2018. 3. FAO, IFAD, UNICEF, WFP and WHO. The State of Food Security\nand Nutrition in the World 2019. In: Safeguarding against econom-\nic slowdowns and downturns. Rome: FAO; 2019. 3. FAO, IFAD, UNICEF, WFP and WHO. The State of Food Security\nand Nutrition in the World 2019. In: Safeguarding against econom-\nic slowdowns and downturns. Rome: FAO; 2019. 4. Drewnowski A, Popkin BM. The nutrition transition: new trends in\nthe global diet. Nutr Rev. 1997;55(2):31–43. 4. Drewnowski A, Popkin BM. 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Conclusion and Recommendations While budgetary dependence on oil revenues and the frag-\nmentation of the federal system will challenge Nigeria’s abil-\nity to implement policies that focus on the common systemic\ndrivers of malnutrition and climate change, Nigeria has made Curr Envir Health Rpt (2020) 7:392–403 401 environment, and reduce climate change for a growing\npopulation. already present in Nigeria’s national policies, budgetary\nguidelines do not explicitly promote double- or triple-duty\nactions. Given Nigeria’s budget constraints and oil revenue\nvolatility, double- or triple-duty actions should become prior-\nity strategies. p p\n10.•• IPCC. Climate Change and Land: an IPCC special report on climate\nchange, desertification, land degradation, sustainable land manage-\nment, food security, and greenhouse gas fluxes in terrestrial ecosys-\ntems [Shukla PR, Skea J, Calvo E, Buendia V, Masson-Delmotte\nHO, Pörtner DC et al (eds.)]. In Press. 2019. This report provides\na detailed analysis of climate change interactions with agricul-\nture and food security and weighs the evidence of high to low\nconfidence for adaptation and mitigation across food systems. Open Access This article is licensed under a Creative Commons\nAttribution 4.0 International License, which permits use, sharing, adap-\ntation, distribution and reproduction in any medium or format, as long as\nyou give appropriate credit to the original author(s) and the source, pro-\nvide a link to the Creative Commons licence, and indicate if changes were\nmade. The images or other third party material in this article are included\nin the article's Creative Commons licence, unless indicated otherwise in a\ncredit line to the material. If material is not included in the article's\nCreative Commons licence and your intended use is not permitted by\nstatutory regulation or exceeds the permitted use, you will need to obtain\npermission directly from the copyright holder. 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https://openalex.org/W2912342079
https://link.springer.com/content/pdf/10.1007/s40626-019-00139-9.pdf
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Metabolomics for understanding stomatal movements
Theoretical and Experimental Plant Physiology
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cc-by
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Metabolomics for understanding stomatal movements David B. Medeiros . Luana M. da Luz . Hellen O. de Oliveira . Wagner L. Arau´jo . Danilo M. Daloso . Alisdair R. Fernie Received: 15 October 2018 / Accepted: 5 January 2019 / Published online: 22 January 2019  The Author(s) 2019 Received: 15 October 2018 / Accepted: 5 January 2019 / Published online: 22 January 2019  The Author(s) 2019 Abstract Stomata control the exchange of CO2 and water in land plants. For this reason, plants evolved to quickly respond the surrounding environment and endogenous cues in order to maintain their photosyn- thetic rates, but avoiding excessive water loss. Although guard cell has been used as model for characterization of signaling pathways, mainly regard- ing abscisic acid (ABA) response, several important questions about its functioning remain elusive. Recently, transcriptomics, proteomics, and metabolo- mics studies carried out using guard cells as model have contributed substantially for our understanding on how guard cells sense and respond to relative air humidity, CO2, ABA, and sucrose. Comparatively, proteomics and metabolomics platforms need sub- stantial improvement to increase the number of analytes detected. However, with the introduction of metabolomics-based technologies, several studies have been published increasing our knowledge on guard cell function. Here, we review these new exciting findings as well as discuss the importance of using these new data to improve prediction when modelling stomatal behavior. Keywords Stomata  Guard cells  Metabolites  Metabolite profiling  Mass spectrometry Theor. Exp. Plant Physiol. (2019) 31:91–102 https://doi.org/10.1007/s40626-019-00139-9 (0123456789().,-volV)( 01234567 89().,-volV) D. M. Daloso Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Ceara´, Fortaleza, Ceara´ 60440-970, Brazil D. B. Medeiros (&)  A. R. Fernie Max-Planck-Institut fu¨r Molekulare Pflanzenphysiologie, Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany e-mail: medeiros@mpimp-golm.mpg.de 1 Introduction Stomata, microscopic pores surrounded by two spe- cialized cells—the guard cells -in the leaf epidermis, control the exchange of carbon dioxide (CO2) and water in land plants. Therefore, they are the main gate for two essential process in plants; photosynthesis and transpiration. For this reason, plants need to adapt quickly and efficiently the environment and endoge- nous cues in order to gain sufficient CO2 to maintain their photosynthetic rates and avoid excessive water loss and thus prevent dehydration (Lawson et al. 2014; Schroeder et al. 2001). D. B. Medeiros (&)  A. R. Fernie Max-Planck-Institut fu¨r Molekulare Pflanzenphysiologie, Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany e-mail: medeiros@mpimp-golm.mpg.de L. M. da Luz  H. O. de Oliveira  W. L. Arau´jo Max-Planck Partner Group at the Departamento de Biologia Vegetal, Universidade Federal de Vic¸osa, Vic¸osa, Minas Gerais 36570-900, Brazil Guard cells have been extensively studied for over a century, and considerable efforts have been made to better understand their structure, development, phys- iology, and metabolism (Bergmann and Sack 2007; Daloso et al. 2017; Dow and Bergmann 2014; Kim et al. 2010; Mott 2009; Santelia and Lawson 2016). These advances have rendered the guard cell as one of D. M. Daloso Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Ceara´, Fortaleza, Ceara´ 60440-970, Brazil 12 3 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 92 Covering the guard cell metabolome is still not technically viable using a single analytical system but recent developments have considerably extended our ability to analyze this cell type. In fact, recent efforts to comprehend the guard cell metabolome have shed considerable light on metabolic regulation of stomatal movement in response to environmental stimuli. In this review we cover the most recent studies applying guard cell metabolomics approaches in order to understand the roles of important players in stomatal movement such as abscisic acid (ABA), CO2, sucrose, organic acids as well as plant cell–microbe interaction. We further discuss the importance of integrating metabolomics data into mathematical modelling in order to optimize predictions of stomatal behavior under challenging conditions. the best studied plant cell models for membrane transport, signaling, and homeostasis (Blatt 2000; Engineer et al. 2016; Hetherington and Brownlee 2004; Hills et al. 2012; Israelsson et al. 2006). Stomatal pores are structurally very simple, but the surrounding guard cells are one of the most specialized cell in land plants. 1 Introduction They are morphologically distin- guished from other epidermal cells and possess a complex signal transduction network, tightly regu- lated membrane ion system, and specialized metabolic pathways which modulate guard cell turgor and subsequently promote opening and closure of stomata (Santelia and Lawson 2016). Generally, the stomatal pores open in response to increases in guard cell volume driven by decreases in the water potential of the guard cell as result of the osmolytes accumulation and subsequently water influx. Conversely, during stomatal closure, efflux of osmolytes from guard cells is required with associated increase in guard cell water potential and in turn efflux of water (Assmann and Wang 2001). Potassium (K?) and chloride (Cl-) act as the main inorganic ions, while malate2- and sucrose (Suc) are considered as the main organic osmolytes during stomatal movements (Kollist et al. 2014; Roelfsema and Hedrich 2005; Vavasseur and Raghavendra 2005). 2 Metabolomics studies in guard cells While earlier studies on guard cells focused on changes in accumulation of sugars, organic acids, and K? in guard cell (Amoedo et al. 1996; Lu et al. 1995; Talbott and Zeiger 1993, 1996), the recent introduction of new high throughput technologies has reignited guard cell metabolism research. Metabolo- mics approaches have been applied to analyze changes in guard cell metabolism, being highly useful in providing a broader understanding of guard cell function. However, despite the improvements in guard cell separation methods, all the studies involving guard cell metabolome still face the bottleneck of high quality and rapid cell isolation, in order to disturb as few as possible guard cell primary metabolism. In the last 5 years several studies were published, in which metabolomics approaches were used for answering several unsolved questions on guard cell metabolism, below we review these exciting new findings. The platforms used in each study are summarized in Table 1. Most of the knowledge we have regarding stomatal movements came from the discovery and characteri- zation of several components of the protein transport- ing system in guard cells (Eisenach and De Angeli 2017; Jezek and Blatt 2017). However, lately guard cell metabolism has been revisited and we have recently witnessed great efforts in assessing the importance of a range of metabolites in the regulation of stomatal function in elegant biochemical and physiological studies. The knowledge from these studies has increased our understanding on how guard cells regulate their primary metabolism through organic acid, sucrose, starch, and lipid biosynthetic and degradative pathways in order to control stomatal movements. Although we discuss some of these studies in the following sections the focus of this review is to give an update on how metabolomics approaches have been applied in guard cells studies rather than providing an overview of the regulation of guard cell metabolism for which several recent reviews document the most important recent findings (Daloso et al. 2017; Lima et al. 2018; Santelia and Lawson 2016). 2.1 Metabolic signatures for ABA response napus in response to ABA Zhu and Assmann (2017) GC–TOF–MS Validation of modeling flux-based predictions using mesophyll and guard cell protoplasts fed with NaH13CO3 Robaina-Estevez et al. (2017) GC–TOF–MS Feeding with [U-13C]-sucrose in EF from Arabidopsis Medeiros et al. (2018b) LC–MRM–MS EF incubated with bacteria (Pst DC3000) and labeled with 13C and 15N isotopes Pang et al. (2018) LC liquid chromatography, MS mass spectrometry, GC gas chromatography, MRM multiple reaction monitoring, TOF time of flight (Zhu and Assmann 2017). Non-targeted metabolomics was used as a complementary approach to targeted metabolomics—providing not only mass/charge ratios but also the mass spectra of all detectable precursor molecules. The platform combination resulted in a metabolite profile of 390 non-redundant metabolites, of which 77 were ABA responsive. On performing an enrichment analysis of the metabolic changes flavone and flavonol biosynthesis and metabolism of amino sugar, nucleotide, sugar, sucrose, and starch were highly affected by ABA (Zhu and Assmann 2017). a time-course experiment utilizing liquid chromatog- raphy–multiple reaction monitoring–mass spectrome- try (LC–MRM–MS) in order to perform targeted analysis of 85 signaling-related metabolites in Ara- bidopsis guard cell protoplasts (Jin et al. 2013). Arabidopsis wild-type plants and gpa1 mutants dis- playing ABA-hyposensitive stomata were evaluated. This targeted metabolomics study revealed, as would perhaps be anticipated, coordinated regulation of signaling metabolites even in unrelated pathways. For instance, hormone related metabolites were more responsive to ABA treatment in wild type than in gpa1 guard cells. Additionally, the majority of hormones also displayed different ABA responses in guard cell metabolite profiles when compared with those in the mesophyll cell metabolome, suggesting that ABA is most likely acts upstream of the other hormones (Jin et al. 2013). Recently, primary metabolism in guard cells has received increased attention mainly regarding the roles of organic acids, sucrose (discussed below) and starch metabolism (we suggest the read of Santelia and Lawson (2016), Santelia and Lunn (2017), and Daloso et al. (2017)). On the other hand, secondary metabo- lism of guard cells is poorly understood. New insights have, however, been published showing the connec- tion between flavonol biosynthesis and ABA signaling (Watkins et al. 2017, 2014). Flavonol accumulation was observed in guard cells of Arabidopsis, but not in surrounding pavement cells (Watkins et al. 2014). 2.1 Metabolic signatures for ABA response Guard cells are well established as a model cell type for deciphering ABA signaling in plants. The regula- tory role of ABA signaling pathway on stomatal regulation has being recently improved by using metabolomics tools. For instance, the metabolic response to ABA treatment was recently followed in 12 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 93 Table 1 Technologies applied for metabolomics studies in guard cell Platform used Remarks References LC–MS Guard cell protoplasts from Columbia-0 and gpa1 Arabidopsis plants subjected in response to ABA Jin et al. (2013) GC–MS/MS and LC–MRM–MS Mesophyll and guard cells from B. napus subjected in response to NaHCO3 Misra et al. (2015b) GC–TOF–MS Feeding with NaH13CO3 in guard cell-enriched epidermal fragments (EF) from tobacco Daloso et al. (2015) GC–TOF–MS Feeding with NaH13CO3 in EF from transgenic tobacco plants Daloso et al. (2016b) GC–MS/MS, LC–MRM–MS, and LC–TOF–MS EF from Arabidopsis under 800 ppm CO2 Geng et al. (2016) GC–TOF–MS EF from tdt Arabidopsis mutant, apoplastic leaflet fluid, and non-aqueous fractionation samples Medeiros et al. (2017) GC–MS/MS and LC–TOF–MS Guard cell protoplasts from B. napus in response to ABA Zhu and Assmann (2017) GC–TOF–MS Validation of modeling flux-based predictions using mesophyll and guard cell protoplasts fed with NaH13CO3 Robaina-Estevez et al. (2017) GC–TOF–MS Feeding with [U-13C]-sucrose in EF from Arabidopsis Medeiros et al. (2018b) LC–MRM–MS EF incubated with bacteria (Pst DC3000) and labeled with 13C and 15N isotopes Pang et al. (2018) LC liquid chromatography, MS mass spectrometry, GC gas chromatography, MRM multiple reaction monitoring, TOF time of flight Table 1 Technologies applied for metabolomics studies in guard cell Platform used Remarks References LC–MS Guard cell protoplasts from Columbia-0 and gpa1 Arabidopsis plants subjected in response to ABA Jin et al. (2013) GC–MS/MS and LC–MRM–MS Mesophyll and guard cells from B. napus subjected in response to NaHCO3 Misra et al. (2015b) GC–TOF–MS Feeding with NaH13CO3 in guard cell-enriched epidermal fragments (EF) from tobacco Daloso et al. (2015) GC–TOF–MS Feeding with NaH13CO3 in EF from transgenic tobacco plants Daloso et al. (2016b) GC–MS/MS, LC–MRM–MS, and LC–TOF–MS EF from Arabidopsis under 800 ppm CO2 Geng et al. (2016) GC–TOF–MS EF from tdt Arabidopsis mutant, apoplastic leaflet fluid, and non-aqueous fractionation samples Medeiros et al. (2017) GC–MS/MS and LC–TOF–MS Guard cell protoplasts from B. 2.2 Response to high CO2 concentration biosynthetic enzyme, was demonstrated to be expressed in guard cells, but not pavement cells. Interestingly, null mutation for the gene encoding CHALCONE SYNTHASE increased the levels of reactive oxygen species (ROS) in the guard cells. Further, guard cells of mutant plants showed faster ABA-induced closure compared to wild type, suggest- ing that flavonols may reduce the ABA-dependent ROS burst that drives stomatal closing (Watkins et al. 2014). Curiously, flavonols levels were positively regulated in guard cells by ethylene treatment in the wild type, but not in an ethylene-insensitive mutant, suggesting an ethylene-regulated mechanism of flavo- nols controlling ROS burst under ABA signaling (Watkins et al. 2014). Similarly, it was also demon- strated that in tomato (Solanum lycopersicum), ABA- induced ROS accumulation is followed by stomatal closure (Watkins et al. 2017). Ethylene treatment of wild-type tomato plants increased flavonol accumula- tion in guard cells; however, no flavonol increases were observed in Neverripe (Nr) plants, an ethylene receptor deficient mutant. Taken together, these results strongly suggest the roles of flavonols as important players during the ABA-dependent ROS production for the regulation of stomatal opening. Intriguingly, the link between flavonols and stomatal functioning was also observed in an independent recent study investigating metabolic components of water stress in a Meditter- anean vineyard (Gago et al. 2017). High ambient CO2 concentrations mediate closure of stomatal pores in plants and conversely low ambient CO2 concentration triggers the opening of stomatal pores. Within the context of the raising atmospheric CO2 concentration to understand how guard cells adjust their physiology and metabolism in response to CO2 is a key step for the development of plants better adapted to the shifting climate condition (Geng et al. 2017). Indeed, although the whole network of CO2 response is not fully clear, recent studies have addressed the molecular and cellular mechanisms mediating CO2 regulation of stomatal movements (Engineer et al. 2016; Zhang et al. 2018). For instance, changes in the metabolite profile in response to increases in CO2 concentration was investigated over time. For this propose mesophyll and guard cells protoplasts from B. napus were fed with sodium bicarbonate (NaHCO3) (Misra et al. 2015b). In this study, targeted metabolite profiling in untreated and NaHCO3 treated mesophyll and guard cell was performed using LC–MRM–MS and GC–MS/MS based technologies, in order to identify 268 metabo- lites related to both, primary and secondary metabo- lism. 2.1 Metabolic signatures for ABA response Accordingly, CHALCONE SYNTHASE, a flavonol More recently, a non-targeted metabolomics study utilizing a combination of gas chromatography–mass spectrometry (GC–MS/MS) and liquid chromatogra- phy–time of flight–mass spectrometry (LC–TOF–MS) was applied to identify the metabolic signatures in response to ABA in B. napus guard cell protoplasts 12 12 3 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 94 2.2 Response to high CO2 concentration 2.3 Understanding the roles of Suc in guard cells Despite the above-discussed evidence, the direct proof that sucrose acts as an energetic substrate during the dark-to-light transition was still missing until earlier this year when the work using a combination of stomatal aperture assays and kinetic [U-13C]-sucrose isotope labelling experiments was published (Medeiros et al. 2018b). This work aimed to directly follow the fate of the carbons released from sucrose breakdown during light-induced stomatal opening. In this study GC–TOF–MS was used revealing rapid and high 13C enrichment in fructose and glucose following dark-to-light transition. Clear 13C enrichment in gly- colysis and TCA cycle intermediates as well as glutamate and glutamine was also observed indicating activation of these pathways during light-induced stomatal opening. These new findings allowed a reevaluation of the current models concerning the role of sucrose in stomatal opening. It is now clear that during stomatal opening sucrose is degraded providing carbon skeletons for glutamine biosynthesis, however, the physiological reason behind this is, as yet, far from being understood (Lima et al. 2018; Medeiros et al. 2018b). For a long time sucrose was believed to exclusively play an osmolytic role in stomatal opening (Daloso et al. 2016a). However, by using metabolomics tools recent findings have not only challenged this claim but also proposed an energetic role of sucrose during light- induced stomatal opening (for a complete overview about sucrose roles during stomatal movements please read Daloso et al. (2016a) and Lima et al. (2018)). The section below compiles interesting reports focused on the application of metabolomics tools to solve the open questions about guard cell metabolism. The metabolic changes in guard cell-enriched epidermal fragments from tobacco (Nicotiana taba- cum L.) during light-induced stomatal opening were evaluated alongside with isotope kinetic labelling experiment by feeding the guard cell-enriched epider- mal fragments with NaH13CO3 under light conditions in order to understand the distribution of photosyn- thetic fluxes in guard cells (Daloso et al. 2015). By using a GC–TOF–MS platform it was observed that concomitant with the decrease in sucrose, there were increases in the level and 13C enrichment of the TCA cycle-related metabolites. This report was the first evidence supporting the hypothesis that sucrose is catabolized within guard cells in order to provide carbon skeletons for organic acid production. 2.2 Response to high CO2 concentration Interestingly, opposing metabolic responses were observed between the two cell types, in that while mesophyll cells presented increases in amino acid, phenylpropanoid, redox metabolite, auxin and cytokinin contents all of which were decreased in treated guard cells. The outcome from both cell type metabolite profile revealed that 26 and 27 metabolites in mesophyll and guard cells were exclusively iden- tified, respectively. Regarding primary metabolism, constant increase of malate was observed across the experiment in NaHCO3 treated guard cells. Addition- ally, in these cells increases in fructose, glucose, and mannose were observed at the early time-points (Misra et al. 2015b). In another study by using three different y g Returning to the hormone response it is clear that hormones are good targets to investigate guard cell metabolomics. Indeed, considerable attention has been paid to ABA, but the crosstalk between ABA and other hormones can potentially also affect stom- atal movements. For instance, brassinosteroid and jasmonate can act concurrently with ABA promoting senescence and programmed cell death (Hossain et al. 2011; Zhang et al. 2009). Other hormones such as cytokinin, auxin, salicylic acid and ethylene have been showed to act antagonistically to ABA under water deficit conditions (Chen et al. 2013; Ha et al. 2012; Meguro and Sato 2014; Tanaka et al. 2005). Therefore, we believe that metabolomics tools for specific cells can be useful in order to evaluate the metabolic changes provoked by the hormone signaling network and improving methods for single cell hormone measurements will likely greatly increase our ability to understand and model hormonal aspects of the control of stomatal movements. In another study by using three different approaches; GC–MS/MS, LC–MRM–MS, and LC– TOF–MS it was able to successfully annotate 358 metabolites (Geng et al. 2016). In this study the metabolic responses in guard cell-enriched epidermal fragments of B. napus were evaluated in response to elevated CO2 (800 ppm) in a time-course experiment (over 1 h). Interestingly, these authors reported changes in primary metabolites such as sugars, fatty acids, amino acids and nucleotides under elevated CO2 123 123 Theor. Exp. Plant Physiol. (2019) 31:91–102 95 treatment. Additionally, ROS production increased and stomatal aperture decreased over the time. 2.3 Understanding the roles of Suc in guard cells Another important finding from this work was the qualitative demonstration that CO2 fixation in tobacco guard cells can be catalyzed both by ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) and phos- phoenolpyruvate carboxylase (PEPc) (Daloso et al. 2015). 2.2 Response to high CO2 concentration Curi- ously, following the previous findings in metabolic guard cell response to ABA (Zhu and Assmann 2017), alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. However, the novelty presented by this work was that after 10 min under elevated CO2 treatment the jasmonic acid biosynthesis pathway was significantly induced (Geng et al. 2016). SYNTHASE 3 (SUS3) in guard cells (Daloso et al. 2016b). A kinetic isotope labeling experiment follow- ing the metabolic fate of NaH13CO3 during the dark- to-light transition was performed using guard cell- enriched epidermal fragments which were analyzed via GC–TOF–MS. Minor changes were observed in the metabolite profile of whole leaves, whereas increased fructose and decreased organic acids levels were observed in the transgenic plants. In good agreement with the results from the previous study, increased labeling of the TCA cycle-related metabo- lites was observed, suggesting that breakdown of sucrose is a mechanism providing carbon skeletons during stomatal opening (Daloso et al. 2016b). SYNTHASE 3 (SUS3) in guard cells (Daloso et al. 2016b). A kinetic isotope labeling experiment follow- ing the metabolic fate of NaH13CO3 during the dark- to-light transition was performed using guard cell- enriched epidermal fragments which were analyzed via GC–TOF–MS. Minor changes were observed in the metabolite profile of whole leaves, whereas increased fructose and decreased organic acids levels were observed in the transgenic plants. In good agreement with the results from the previous study, increased labeling of the TCA cycle-related metabo- lites was observed, suggesting that breakdown of sucrose is a mechanism providing carbon skeletons during stomatal opening (Daloso et al. 2016b). 2.4 Metabolic changes due to altered organic acids transport in guard cell Malate and fumarate have been highlighted as impor- tant players during stomatal movements. Given their importance in controlling stomatal movement, chan- nels and transporters were recently identified and functionally characterized to be involved with organic acid transport at both plasma membrane and tonoplast of guard cells (Eisenach and De Angeli 2017; Marti- noia 2018). In this context metabolomics approaches A follow-up study was performed using transgenic tobacco plants specifically overexpressing SUCROSE 12 3 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 96 fragments, intermediates of the tricarboxylic acid cycle were altered differently in the two tissues. For instance, lower malate and fumarate accumulation was observed in leaves but not in epidermal fragments (Medeiros et al. 2017). These findings confirmed the previous hypothesis of functional redundancy for organic acids transport at the tonoplast in guard cells (Meyer et al. 2011). have been used in order to evaluate changes in the metabolism of both leaves and guard cells (Medeiros et al. 2016, 2017). QUAC1 (Quick activating Anion Channel 1), also known as ALMT12, is a member of ALMT (Alu- minum activated malate transporter) family, was identified as an ABA-dependent anion-selective chan- nel at the guard cell plasma membrane responsible for malate efflux during stomatal closure in a voltage- dependent manner (Meyer et al. 2010). Loss-of- function of QUAC1 resulted in an impaired kinetic of stomatal closure in response to dark and high CO2 levels as well as reduced ABA-induced stomatal closure coupled with significant changes in organic acid accumulation as well as increases in both stomatal and mesophyll conductance (Medeiros et al. 2016). By using the GC–TOF–MS system 40 metabolites related to primary metabolism were successfully annotated, showing relatively few changes in the metabolite profile of mutant leaves. Interestingly, looking at the organic acids levels, it was observed that maleic acid, malate, fumarate, succinate increased in the knockout lines. These results indicate that changes in organic acids homeostasis in guard cell can affect the whole leaf metabolism highlighting the coordination between mesophyll and guard cell metabolism. Recently the report of a new ALMT member (ALMT4) demonstrated it to be a phosphorylation- dependent anion channel, releasing malate from the vacuole during stomatal closure in response to ABA (Eisenach et al. 2017). 2.4 Metabolic changes due to altered organic acids transport in guard cell almt4 knockout mutants displayed normal stomatal opening, but by contrast to other organic acids transporters at the tonoplast, almt4 plants presented impaired stomatal closure in response to ABA, but not to darkness (Eisenach et al. 2017). Altogether these exciting findings suggest that independent mechanisms may be activated under specific conditions and individual organic acid trans- porters are regulated independently in a stimulus- dependent manner at the tonoplast (Medeiros et al. 2018a). 2.5 The guard cell metabolome during plant cell– microorganism interaction A vacuolar malate transporter (tonoplast Dicar- boxylate Transporter—tDT) was also recently char- acterized in Arabidopsis. tDT was considered the main malate transport system at the tonoplast and required for the proper accumulation of malate in Arabidopsis leaves (Emmerlich et al. 2003; Hurth et al. 2005). tDT was only recently functionally characterized (Frei et al. 2018). Highly purified tDT protein showed transport activity as malate and citrate 1:1 in an antiport mode, also accepting fumarate and succinate as substrates (Frei et al. 2018). However, the func- tional role of tDT in guard cells remained unclear until recently when the metabolic impact of the tDT absence was studied (Medeiros et al. 2017). This study also used GC–TOF–MS technology to assess metabolic changes in leaves and guard cell-enriched epidermal fragments of wild type and tdt knockout lines. This study showed that manipulating vacuolar organic acid transport by suppressing tDT greatly impacts mitochondrial metabolism but has minor effects on stomatal and photosynthetic capacity. Interestingly, by comparing the metabolite profiles of leaves and guard cell-enriched epidermal The interaction between plants and microorganisms is known to triggers signaling cascade and metabolic responses. However, the main issue of analyzing changes in the plant metabolic profile during the interaction plant cell–microorganism is avoiding con- tamination with metabolites from the coexisting microorganism (Pang et al. 2018). A very recent work addressed this issue by implementing an effective stable isotope labeling approach coupled to LC– MRM–MS measurements (Pang et al. 2018). The plant bacterial pathogen Pseudomonas syringae pv. tomato DC3000 was incubated in isotope-labeled medium (Celtone complete medium with [ 98% 13C and [ 98% 15N). This procedure was used in order to differentiate plant metabolites from bacterial metabo- lites. This study suggested that bacterial infection may affect stomatal movement by reprograming the guard cell signaling network and primary metabolism. It was observed that at 30 min after bacterial infection the most affected pathways were related to amino acid metabolism. Whereas at 180 min, aminoacyl-tRNA biosynthesis, the tricarboxylic acid (TCA) cycle, and 123 123 Theor. Exp. Plant Physiol. (2019) 31:91–102 97 Despite the fact that mass spectrometry-based metabolomics platforms have been widely used in plant biology, these platforms have only been recently used to investigate guard cell metabolism (Medeiros et al. 2015; Misra et al. 2015a). 2.5 The guard cell metabolome during plant cell– microorganism interaction The establishment of a reproducible and reliable analytical platform is crucial for the performance of -omics studies, which can subsequently aid in the generation of mathematical models. Some models for leaf metabolism of C3, C4, and CAM plants have been published (Arnold and Nikoloski 2014; Cheung et al. 2014, 2015; Chew et al. 2014; Masakapalli et al. 2010, 2013; Pfau et al. 2018; Seaver et al. 2015; Shameer et al. 2018; Sweetlove et al. 2013; Wang et al. 2012). Recently the AraCORE model created for leaf primary metabolism (Arnold and Nikoloski 2014) has been adapted to model guard cell metabolism (Robaina-Estevez et al. 2017). Large- scale omics data have been used to constrain different models and thus more faithfully predict metabolic fluxes through metabolic networks (Nikoloski et al. 2015; Orth et al. 2010). This is part of a systemic characterization of plants, in which the data generated is used either to create or to improve mathematical models, following a ‘‘design–build–test–learn’’ cycle (Sweetlove et al. 2017). carbon fixation as well as the biosynthesis of pheny- lalanine, tyrosine, and tryptophan were dramatically affected. In summary, metabolite profiling has aided the identification of several novel interactions between the TCA cycle, photosynthesis and stomatal function as well as guard cell responses to both biotic and abiotic stresses. Due to the vast complexity of the metabolic and molecular regulatory pathways underlying guard cell function they remain not fully understood. How- ever, the improvements of new technologies toward metabolomics approaches will help us to better understand stomatal responses. Furthermore, the usage of metabolic data into mathematical models can optimize predictions of stomatal behavior in a changing environment. Studies in this direction have been performed and are discussed in the following section. 3 Metabolomics data for improving stomatal behavior predictions Transcriptomics, proteomics, and metabolomics stud- ies carried out using guard cells as model have contributed substantially for our understanding on how plant cells sense and respond to environmental and endogenous signals such as relative air humidity, CO2, ABA, and sucrose (Medeiros et al. 2015). All of the knowledge gained from these studies are useful for the establishment of systems biology approaches. Transcriptomic data has advantage over other omics platforms for the use in mathematical models given the higher coverage of transcriptomic techniques (Alseekh and Fernie 2018). Comparatively, pro- teomics and metabolomics platforms need substantial improvement to increase the number of analytes detected. This is an important issue for the creation of genomic scale metabolic models, in which several reactions of the metabolic network are included in the model (Hyduke et al. 2013). However, it is noteworthy that gene expression does not always correlate to protein expression/activity and subsequently to meta- bolic changes (Fernie and Stitt 2012; Lehmann et al. 2009). In this scenario, metabolomics approaches provide vital information by which to understand the structure and the regulation of metabolic networks as well as to enhance the predictive power of metabolic models. Existing models and their combinations have clearly contributed to the predictive modelling and refinement of the relationship between stomatal con- ductance and the players regulating stomatal function. Unfortunately, limited success in clarifying how changes in stomatal movements, by consequence water loss and CO2 gain, are affected by the interplay of environment, cellular levels, as well as the coordi- nation between mesophyll and guard cells. The OnGuard platform is one of the first modelling approaches created specifically for guard cells (Hills et al. 2012). Although the possibility to analyze guard cell signaling pathways using the theory of biological networks was postulated much earlier (Hetherington and Woodward 2003). OnGuard is a software built based on the knowledge of transport and accumulation of important molecules for guard cell osmoregulation such as K?, Cl-, Ca?2, malate, and sucrose. Simula- tions of the transport of these molecules through both plasma and tonoplast membranes of guard cells can be now performed by OnGuard (Chen et al. 2012; Hills et al. 2012; Wang et al. 2014). Despite its great contribution to understand important process that regulates stomatal movements, OnGuard software is 12 3 3 Theor. Exp. Plant Physiol. 3 Metabolomics data for improving stomatal behavior predictions (2019) 31:91–102 98 output of the model can be manipulated according to the aim of the study (Medeiros et al. 2015; Orth et al. 2010). The AraCOREred model indicates that tran- scriptome data can be used to predict cell-specific metabolic changes. This model can then be used in the future to predict guard cell metabolic responses to ABA and sucrose, given the availability of transcrip- tome data under ABA and sucrose treatment (Bates et al. 2012; Bauer et al. 2013; Leonhardt et al. 2004; Pandey et al. 2010). However, it is important to highlight that AraCOREred model was created assum- ing the guard cell metabolism at steady states. Due to the dynamic nature of the guard cell metabolism, the current challenge is to model guard cells under non- steady state condition, which would seem to be more physiologically relevant given the high plasticity of guard cell metabolism under varying conditions. Modelling strategies such as dynamic flux balance analysis seems to be great option to fulfil this gap. The establishment of such non-steady state modelling, while challenging, may increase the reliability of predictions by the current available models for guard cell functioning. not able to predict metabolic alterations in guard cells. This is partially due to the relative scarcity of guard cell metabolomics data. However, given that recent metabolomics approaches (reviewed above) being adapted to study guard cells, this hurdle is no longer valid. LC–MS and GC–MS-based metabolomics studies have provided important information regarding the guard cell response to ABA, bicarbonate, and sucrose (Daloso et al. 2015; Jin et al. 2013; Medeiros et al. 2018b; Misra et al. 2015b; Zhu and Assmann 2017). Furthermore, the establishment of a methodology to perform 13C-feeding experiments with the use of labelled substrates resolve a long debate regarding the role of sucrose for guard cell regulation and the mode of CO2 fixation found in guard cells (Daloso et al. 2015; Medeiros et al. 2018b). RubisCO-mediated CO2 fixation has long been thought to be absent in guard cells (Outlaw and Manchester 1979). However, the 13C distribution found in guard cells subjected to light indicate a clear CO2 fixation mediated by both RubisCO and PEPc (Daloso et al. 2015). The results of this study were substantially different from those observed in the Arabidopsis rosette which is largely composed of mesophyll cells (Szecowka et al. 2013). 3 Metabolomics data for improving stomatal behavior predictions Taking this into account, a systems biology study compared the NaH13CO3 distribution in mesophyll and guard cell protoplasts by combining flux predic- tions using a genome scale metabolic model and the 13C distribution measured in both cells via GC–TOF– MS (Robaina-Estevez et al. 2017). The modelling strategy of this work was based in previous published transcriptome data (Bates et al. 2012; Pandey et al. 2010) that were integrated into the AraCORE model (Arnold and Nikoloski 2014). As output, the new model, namely AraCOREred, provides cell-specific metabolic predictions of 455 reactions which com- prise 374 primary metabolites of mesophyll and guard cells (Robaina-Estevez et al. 2017). Interestingly, the results of this work confirmed that guard cells have higher anaplerotic CO2 fixation by PEPc than meso- phyll cells as well as regarding the source of sucrose and malate within guard cells. 123 4 Concluding remarks Over the past 5 years the application of metabolomics approaches in order to better understand guard cell metabolism and the mechanisms underlying the stomatal responses to environmental and endogenous cues has increased. Metabolite profiling of isolated or enriched-guard cells extracts has aided the identifica- tion of several novel interactions between the TCA cycle, photosynthesis, and stomatal function. Further- more, guard cell metabolomics studies have fulfilled some important unsolved questions regarding the roles of sucrose and organic acids transport and metabolism during stomatal movements as well as the guard cell responses to water deficit, high CO2 concentrations, and plant defense against pathogens. Alongside other omics technologies, metabolomics studies of the highly specialized guard cell have proven to be a powerful tool for understanding the hormonal and metabolic regulation of stomatal move- ments. This is particularly important since plant growth, development, and yield depend greatly of the fine control of plant–environment gas exchanges. Given the complexity of the interconnected networks The use of such genome scale, constraint-based metabolic models is important to understand the regulation of plant metabolism (Sweetlove et al. 2017). Mainly because the model can predict meta- bolic changes at subcellular level under different conditions, given that the input of signals and the 123 Theor. Exp. Plant Physiol. (2019) 31:91–102 99 governing stomatal movements under varying condi- tions, we argue that metabolomics approaches are highly valuable. 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The sustainability of human resource planning for construction projects
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The sustainability of human resource planning for construction projects Adegbenga Raphael Ashiru1 and Adebimpe Lucia Ashiru2 1 Lecturer- Department Of Building Technology, Federal Polytechnic, Kaura - Namoda. Zamfara - State. Nigeria 2 Lecturer- Department Of Business Administration and Management, Federal Polytechnic, Kaura - Namoda. Zamfara - State. Nigeria Abstract. Human Resource planning for construction project is described as a way of managing human resources within a growing nature of construction industry. Nonetheless, management of human resource is an extremely stimulating task. However, in spite of the substantial resources allocated to construction planning, there are rising concerns over its failure to achieve its goals and different application of planning strategies. It is against this backdrop that this research was carried out to examine the sustainability of Human resource planning for construction projects to justify the aforesaid statement. Furthermore, the researchers adopted a quantitative approach that provided a broader understanding of the research. SPSS Version 20 was used to obtain the descriptive and inference statistical analysis. Interestingly, majority of the literature findings were justified by the empirical findings noting that there is correlation between the two findings except where the respondent chose combination of the causes of neglect of HR planning on construction projects stressing that one cause cannot significantly neglect the process on construction projects. Conclusively, the research results indicated that through improving the practicality of Project HR planning by construction organisations, it would contribute significantly towards improving performance both at organisational and project level. The findings recommended guidelines for improving the visibility and adaptability of sustainable HR planning for construction projects and further provided justification for implementing its policies and procedures. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding Author: ashiruadegbenga@fedponam.ed.ng , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 https://doi.org/10.1051/matecconf/201925802018 P Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 es/by/4.0/). ces. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 /) 2.1.1 Evaluation of Current Resources Evaluation current human resource is another stage in HR planning through assessing employee’s interest, abilities, skills and experiences. After comparing these with SHRM plans, resource shortage that required filling of skills gaps will be outlined [17]. Through this, efficient database must be created to accommodate information about employees’ qualification, interests, training and experience during employment lifespan. 2.1.3 Internal Availability Analysis Before carrying out external recruitment, the organisation may check the availability of existing employee through employee future movement analysis in relation to transfers, promotions, resignations, terminations and retirements. However, organisations can predict these trends to prevent temporary shortage of employee [18]. An organisation could compare internal resources availability with the future needs through HR approach. Through this, internal employee-development and external recruitment could be determined. 1 Introduction of sustainable HR planning for construction projects. of sustainable HR planning for construction projects. • To draw conclusions on the relevance of sustainable HR planning for construction projects and makes recommendations to practitioners and for future research. As human resource (HR) is significant to all industries, including the construction industry, maintaining and attracting skilled employees is a priority. Hence, complexities in human contacts within construction industry will bring about construction-based issues of HR planning in such organisations [7]. However, it is against this backdrop that this research was carried out to answer the following fundamental research questions: Nonetheless, construction firms must ensure that current and future market analysis are carried out to meet functions of HR planning thereby ensuring that right percentage of individuals with right skill are considered [15]. By analysing the current human resources as well as linking these to potential opportunities in future market may allow recruitment and employee- development plans decisions to be made [16]. Subsequently, Leng suggested HR planning approaches that could use to ease HR problems as follows [15]; • What is the relationship between HR planning and construction project productivity; • How can sustainable Human Resource planning be used to improve individual and team performance at both construction organisation and project levels To ease the problem which underperformance have placed on construction industry, therefore, researcher will discuss basic ways in which HR planning could be used to manage construction projects to justify Loosemore’s opinion that team or individual behavior have posed a rising concern on risks in construction projects and recommended that effective human resource planning is the possible way to alleviate those construction risks [8]. 2 General Overview of Construction Industry Construction industry offers opportunity to mankind through job creation and the industry offers substantial responsibilities for those who work in it [9]. Nonetheless, the industry creates built environment as it designs, produce, conserve and demolishes structures [10]. A main contractor (Builder) tenders for construction contracts and when contract is awarded, the Builder further award subcontract to specialised contractors. Furthermore, micro organisations dominates construction sector with various organisation merging together to form teams to carry-out a project [11]. Those involved in project delivery include professionals such as clients/owner and general contractor, suppliers and subcontractors forms a team of contractor. Also, Architects, Civil Mechanical, Structural, Electrical Engineers, Land and Quantity Surveyors forms Consultant’s team [12]. However, construction industry remains a poorly understood sector despite its contribution to economic and social growth of nations [11]. Finally, the agenda of performance improvement has not been discharging by management of workforce within the sector. 2.1.2 Job Analysis and Design Job analysis is a process of analysing organisational restructuring methods to meet current and future business needs through collection of information of existing human resource and this further includes the following: • Assigning individual responsibilities to future needs by examining the organisational structure; • Identifying shortages or misfits; Redesigning misfit positions and individuals • Redesigning misfit positions and individuals responsibilities to suit the firm’s future needs. responsibilities to suit the firm’s future needs. 1 Introduction of each employee performance measurement through effective training [4]. A system that measures performance is needed in order to monitor improvements within construction teams [5]. Also, Langford et al argued that construction industry has a habit to shed personnel as part of a survival plan, retraining those whose skills are replaceable [6]. To justify the aforesaid statement, the research aimed to examine and promote the sustainability of HR planning for construction projects and to achieve this, the under listed objectives are set to achieve the aim of the research: The word planning is defined as a detailed system for achieving an objective meaning. However, a detailed system includes the method, the activities to carryout, time of those activities and resources including finance [1]. Moreover, an organisational planning creates efficiency and effectiveness in an organisation. Its provides clearer focus which bonds managers and subordinates in creating strong teams and produces limitless fulfillment among planners with common vision. Nonetheless, effective planning before implementation is significant for successful delivery of every project but there are indications of inadequate effort and time in planning construction projects especially at tender and pre-contract stages [2]. • To critically review literature relating to challenges of HR planning for construction projects and develop proper understanding of current situation in Construction industry; • To collect data from contracting organisations including Wolverhampton city council on challenges of HR planning for construction projects; Furthermore, in spite of the substantial resources allocated to construction planning, there are rising concerns over its failure to achieve its goals and the different application of planning strategies [3]. Nevertheless, the significant factor in implementing organisation’s planning policy in construction is the need • To analyse data collected and validate a framework that could help stakeholders focuses their priorities , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 https://doi.org/10.1051/matecconf/201925802018 Planning. However, HR planning should be the link between HRM and the overall organisational strategic plan. Nevertheless, Bulla and Scott argued that HR planning is the process of acknowledging organisational HR requirements and plans to satisfy those requirements are made [13]. Furthermore, Reilly maintained that HR planning is a way that an organization estimates the demand for workforce and evaluates its nature, size and sources of supply to meet the demand [14]. HR planning further includes creation of strategies in recruitment and selection, retention, talent management, absence management, flexibility and employer brand. 2.1.4 Employee Turnover Analysis 2.1.4 Employee Turnover Analysis 2.1.4 Employee Turnover Analysis Employee turnover analysis can be carried out for two purposes, namely; to ascertain possible future employee replacement and identify purpose for employee transient and best action to retain them. In fact, an assessment could be carried out on the effect of promoting and transferring of individuals in an organisation through HR planning [19]. • Interpersonal interfaces – this could be formal or informal relationships between various individuals on a site. (II) Labour requirement- This required individual or team competencies at different time frame. General resource requirements may involve labour requirement which form part of resource planning. (III) Organisational Constraints For efficiency, effectiveness and upward growth in an organisation, there must be agreed objectives to be able to manage risks with establishment of goals and targets through organisational strategic level [6]. Apparently, the effective use of individuals involved in the project is the essence of project HRM. This revolves round identifying, assigning project responsibilities, documenting and reporting relationship. Although, necessity for planning is very vital in construction due to; • Collective bargaining– prescribed agreements with labour union may require assured responsibilities or relationships; • Project Team Preferences– Success of a team in previous project can probably be recommended for similar project in future. B) Techniques And Tools For Project Organisational Human Resource Planning B) • project complexity which requires a huge number of human input; • The overall competitive approach in an unstable market and aggressive environment. • Project Templates- Since every project is distinctive, definitions of responsibility of a comparable project can help hasten the process of organisational planning in a new project. Therefore, project HR manager must possessed wide knowledge about market, product and method the organisation is operating. This will allow project work analysis and labour requirement be carried out. However, Leng identified in Table 1, a synopsis of project HR planning as follows [15]: • Human resource procedures and practices – Many organisations operates different guidelines, policies and procedure with series of facets of organisational planning that can support project team. Example is where organisations allow a manager to perform his duties like a coach. • Human resource procedures and practices – Many organisations operates different guidelines, policies and procedure with series of facets of organisational planning that can support project team. Example is where organisations allow a manager to perform his duties like a coach. Table 1- Overview of Project HR Planning INPUT TOOLS & TECHNIQUES OUTPUT Interfaces in the project Project Templates Roles and responsibility Labour Requirement Human resource procedures & practices Labour management plan Organisational Constraints Analysis of stakeholder Organisational chart and Supporting Documents ) Inputs to Project Human Resource Planning Table 1- Overview of Project HR Planning • Analysis of Stakeholder– The needs of stakeholders should be identified, analysed and met. C) Outputs from the project organisational human resource planning C) Outputs from the project organisational human resource planning (I) Roles and Responsibility – This is about who does what and who decides what. This should be allocated to suitable project professional. Most roles and responsibilities may vary over time and allocated to active professionals involved in project activities. (I) Roles and Responsibility – This is about who does what and who decides what. This should be allocated to suitable project professional. Most roles and responsibilities may vary over time and allocated to active professionals involved in project activities. A) Inputs to Project Human Resource Planning A) 2.1 Human Resource Planning in Construction A process that ascertains current and future organisational human resources needs to achieve organisational goals is described as Human Resource 2 , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 https://doi.org/10.1051/matecconf/201925802018 • Technical interfaces- Technical interfaces could be formal or informal relationship between professionals on a project and this happen within project phases where civil engineer work could be compatible with design of structural engineer. 3 Methodology • Training needs- an employee skill is very important on a project and those skills could be developed as part of the project. However, the nature of work, organisation strategy, economic and social changes are vital HR planning factors influencing for labour force supply in construction industry and it is very vital for the management of the organisation to ensure the effective use of HR planning and avoid inconsistency so that it can bring coherence and compatibility between project team members as well as the different group within the structure of the firm [20]. The researcher distributed twenty (20) well- structured questionnaires to selected respondents who are managers involved in decision making at strategic level for their respective organisations [6]. The researcher recovered twelve (12) responses achieving 60% of the feedback. The data collected was analysed by descriptive and inference statistics. The research instrument also measures a reliability and validity test of 0.766. Furthermore, data relating to challenges of HR planning were extracted from four questions which were adopted from [15] to develop varying challenges of HR planning for the research work. Indeed, the paper presented various improvement methods that can be adopted to encourage the practicality of the subject matter as SPSS levene test was used to ascertain the equality of variance for the research work. 3 Methodology This research was carried out in Wolverhampton West Midlands, United Kingdom to identify the various impacts of sustainability of Human Resource planning on construction projects in Wolverhampton city. Nonetheless, non-probability sampling was adopted as this enables us to select participants based on the research area in Wolverhampton city. This further assumed a purposive sampling strategy where respondents were selected at the discretion of the researcher but with the sole purpose to be typical of the whole population and the research question will be best examined to evaluate respondent engagement. The researcher adopted a quantitative approach that provided a broader understanding of the research problem [20] and [21]. SPSS Version 20 was used to obtain and analyse the descriptive and inference statistical analysis. The researcher distributed twenty (20) well- structured questionnaires to selected respondents who are managers involved in decision making at strategic level for their respective organisations [6]. The researcher recovered twelve (12) responses achieving 60% of the feedback. The data collected was analysed by descriptive and inference statistics. The research instrument also measures a reliability and validity test of 0.766. Furthermore, data relating to challenges of HR planning were extracted from four questions which were adopted from [15] to develop varying challenges of HR planning for the research work. Indeed, the paper presented various improvement methods that can be adopted to encourage the practicality of the subject matter as SPSS levene test was used to ascertain the equality of variance for the research work. This research was carried out in Wolverhampton West Midlands, United Kingdom to identify the various impacts of sustainability of Human Resource planning on construction projects in Wolverhampton city. Nonetheless, non-probability sampling was adopted as this enables us to select participants based on the research area in Wolverhampton city. This further assumed a purposive sampling strategy where respondents were selected at the discretion of the researcher but with the sole purpose to be typical of the whole population and the research question will be best examined to evaluate respondent engagement. The researcher adopted a quantitative approach that provided a broader understanding of the research problem [20] and [21]. SPSS Version 20 was used to obtain and analyse the descriptive and inference statistical analysis. • Job descriptions- an outlines of job title, responsibilities, Skills, authority, physical environment, and other requirements to execute a job. 4 Findings The literature sources agreed to varying challenges of HR planning on construction projects. The four identified factors considered are presented in table 2, 3, 4 and 5. • Increases Personal Growth- Project team commitment towards professional development and challenges; Table 2. Factors HR Planning Factors to look into when performing HR planning operation for construction projects Mean Std. Deviation Does your organisation appraise labour resources such as interest, abilities, skills and experience? 3.99 1.187 Does your organisation designs job specifications to make sure right individuals are in right positions 3.76 1.404 Source: Field Survey, 2017 • Boost Business Performance- Ability of business activities to perform through achieving low operational control, employee turnover and repetitive business. (I) Interfaces in the project • Organisational interfaces- Organisational interfaces can be formal or informal relationships between departments and could be extremely complex or simple. Example could be an emergence compound telecommunications system to coordinate different subcontractors on a project. (II) Labour Management Plan – It described project team take off time and defines when and how labour will be utilised. The labour plan could be very detailed and be formal or informal which is subjected to project requirements. 3 , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 https://doi.org/10.1051/matecconf/201925802018 • Poor management and leadership commitment • Poor organisation’s management support; • Poor commitment of stakeholders concerning HR practices • Resistance to change in new HR techniques and tools • Neglect of significance of HR involvement in a project (III) Organisational chart- This is a project reporting relationships in graphical representation. An organisational chart is a typical example of organisational breakdown structure (OBS) which could be formal or informal and detailed framed that shows departments in an organisation and their responsibilities depending on project necessities. (III) Organisational chart- This is a project reporting relationships in graphical representation. An organisational chart is a typical example of organisational breakdown structure (OBS) which could be formal or informal and detailed framed that shows departments in an organisation and their responsibilities depending on project necessities. (IV) Supporting document – Organisational planning supporting detail is subjected to project size and area. Supporting detail information may include the following, 2.3 Impacts of Sustainable HR Planning on Performance for Construction Projects • Enhance Technical Performance- To achieve technical performance of a project, technical requirements must be specified at the commencement of the project in order to eliminate human error, possible delay and low skill level. • Increases Technical Innovations- Effective ways of identifying technical problems during the project with ability of solving them. Such innovation includes ways of enhancing critical thinking and problem solving abilities. • Improves Effectiveness In execution Of Project- This is an effective way of meeting time and cost targets. This involves achieving quality standard, time and cost targets. 2.4 Causes of Failure in HR Planning for Construction Projects Causes of failure in Project HR Planning are highlighted by [15] as follows: 4 , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 https://doi.org/10.1051/matecconf/201925802018 a considerable benefit over off-job training in practice and widely practice by construction organisations. The empirical result indicated that most of the respondents Strongly Agree that evaluation the current and existing resources is paramount important for their organisation where individual interest, abilities, skills, or experience in HR Planning could be carried out before beginning of any construction projects with table 2 reflecting opinion with the mean 3.99. Also, job specification as a major factor is considered at an Agreeable rate. This indicated that efficient design of job specifications could make responsibilities be allocated right individuals reflecting a mean of 3.76. Therefore, the literatures justified empirical result. Similarly, [23] opined that through effective HR technical innovation and performance could be improved to reduce human error, delays and enhance team problem solving skills. It is strongly agree that effective Project HR planning in practice will improve project deliveries to cost, time and standard quality through selection of right individuals for the right job reflecting mean 3.69. Continuous training of teams to enhance competencies will reduce chance of delay in project team task performance, human error and further improve problem solving abilities. Table 3. Techniques and Tools in HR Planning Application of techniques and tools in HR planning Mean Std. Deviation Does your organisation practices HR procedures and guidelines in retaining project teams 3.53 1.605 Does your organisation enhance labour management plan to reduce cost and increase motivation 3.71 1.456 Source: Field Survey, 2017 Table 3. Techniques and Tools in HR Planning Table 5. Failure in HR Planning Causes of failure in HR planning for construction Mean Std. Deviation A, B, C, D, E 3.90 0.508 B,D 3.09 1.023 B,E 3.06 0.799 A,D 3.07 1.995 A,C,D,E 3.79 1.478 A,B,D 3.57 1.282 B,D,E 3.48 1.285 A,D,E 3.18 1.240 A,E 3.89 0.891 Source: Field Survey, 2017 Analysis of table 3 shows that respondents strongly agreed that organisation set standard practices for HR procedures in allocating team responsibilities and producing guidelines for job activities reflecting mean of 3.53. 2.4 Causes of Failure in HR Planning for Construction Projects This shows that job guidelines ought to obviously outline the work scope, job function, what to be carried out with essential skills and capabilities required from project team to successfully carryout the construction project. Eventually, the scale with mean 3.71 shows a strong positive approach practices when an organisation utilise standard HR procedures and guidelines and the feedback from labour management plan shows that respondents justified the literature that organisation should look into a suitable rewarding plan that would enhance level of employee’s motivation and cost reduction to improve effectiveness. Source: Field Survey, 2017 Identified failures are represented in table 5 with alphabet A-E and define as - A- Poor of effective management and leadership commitment; B- Poor organization’s management support; C- Poor stakeholders commitment in regards to HR practices; D- Resistance to change in new HR techniques and tools; E- Neglect of important of HR involvement in a project). g j Nevertheless, the scales with mean 3.90; 3.09; 3.06; 3.07; 3.79; 3.57; 3.48; 3.18 and 3.89 shows that the entire occurrence have greater contribution to the neglect of important construction Project HR planning. Table 4. HR Planning on Performance Effect of HR planning on performance for construction projects Mean Std. Deviation Does your organisation considers improving team performance to improve efficiency on the construction project through in individual skills, competency and team behaviour. 3.08 1.569 Does your organisation retrain project team to enhance efficiency and performance 3.18 1.368 Does effective Project HR planning aids technical innovation improvement and efficient performance by eliminating delays, labour error and upsurge manner of problem solving 3.69 1.625 Source: Field Survey, 2017 Table 6- Independent Sample t Test for Perceptions Factors in HR Planning Variables M SD T df P- VALUE Does your organisation undertakes labour resources appraisals such as interest, abilities, skills or experience. Project managers 1.80 0.76 -3.21 24 0.004* Non Project managers 3.10 1.37 Does your organisation design job specifications to make sure that responsibilities are given to right individuals. Project managers 1.70 0.66 -2.096 5.765 0.087 Non Project managers 2.69 1.26 *significant at 0.05 Level of P-Value Table 6- Independent Sample t Test for Perceptions Factors in HR Planning Variables M SD T df P- VALUE Does your organisation undertakes labour resources appraisals such as interest, abilities, skills or experience. 2.4 Causes of Failure in HR Planning for Construction Projects Project managers 1.80 0.76 -3.21 24 0.004* Non Project managers 3.10 1.37 Does your organisation design job specifications to make sure that responsibilities are given to right individuals. Project managers 1.70 0.66 -2.096 5.765 0.087 Non Project managers 2.69 1.26 *significant at 0.05 Level of P-Value Table 6- Independent Sample t Test for Perceptions Factors Source: Field Survey, 2017 The observation of [23] argued that to improve efficiency and performance on construction projects, organisations need to place project team on formal training either on-job or off-job training. Nevertheless, the scenario as seen in table 4 having mean 3.08 and 3.18 specifies that formal developmental training of project team may improve efficiency and effectiveness of Human Resource. Nonetheless, on-job training offers 5 , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 https://doi.org/10.1051/matecconf/201925802018 Table 6 is the result of perception factors on Project Managers and Non-Project Managers. At 0.05 P- value, both Project managers and non- Project managers seems to strongly agreed on factors to look into when performing HR planning operation for construction projects such that their respective organisations create job specifications to ensure that right responsibilities are given to right individuals (t= -2.096, df = 5.765). On the other hand, non-Project managers hold stronger perception than Project managers for their respective organisations to undertake employee resources appraisals such as skills, abilities, experience and interests (t = -3.21, df = 24). The implication of this is that non-Project managers agree more than Project managers that their respective organisations always undertake employee resources appraisals such as skills, abilities, experience and interests. management and avoid inconsistency so that HR planning for construction project can bring sustainable coherence and compatibility between project team members as well as the different group within the structure of the organisation. Lastly, transformational leadership from the organisation top management is necessary to help in creating a positive organisational- project culture. 5 Conclusion Spearman’s Rho Correlations Between Five Principal Causes of Neglect of HR Planning HR Tools, Techniques And Output Poor effective management and leadership commitment Poor organisation’ s management Support Poor stakeholde rs commitme nt in regards to HR practices Resistance to change in new HR techniques and tools Neglect of important of HR involvement in a project Project Templates Correlation 0 .006 0.269 0.044 -0.098 -0.105 Sig (p<0.05) 0.976 0.169 0.848 0.634 0.616 Analysis of stakeholder Correlation -0.066 -0.136 0.087 0.014 0.284 Sig (p<0.05) 0.742 0.500 0.682 0.938 0.157 Human resource procedures and practices Correlation 0.064 0.005 0.146 0.371(*) 0.273 Sig (p<0.05) 0.762 0.982 0.485 0.048 0.181 Roles and responsibility Correlation -0.312 0.026 0.334 -0.124 0.032 Sig (p<0.05) 0.125 0.904 0.096 0.555 0.888 Employee management plan Correlation -0.113 0.065 0.111 0.188 0.234 Sig (p<0.05) 0.579 0.757 0.584 0.379 0.247 *Correlation significant at 0.05 Level of P-Value Table 7. Spearman’s Rho Correlations Between Five Principal Causes of Neglect of HR Planning *Correlation significant at 0.05 Level of P-Value Spearman’s Rho correlation findings in Table 7 for five principal causes of HR planning neglect indicated that at 0.05 level of P-value the research shows that there is a strong correlation between resistance to change in new HR techniques and tools and Human resource procedures and practices (correlation = 0.371 and p- value= 0.048) and was found to have better effect on site productivity since correlation significant is less than 0.05. The implication of this correlation is that the more a project manager resists changing to adopt new HR techniques and tools, the more the neglect of human resource procedures and practices exist on construction project. Therefore, to achieve optimal productivity on construction project, it is advisable that an organisation allows the project manager to perform his responsibility as a coach to create a positive approach to people based 5 Conclusion The strategic finding established factors to look into when performing HR planning operations for construction projects. Initially, effective utilisation of labour appraisals to assess interest, abilities, skills and experience and design job specifications to ensure the responsibilities are given to right individuals were identified in the literature review by different scholars as part of causes of desertion of HR planning on construction projects. However, the key consideration factors for Sustainable Project HR Planning (Organisation’s structure with responsibilities given to right individuals and assessment of current resources) will bring about putting into practice of new HR Planning technique and tools for construction project. Essentially, the main reoccurring theme identified was that management of the construction organisations needs to look into the essential factors needed to be considered at the strategic level and the leaders leading a construction project team to consider those essential factors needed at operational level to clarify the numerous functions of HRM in the construction organisations and avoid inconsistencies among several practices on construction projects. Nevertheless, the impact of sustainable HR planning towards performance should include its efficient practices as advocated and strongly considered by both literature and empirical. Lastly, objectives of the paper were met as the sustainability of HR planning for construction projects were examined through literature review and substantiated with the responses from participant to obtain empirical findings which were justified in the research work. Table 7. Spearman’s Rho Correlations Between Five Principal Causes of Neglect of HR Planning HR Tools, Techniques And Output Poor effective management and leadership commitment Poor organisation’ s management Support Poor stakeholde rs commitme nt in regards to HR practices Resistance to change in new HR techniques and tools Neglect of important of HR involvement in a project Project Templates Correlation 0 .006 0.269 0.044 -0.098 -0.105 Sig (p<0.05) 0.976 0.169 0.848 0.634 0.616 Analysis of stakeholder Correlation -0.066 -0.136 0.087 0.014 0.284 Sig (p<0.05) 0.742 0.500 0.682 0.938 0.157 Human resource procedures and practices Correlation 0.064 0.005 0.146 0.371(*) 0.273 Sig (p<0.05) 0.762 0.982 0.485 0.048 0.181 Roles and responsibility Correlation -0.312 0.026 0.334 -0.124 0.032 Sig (p<0.05) 0.125 0.904 0.096 0.555 0.888 Employee management plan Correlation -0.113 0.065 0.111 0.188 0.234 Sig (p<0.05) 0.579 0.757 0.584 0.379 0.247 *Correlation significant at 0.05 Level of P-Value Table 7. 5.2 Recommendations for Future Research The results from the research showed that it is preferable to explore all HR planning for construction project best practices and be adopted by construction organisations and leaders leading construction projects. However, further research can be carried out on implementation of Project HR Planning by stakeholders for its success. 11. A. Dainty, I. Grugulis and D. Langford, Understanding Construction Employment: The Need for a Fresh Research Agenda Personnel Review. 36, 501-508 (2007) 12. G. Dessler, “Human Resource Management”, 8th Ed. Upper Saddle River, New Jersey: Prentice Hall Incorporated. (2000) 5.1 Recommendations Obviously, the paper findings suggested that the inconsistency in the various practices of HRM have led desertion of planning for construction project. Critically empirical findings results highlighted that construction industry recognised the use HR planning, therefore, adaptability and visibility of implementation of guidelines, policies and procedures of sustainable HR planning on construction project should be justified and improved. The following are recommended: • Strategic plan should be incorporated into construction organisation system; • HR planning objective should be incorporated into the construction organisational strategic plans; 6 , 0 (2019) MATEC Web of Conferences 258 SCESCM 2018 2018 https://doi.org/10.1051/matecconf/201925802018 7. A. Dainty and M. Loosemore, Human Resource Management in Construction: Critical perspectives”, London: Spon Press, (2012) • Concerted efforts should be maintained by strategic decision makers in construction organisations to encourage contemplative learning and incessant improvements through training for individuals and teams; 8. M. Loosemore, Construction Crisis Management, American Society of Civil Engineers, New York. (2000) • Sustainable application of HR technique and tools application should be encouraged to assess the need organisation and projects. 9. |J. Schervayder and R.S. Mayo, Construction Fundamentals, 2nd Ed. New Yoon: McGrew Hill. (2004) 10. L. J. Mullins, “Management and Organizational Behaviour”, 5th Edition, Essex: Pearson Education. (1999) References 13. D N. Bulla and P M. Scott, Manpower requirements forecasting: a case example, in Human Resource Forecasting and Modelling, 1st Ed, Human Resource Planning Society: New York (1994) 1. A.R.J. Dainty, B.M. Bagilhole and R.H. Neale ‘The Compatibility of Construction Companies’ Human Resource Development Policies with Employee Career Expectations,’ Engineering, Construction and Architectural Management 7, 169-78, (2000) 14. P. Reilly, Guide to Workforce Planning in Local Authorities, Employers Organization for Local Government, London (2003). 2. A. Nagendra, and M. Deshpande, Human Resource Information Systems (HRIS) in HR planning and development in mid to large sized organizations” Proceedia of Social and Behavioural Science, 133, 61-67 (2014) 15. T. H. Leng, Human resource planning and human resource development in construction. Master’s Thesis, University Technology of Malaysia (2005) 16. P. Boxall and J. Purcell, Strategy and Human Resource Management, London: Palgrave Macmillan (2016) 3. C. Fogarassy, K. Szaboand, and J. Poor, Critical issues of human resource planning, performance evaluation and long-term development on the central region and non-central areas: Hungarian case study for investors. International Journal of Engineering Business Management 9, 1–9 (2017) 17. K. Akingbola, "A Model Of Strategic Non-profit Human Resource Management." Voluntas: International Journal of Voluntary & Non-profit Organizations 24, 214-240 (2013) 18. M. Loosemore, A. Dainty and H, Lingard, “Human Resource Management in Construction Projects- Strategic and Operational Approaches”, London: Spon Press (2003) 4. A. Laufer and R. Tucker, “Is Construction Planning Really Doing its Job? A Critical Examination of Focus, Role and Process” Construction Management and Economics, London, E. & F. N. Spon, 5, 243-66 (1987) 19. S.G. Naoum, Peoples and organisational management in construction: 1st ed. London: Thomas Telford Publishing (2001) 5. L.Nesan, H. Jawahar and D. Gary. Empowerment in Construction: The way Forward for Performance Improvement, Baldock, Hertfordshine, England: Research Studies Ltd, (1999). 20. P. Farrel, Writing a built environment dissertation. West Sussex, UK: Blackwell. 79. (2011). 21. S.G Naoum, Dissertation Research and Writing for Construction Students, 3rd ed Abingdon, Oxon: Routledge. 67-97 (2013). 6. D. A. Langford R. Fellows, M. Hancock and A. Gale, Human Resource Management in Construction, 2nd ed. London: Longman, (1995) 7 7
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Audit Analysis of Abnormal Behavior of Social Security Fund Based on Adaptive Spectral Clustering Algorithm
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Hindawi Complexity Volume 2024, Article ID 9873842, 1 page https://doi.org/10.1155/2024/9873842 Retraction Retracted: Audit Analysis of Abnormal Behavior of Social Security Fund Based on Adaptive Spectral Clustering Algorithm Complexity Received 23 January 2024; Accepted 23 January 2024; Published 24 January 2024 Copyright © 2024 Complexity. Tis 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. Tis article has been retracted by Hindawi following an investigation undertaken by the publisher [1]. Tis investigation has uncovered evidence of one or more of the following indicators of systematic manipulation of the publication process: (1) Discrepancies in scope (2) Discrepancies in the description of the research reported (3) Discrepancies between the availability of data and the research described (4) Inappropriate citations (5) Incoherent, meaningless and/or irrelevant content included in the article (6) Manipulated or compromised peer review Te presence of these indicators undermines our confdence in the integrity of the article’s content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity. We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation. Te corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received. References [1] Y. Wu, Y. Chen, and W. Ling, “Audit Analysis of Abnormal Behavior of Social Security Fund Based on Adaptive Spectral Clustering Algorithm,” Complexity, vol. 2021, Article ID 4969233, 11 pages, 2021. Hindawi Complexity Volume 2021, Article ID 4969233, 11 pages https://doi.org/10.1155/2021/4969233 D E Research Article Audit Analysis of Abnormal Behavior of Social Security Fund Based on Adaptive Spectral Clustering Algorithm Yan Wu,1 Yonghong Chen,2 and Wenhao Ling T C 3 1 School of Business, Xi’an University of Finance and Economics, Shaanxi, Xi’an 710100, China School of Public Administration, Xi’an University of Finance and Economics, Shaanxi, Xi’an 710061, China 3 Institute of Local Government and Societal Governance, Henan University, Kaifeng, Henan 475004, China 2 Correspondence should be addressed to Wenhao Ling; lwh@henu.edu.cn Received 7 April 2021; Revised 27 April 2021; Accepted 10 May 2021; Published 19 May 2021 A R T E Academic Editor: Zhihan Lv Copyright © 2021 Yan Wu et al. 'is 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. Abnormal behavior detection of social security funds is a method to analyze large-scale data and find abnormal behavior. Although many methods based on spectral clustering have achieved many good results in the practical application of clustering, the research on the spectral clustering algorithm is still in the early stage of development. Many existing algorithms are very sensitive to clustering parameters, especially scale parameters, and need to manually input the number of clustering. 'erefore, a density-sensitive similarity measure is introduced in this paper, which is obtained by introducing new parameters to transform the Gaussian function. Under this metric, the distance between data points belonging to different classes will be effectively amplified, while the distance between data points belonging to the same class will be reduced, and finally, the distribution of data will be effectively clustered. At the same time, the idea of Eigen gap is introduced into the spectral clustering algorithm, and the verified gap sequence is constructed on the basis of Laplace matrix, so as to solve the problem of the number of initial clustering. 'e strong global search ability of artificial bee colony algorithm is used to make up for the shortcoming of spectral clustering algorithm that is easy to fall into local optimal. 'e experimental results show that the adaptive spectral clustering algorithm can better identify the initial clustering center, perform more effective clustering, and detect abnormal behavior more accurately. R 1. Introduction 'e development history of the social security fund market is less than 20 years, but it has developed rapidly and has become an indispensable part of the national economic life [1, 2]. 'eoretical and empirical studies have shown that, in a market dominated by individual investors, investment decisions lack sufficient rationality due to limited information acquisition capabilities. From the perspective of the domestic securities market, individual investors are obviously speculative, often fast in and out for short-term combat, are susceptible to market sentiment, and often blindly follow the trend, causing disorderly volatility in my country’s securities market, leading to significant market price fluctuations. Divorced from the actual situation of the operation of the national economy, it has failed to effectively guide resource allocation and industrial structure optimization, and it is also difficult to use market forces to evaluate the benefits of listed companies [3, 4]. 'erefore, the detection of abnormal behaviors of social security funds is beneficial to the development of the fund market [1, 5]. Cluster analysis is a statistical method for studying sample classification, as well as a method of data mining, which can effectively realize the exploration of the internal connections between things [6, 7]. 'e fundamental purpose of the clustering algorithm is to automatically classify the given samples into corresponding classes under certain standards. Manual classification of data has great limitations in practical applications [8]. Clustering belongs to the category of unsupervised classification. In the process of classification, the degree of similarity between the given classification objects is distinguished according to some characteristics of the objects themselves. Clustering algorithms have received more and more attention from 2 Complexity researchers and are currently widely used in many fields. With various abnormal behaviors in the fund market in the past two years, many researchers have used clustering algorithms to detect such abnormal behaviors [9, 10]. 'e spectral clustering algorithm is a relatively new research direction in clustering algorithms, which uses the properties of the feature vector in the similarity matrix of the data set to cluster the data set. 'e idea of the spectral clustering algorithm is to use the similarity between data points to cluster the data set. 'is method can also be applied to the cluster analysis problem in the nonmeasure space [11]. Many methods based on spectral clustering have achieved many good results in the practical application of clustering, but the study of spectral clustering algorithms is still in the early stage of development. Many existing algorithms are very important for clustering parameters, especially scale parameters. It is sensitive and requires manual input of the number of clusters. 'erefore, this paper proposes an adaptive spectral clustering algorithm. 'e main contributions of the algorithm are as follows: (1) 'is paper introduces a density-sensitive similarity measure, which is obtained by introducing new parameters to deform the Gaussian function. Under this metric, the distance between data points belonging to different classes will be effectively enlarged, while the distance between data points belonging to the same class will be reduced, and finally the distribution of data will be effectively clustered. (2) 'e idea of the intrinsic gap is introduced into the spectral clustering algorithm, and the intrinsic gap sequence is constructed on the basis of the Laplacian matrix to solve the problem of the number of initial clusters. (3) 'e advantage of the artificial bee colony algorithm’s strong global search ability is used to make up for the shortcoming of the spectral clustering algorithm that it is easy to fall into the local optimum. At the same time, in order to prevent the premature phenomenon of artificial bee colony algorithm, its location search formula is improved. and accurately identify abnormal and nonabnormal behaviors, which is also one of the current research hotspots [14–17]. Spectral clustering algorithms are derived from the theory of spectrograms. Based on different segmentation methods, researchers have proposed some more classic spectral clustering algorithms. PF algorithm [18], as the original prototype of spectral clustering, has been extensively studied in the field of machine learning. Wang et al. [19] proposed a canonical cut set to divide the graph and proposed the famous SM algorithm. 'e clustering result of the SM algorithm is also significantly higher than that of the PF algorithm. Xu et al. [20] proposed the famous SLH algorithm. After the algorithm constructs the similarity matrix according to the multipath canonical cut set criterion, the data of eigenvalues and eigenvectors is determined by the number of cluster inputs. If the number of clusters is 3, then the number of feature vectors is 3. Although its consideration is comprehensive, its high complexity affects the calculation speed and the efficiency is poor, but it still achieves a good clustering effect. Ding et al. [21] proposed the NJW algorithm. 'e algorithm is the same as the SLH algorithm. 'e number of eigenvalues is determined by the number of clustering groups after obtaining the eigenvectors. Shiotsuka et al. [22] proposed the Mcut algorithm. 'is algorithm deeply studies the vector and uses this vector as a criterion as a feature vector. 'e clustering results obtained tend to be in a balanced state, and it takes a long time for larger data sets. Zhu et al. [23] proposed the MS algorithm. Similar to the SLH algorithm and the NJW algorithm, the selection method of the feature vector is similar. Contrary to Mcut, it has a poor clustering effect when the data set is small or when the segmented image is required to be small. At present, many scholars have carried out extensive research on spectral clustering, mainly focusing on the determination of the number of clustering groups, the construction of feature vectors, and the construction of similarity matrices. Aiming at the adaptive problem of the number of clustering groups, literature [24] converts large data sets into small data sets, uses correlation to merge the grouped data, and after fully studying its principle, proposes a new spectral clustering algorithm; the algorithm can automatically obtain the number of clustering groups. Literature [25] calculates the difference between adjacent eigenvalues after arranging the eigenvalues by calculating the eigenvectors and eigenvalues of the data similarity matrix. If the previous feature value minus the current feature value is the largest, the number of current feature values counted is the number of cluster groups. Literature [26] first studied the distribution of data, estimated the proportional parameter of each data point based on this, and proposed the STSC algorithm, but the calculation consumes too many resources and runs slowly. Aiming at the problem of feature vector selection and similarity matrix construction, literature [27] sets a threshold based on the feature requirements of the data and runs the NJW algorithm multiple times. 'e number of runs is manually set, and the adaptation of the threshold A R T E T C R 'e organizational structure of this article is as follows. 'e second part shows the relevant research content of this article. 'e third part shows the research algorithm of this article. 'e fourth part is the experimental results. 'e fifth part is the conclusion. 2. Related Knowledge At this stage, the computer network has basically realized globalization, and the huge network system facilitates the exchange and transmission of fund information. Facing the increasingly complex network environment, it is of great significance to accurately detect the abnormal behavior of fund investment [12, 13]. In this regard, experts and scholars apply clustering algorithms to detection technology, relying on unsupervised and semisupervised methods to quickly D E Complexity 3 parameter a is obtained, which eliminates the interference of manual input on the construction of the similarity matrix, and improves the clustering effect. Literature [28] uses the rough clustering method to cluster the constructed left and right singular vectors and proposes rough spectral clustering, which is successfully applied in text data mining. Literatures [29–31] proposed that the existing feature vectors do not fully reflect the characteristics of the data itself, and these features themselves are also difficult to extract. 'erefore, some control information is added to the feature vector selection of the spectral clustering algorithm, and a semisupervised spectral clustering feature vector selection algorithm is proposed. Some supervision information is added to the algorithm, and the feature vector is selected on this basis. control parameters, simple and easy to implement algorithm, and strong robustness. In the artificial bee colony algorithm, the members of the algorithm are nectar, lead bee, follow bee, and scout bee, among which scout bee is also called hire bee. Among the four members, the nectar source and the leading bee have a one-to-one correspondence in number. 'e three types of bees search in their respective search neighborhoods and compare their search results continuously to obtain the optimal solution. “Swing dance” is an important way for these three bees to transmit information. Figure 1 is a schematic diagram of the artificial bee colony algorithm. 'e realization process of the artificial bee colony algorithm is as follows. 3. Adaptive Spectral Clustering Algorithm 3.2.1. Initialize Parameters. Suppose that the total number of nectar sources is n, the maximum number of loops of the algorithm is max_m, the maximum number of iterations is max_it, the maximum number of searches is max_s, the number of bees is collected m, and the number of honey bees staying in a nectar source is recorded, and m � 0. By using formula (5), we can randomly generate n honey source information. 3.1. Spectral Clustering Algorithm. 'e spectral clustering algorithm is based on the division of spectral graphs, and its essence is to transform the clustering problem into the optimal segmentation problem of graphs [32–35]. 'e spectral clustering algorithm regards the data samples as the vertices in the graph, which is represented by the set J, the vertices are connected by edges, and the edges are represented by the set B. Assign a weight value w according to the similarity between samples, and an undirected weighted graph T � (J, B) based on the similarity of the samples can be obtained. In this way, in the graph T, the clustering problem is transformed into the optimal graph segmentation problem on the graph T, so that the internal similarity of each subgraph after division is very high, and the similarity between different subgraphs is very low. Let Di denote the i-th data sample point, and let dis (di, dj) denote the distance between di and dj. 'is distance is usually expressed by the European distance ‖di-dj‖, and σ is expressed as a scale parameter. We can get the similarity matrix: T C A R T E 2 − 􏼐dis di ,dj 􏼁 /2σ 2 􏼑 Sij � e D E R . (1) 'e distribution of surrounding data can be effectively reflected by the degree of the data point. 'e degree matrix is a diagonal matrix composed of diagonal elements with all degree values, expressed as Duiij � 􏽘 Sij . (2) 'e noncanonical Lagrangian matrix is expressed as Feiij � Duiij − Sij . (3) Iij � min i × rand(max j − min j) + min j. (5) In the formula, j ∈ {1, 2, . . ., m}, rand is a random number between (0, 1), and max_j and min_j are the upper and lower limits of the j-th dimension. 3.2.2. Lead the Bee Stage. Lead the bee to search for the local nectar source location based on the greedy selection strategy in the nectar source neighborhood. In the process of searching for a nectar source, if a new and better nectar source is found, the lead bee will compare the nectar source with the best nectar source searched in history. If F (Jij) > F (Iij), select the nectar source and pass the formula (6) to update the position of the nectar source, change the position of the nectar source from Iij to Jij, and lead the bee position to update the position of the food source by the following formula: Jij � fij + λ􏼐fij − fkj 􏼑. (6) In the formula, k is generated randomly. Jij represents the new nectar source position generated near fij, λ ∈ (− 1, 1) is a random number, and the new nectar source position is constrained by λ to be generated near the original nectar source fij. 'e canonical Laplace matrix is expressed as Feiij � Dui−ij(1/2) Sij Dui−ij(1/2) . (4) 3.2. Artificial Bee Colony Algorithm. 'e artificial bee colony algorithm is an artificial intelligence bionic algorithm with strong searchability, which has the advantages of fewer 3.2.3. Follow the Bee Phase. 'e leading bee transmits the nectar source information it carries to the follower bee in the form of “swing dance,” and the follower bee selects a higherquality nectar source to follow according to the principle of roulette. Calculate the probability pi selected by the bee by the following formula. Fi represents the fitness value of the nectar source Fi. 'e larger the fitness value of Fi, the higher 4 Complexity Hiring bee stage: multiple search mechanisms Start Cg Initialization Bg Group initialization Rg D E Individual selection; search method Train data set Test data set Constructor group Train data Cg Bg Rg Val data Test data Feature extraction Nectar or nonnectar UF Bee S R S Nectar or nonnectar R Unloading UF honey Recruit a dancing room A area for A EF1 Recruit a dancing area for B UF S T C Nectar A EF1 S EF1 UF Unloading honey room B EF1 Nectar EF1 Calculate population fitness A R T E Multiple bee colony optimization model Record the optimal value Nectar B EF1 Yes Whether there is a scout bee Yes End of iteration? Scout bee search No End No Figure 1: Artificial bee colony algorithm. the probability of being selected by the bee. Fi is generated by the formula (8). pi � fi , 􏽐 fi 1 ⎪ ⎧ ⎪ , ⎪ ⎪ ⎪ 1 + f Fi 􏼁 ⎨ Fi � ⎪ ⎪ 1 ⎪ ⎪ ⎪ ⎩ 1 + ����f F 􏼁����. i R (7) (8) 3.2.4. Reconnaissance Bee Stage. If the nectar source Fi has not changed after mining, the nectar source is abandoned, a new nectar source is generated from equation (6), and a new search is continued. At this time, the lead bee attached to the nectar source Fi becomes a scout bee. 3.2.5. Record the Optimal Solution. Compare the fitness values of the current n feasible solutions, select the optimal solution, and judge whether max_it is greater than max_m at this time, and if it is greater than max_m, the algorithm ends. Because the artificial ant colony algorithm is a parallel algorithm in nature, the search process of each ant is independent of each other and communicates only through pheromone. 'erefore, the artificial ant colony algorithm can be regarded as a distributed multiagent system. It starts independent solution search at multiple points in the problem space at the same time, which not only increases the reliability of the algorithm but also makes the algorithm have a strong global search capability. 3.3. Overview of Density-Sensitive Similarity Measures. In general, clustering is an unsupervised machine learning process. Using some prior knowledge of the data set can improve the effectiveness of clustering. 'e most important thing is the consistency assumption of the data set, that is, local consistency and global consistency. (1) Local consistency: adjacent data points have higher similarity in spatial position (2) Global consistency: data points located on the same structure have a higher similarity For example, in Figure 2, there are two types of points, point a belongs to one of them, and points b, c, d, and e belong to the other. Local consistency is reflected in that the similarity between point d and points b and e is higher than the similarity between point d and points f and c. Global consistency is reflected in that the similarity between point c and point d is higher than the similarity between point c and point a. However, in this example, the traditional Euclidean distance can only reflect the local consistency of the data, not the global Complexity 5 10 8 c 6 4 b a 2 0 –10 –8 –6 –4 –2 –2 0 2 4 6 8 10 d –4 e –6 f –8 –10 Figure 2: Data points on different trends. consistency of the data. Assuming that in Figure 2, points c and f belong to the same class, and point a belongs to another class. 'en, we expect the similarity between c and f to be greater than the similarity between c and a, but the point that is under the Euclidean distance measure c is closer to point a. Based on the above problems, we design a similarity measure that can satisfy both local consistency and global consistency density-sensitive similarity measures. 'is metric can shorten the distance between data points in the same class and, at the same time, enlarge the distance between data points in different classes and effectively describe the actual distribution of data points, so as to achieve a good clustering effect. Define the following similarity measures with adjustable density: Sij � e− number of clusters before clustering. However, in practical applications, the value of the number of clusters k cannot be determined in advance under normal circumstances. At the same time, the calculation of similarity in traditional spectral clustering algorithms is also greatly affected by parameter values. Aiming at these two problems, this paper proposes an adaptive spectral clustering algorithm by introducing density-sensitive similarity measures and an artificial ant colony algorithm. 'e algorithm uses the characteristics of the size of the intrinsic gaps in the cluster to automatically determine the number of initial clusters and then uses the orthogonal feature vector to classify the data. Suppose that, in an ideal state, there are k separable classes for a given data set S. For the normalized similarity matrix, there is a conclusion: the first k largest eigenvalues of the matrix are 1. At the same time, the k + 1 eigenvalue is less than 1, and the actual distribution of the k clusters determines the size of the difference between the two eigenvalues. 'e more obvious the distribution, the greater the difference in eigenvalues, and conversely, the smaller the difference. At the same time, this difference is defined as the intrinsic gap. According to the perturbation theory of the matrix, the larger the value of the intrinsic gap, the more stable the nature of the subspace formed by the selected k eigenvectors. 'e idea of the Eigen gap is developed based on the matrix disturbance theory. Based on the obtained Laplace matrix, the idea of eigenvalues λ is arranged in descending order, that is, λ1 > λ2 > . . .> λn. 'e gap sequence G represents the difference between the k and k + 1 eigenvalues, that is, T � λk − λk+1. Among them, the larger the intrinsic gap, the more stable the subspace constructed by the selected k feature vectors. Usually, the number of clusters in the original data set is determined according to the first maximum value of the gap sequence in this card. 'e initial number of clusters k is generated as shown in D E T C A R T E αdis di ,dj 􏼁/2σ 2 􏼁 − 1. (9) In equation (9), dis (di, dj) represents the Euclidean distance between data points di and dj. 'e variable α is the density parameter. For simple data sets, α generally takes a natural number greater than 1. When the data set is complex and its probability distribution function is not convex, the value of α can be smaller, generally set to 0.2. 'e given data point corresponds to a vertex set J of an undirected weighted graph T � (J, B). Since the density-sensitive similarity measure does not satisfy the triangular inequality, it cannot be directly used to construct the similarity matrix. 'erefore, we redefine a distance measure based on this measure. Use L � {L1, L2, . . ., Ln} to represent the path between the connecting points L1 and Ln with n vertices on the graph, where Lk ∈ J, (Lk, Lk+1) ∈ J. Use Lij to represent the set of all paths between the data point pair di and dj, and define the distance sensitive to the density between di and dj: R D Sij � min 􏽘 Lk , Lk+1 􏼁. (10) 3.4. Adaptive Spectral Clustering Algorithm. 'e traditional spectral clustering method requires manual input of the k � max T1 , . . . , Tn 􏼁. (11) In the follow-up phase, the follower bee generates a new nectar source in its vicinity according to equation (6) and makes comparison choices. But in formula (6), Fij represents the nectar source near fij that is superior to fij in the previous nectar source. Since the parameter λ constrains Fij near fij, there is a lack of overall search and comparison. 'erefore, when the nectar source location is updated by formula (6), the algorithm is easy to fall into the local optimum during operation. 'is article will introduce the concept of a globally optimal solution into this formula. Experiments show that the improved position update formula (12) has a strong purpose and directionality in the search process, and the algorithm has a fast convergence speed, which makes it easy to jump out of the local optimal phenomenon. Fij � fij + λ􏼐fij − fkj 􏼑 + α􏼐fbest,j − fij 􏼑, (12) where Fij is the location of the new nectar source near fij. Both k and j are random numbers generated by random formulas. 'e algorithm implementation process is shown in Figure 3. 6 Complexity Train data set Introduce improved algorithm for clustering Test data set D E Initialize parameters set related parameters Enter the number of clusters k Nectar Global search factor introduces update formula of the follower bee position Construct similarity matrix Find the laplace matrix and normalize it Find the eigenvalues and eigenvectors of the matrix Construct an eigenvector matrix and arrange the eigenvectors in descending order Use feature vector space as search domain T C Improve ABC algorithm to search Construct the intrinsic gap sequence T and determine the value of k Whether to reach the maximum number of iterations If the i-th row in the matrix is divided into the j-th category, then the i-th row is marked as the j-th category. Treat each row in the eigenvector matrix as a point in the clustering space A R T E Output clustering results Figure 3: Algorithm flow. 700 4.1. Clustering Criterion Function. In order to verify the rationality of the initial clustering center selected by the algorithm, the experiment uses the first clustering criterion function value after the initial clustering center is selected to judge. If the function value is smaller, it means that the selection of the initial cluster center is more reasonable and closer to the true cluster center. In addition, due to the decrease of the function value of the clustering criterion, the quality of the clustering is improved, and the algorithm is more efficient. 'e experimental results are shown in Figure 4. It can be seen from Figure 4 that when k � 2, the clustering criterion function value of the improved algorithm is significantly higher than that of the contrast algorithm. However, starting from k � 3, the clustering criterion function value of the improved algorithm has dropped significantly. As the number of clusters k increases, the clustering criterion function values of the comparison algorithm and the improved algorithm tend to be parallel to each other, and there is always a certain gap. 'is is because the improved algorithm pays more attention to the distribution of the initial cluster centers. When k � 2, the improved algorithm selects the least tight data point as the second initial cluster center. Due to the small number of clusters, the selection of the second initial cluster center is far from the true cluster center, resulting in an increase in the value of the clustering criterion function. However, the improved algorithm takes into account the distribution of R Clustering criterion function value 4. Results and Discussion 600 500 400 300 200 100 0 1 2 PF Mcut SM 3 4 5 6 7 8 Number of cluster centers MS SLH STSC 9 10 NJW Our Figure 4: Clustering criterion function. real cluster centers and pays more attention to the uniform distribution. When the number of clusters k increases, the uniformly distributed data points are more in line with the distribution of the optimal clustering center, which effectively reduces the clustering criterion function value of the improved algorithm, thereby improving the clustering quality. It can be seen from Figure 4 that when k � 8, the algorithm in this paper can achieve the best performance. 'erefore, the initial number of clusters in this paper is selected as 8. Complexity 7 4.2. Analysis of Convergence Time. In order to verify the execution efficiency of the algorithm, the experiment uses the convergence time to judge. If the convergence time is shorter, the algorithm runs faster and the execution efficiency is higher. In addition, because the convergence time is reduced, the processing efficiency of the algorithm is increased, and the effect of clustering is improved. 'e experimental results are shown in Figure 5. It can be seen from Figure 5 that as the number of clusters k increases, the convergence time of the improved algorithm is the fastest. 'is is because the improved algorithm takes into account the distribution of the true clustering centers and selects the initial clustering centers from the data points with a low degree of compactness, which greatly reduces the amount of data for distance calculations, and greatly improves the algorithm’s performance while ensuring the clustering effectiveness. Although, when the number of clusters k increases, the initial cluster center selection process of the two algorithms becomes similar, and the convergence time gradually approaches, the convergence time of the improved algorithm is still lower than that of the comparison algorithm. the selection result is more reasonable. In addition, due to the reduction in the number of iterations, the accuracy of clustering increases, and the algorithm is more efficient. 'e experimental results are shown in Figure 7. It can be seen from Figure 7 that the improved algorithm takes into account the distribution of real cluster centers and pays more attention to data points with low tightness. When the number of clusters k increases, the data points with low tightness are closer to the clusters of the new cluster, which effectively reduces the number of iterations of the improved algorithm, resulting in an increase in the number of iterations. 'e convergence time is shown in Figure 8. It can be seen from Figure 8 that as the number of clusters k increases, the convergence time of the improved algorithm is significantly less than that of the contrast algorithm. 'is is because the improved algorithm proposed in this paper guarantees that there is a significant distance between the selected data points, so that the selected data points belong to different clusters to the greatest extent while ensuring the efficiency of the algorithm execution, which improves the execution efficiency of the algorithm, without losing a good initial clustering center; this also significantly reduces the convergence time of the algorithm, accelerates the convergence of the algorithm, and improves the execution efficiency of the algorithm. D E A R T E T C 4.3. Performance Analysis of Clustering Algorithm. 'e clustering results of the improved new algorithm in this paper are given below and compared with the clustering results of the traditional spectral clustering method (STSC). 'e experimental results are shown in Figure 6, where Figures 6(a) and 6(c) are the clustering results of the STSC algorithm. Figures 6(b) and 6(d) are the results of the algorithm in this paper. Figure 6(a) contains three irregularly shaped data sets. It can be seen from the classification results that, for a simple data set, both the STSC algorithm and the algorithm in this paper have achieved ideal clustering results. Figures 6(c) and 6(d) show two concentric circular data sets. 'e traditional spectral clustering algorithm got the wrong classification result for the intersection of the parabola, while the algorithm in this paper got the correct classification result. From the above experimental analysis, we can see that for some simple data sets with obvious classification, both the STSC algorithm and the algorithm in this paper can get the correct classification results. However, for some complex data sets, such as concentric circles, the clustering results of the STSC algorithm have large errors. Because the algorithm in this paper introduces a density-adjustable similarity measure, the similarity between different types of data points is reduced, and the ideal clustering result is obtained, and the number of clusters is accurately calculated. 'e STSC algorithm must manually input the number of clusters and use Euclidean distance as the similarity measure, which does not accurately and effectively reflect the actual clustering distribution of the data, so the clustering effect is relatively poor. To verify the clustering quality of the algorithm, the experiment uses the number of iterations to judge. If the number of iterations is less, it proves that the initially selected cluster center is closer to the real cluster center, and R 4.4. Abnormal Behavior Analysis. We injected outliers into the data set to generate a synthetic data set to evaluate the effect of anomaly detection. 'e specific detection effect is shown in Figure 9. As shown in Figure 9, as the number of clusters k increases, the detection rate of the algorithm in this paper gradually increases. 'e improved algorithm proposed in this paper first selects the points with the largest and smallest compactness, so as to ensure that the two initial cluster centers belong to different clusters. In addition, the initial cluster centers are randomly selected to ensure that the selected data points are evenly distributed to the greatest extent. When the number of clusters k increases, the uniform distribution of data points is more consistent with the distribution of the optimal clustering center, thereby improving the clustering performance and the effect of anomaly detection. Finally, despite the slight increase in execution time, the detection effect of the improved algorithm is still better than the comparison algorithm. DE algorithm has strong global convergence ability and robustness and does not need to rely on the characteristic information of the problem. It is suitable for solving some optimization problems in complex environments that cannot be solved by conventional mathematical programming methods. 'erefore, this article will compare the use of DE optimization, no DE optimization, no artificial ant colony algorithm, and artificial ant colony algorithm. 'e experimental results are shown in Figure 10. It can be seen from Figure 10 that the artificial ant colony algorithm used in this paper has the best performance. 8 Complexity 30 Convergence time 25 D E 20 15 10 5 0 1 2 3 4 5 6 7 8 Number of cluster centers PF Mcut SM T C 9 10 NJW Our MS SLH STSC Figure 5: Convergence time. 10 A R T E 8 6 4 2 10 8 6 4 2 0 0 –10 –8 –6 –4 –2 –2 0 2 4 6 8 10 –10 –8 –6 –4 –2 –2 –4 –4 –6 –6 –8 –8 –10 –8 R –6 –4 –2 10 8 8 6 6 4 4 2 2 0 –2 2 4 6 8 10 –10 –8 –6 –4 6 8 10 2 4 6 8 10 0 –2 –2 –4 –4 –6 –6 –8 –8 –10 –10 (c) 4 (b) 10 0 2 –10 (a) –10 0 0 (d) Figure 6: Comparison of clustering results between SC algorithm and this algorithm. Complexity 9 1 0.9 Accuracy of detection 0.8 D E 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 7 Number of cluster centers PF Mcut SM T C 8 9 MS SLH STSC 10 NJW Our Figure 7: Analysis of the number of iterations and clustering performance. 30 A R T E Convergence time 25 20 15 10 5 0 1 2 3 PF Mcut SM 4 5 6 7 8 Number of cluster centers 9 MS SLH STSC 10 NJW Our Figure 8: Convergence time. 0.8 Accuracy of detection R 1 0.9 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 PF Mcut SM 3 4 5 6 7 8 Number of cluster centers 9 MS SLH STSC Figure 9: Detection effect of abnormal behavior. 10 NJW Our 10 Complexity References 1 Accuracy of detection 0.98 0.96 0.963 0.949 0.936 0.94 0.92 0.9 0.892 0.88 0.86 0.84 0.82 With-DE Without-DE With-ABC Different techniques Without-ABC Figure 10: Detection effect of different optimization techniques. 5. Conclusion 'is paper first introduces a density-sensitive similarity measure, which is obtained by introducing new parameters to deform the Gaussian function. Secondly, in view of the defect that the number of clusters cannot be automatically determined, this paper quotes the idea of the Eigen gap, constructs an Eigen gap sequence, calculates the first maximum value, and determines the number of clusters k, which solves the problem that the spectral clustering algorithm has the problem of insufficient sensitivity of cluster centers. Finally, in order to improve the global search ability of the algorithm, it is combined with the artificial bee colony algorithm with strong global searchability. In order to further enhance the algorithm’s global searchability, the global search factor is introduced into the artificial bee colony algorithm, which effectively improves the artificial bee colony algorithm’s tendency to fall into local optimality, appear premature, and slow the convergence speed of the algorithm. Experimental results show that the improved algorithm in this paper has a good improvement in stability and optimization and can achieve a good clustering effect. However, the improved algorithm in this paper also has limitations, such as the problem that the algorithm takes a long time in the clustering process. 'erefore, how to combine the advantages of artificial bee colony algorithm and spectral clustering algorithm and reduce the time complexity of the algorithm will be the problem to be studied and solved in the next step. [1] W. Han and L. Longzhu, “Analysis of the operation mechanism of American social security fund,” in Proceedings of the 2019 International Conference on Economic Management and Cultural Industry (ICEMCI 2019), pp. 26–29, Atlantis Press, Paris, France, 2019. [2] Y. 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https://openalex.org/W4391551691
http://journal.universitaspahlawan.ac.id/index.php/ners/article/download/16783/12697
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Analisis Kadar Zat Besi (Fe) Pada Tepung Kulit Kentang
Jurnal Ners
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Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008 JURNAL NERS Research & Learning in Nursing Science http://journal.universitaspahlawan.ac.id/index.php/ners Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008 JURNAL NERS Research & Learning in Nursing Science http://journal.universitaspahlawan.ac.id/index.php/ners Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008 JURNAL NERS Research & Learning in Nursing Science http://journal.universitaspahlawan.ac.id/index.php/ners ANALISIS KADAR ZAT BESI (FE) PADA TEPUNG KULIT KENTANG Ifa Nurhasanah Program Studi Sarjana Kebidanan Fakultas Ilmu Kesehatan Universitas Ibrahimy nurhasanah_ifa@yahoo.com Ifa Nurhasanah Program Studi Sarjana Kebidanan Fakultas Ilmu Kesehatan Universitas Ibrahimy nurhasanah_ifa@yahoo.com Abstrak Kentang adalah salah satu buah yang tumbuh di negara Indonesia. Buah ini memiliki manfaat yang baik bagi tubuh bila mengkonsumsinya. Beberapa bagian dari buah ini juga dapat dimanfaatkan dengan biak, salah satunya adalah pada kulitnya. Kulit kentang yang biasanya sering dibuang dan tidak digunakan, memiliki kandungan zat besi yang diperlukan oleh tubuh. Tujuan penelitian ini adalah untuk mengetahui dan menganalisis kandungan kadar zat besi pada tepung kulit kentang. Metode yang digunakan pada penelitian yaitu dilakukan pada uji analisis kuantitatif dan kualitatif. Alat yang digunakan untuk menganalisis kadar zat besi dalam tepung kulit kentang adalah Spektrofotometer Serapan Atom (SSA) yang dilakukan di Laboratorium Sucofindo Surabaya pada bulan Maret-juli 2023. Hasil penelitian ini menunjukkan bahwa terdapat kandungan zat besi pada tepung kulit kentang sebanyak 111, 8 dalam 100 gr. Kesimpulannya bahwa kulit kentang dapat dimanfaatkan sebagai bahan makanan karena memiliki kandungan zat besi yang tinggi bila dibandingkan dengan daging kentang, sehingga dapat meningkatkan kadar hemoglobin dalam tubuh seseorang agar dapat mencegah atau sebagai penanganan dari penyakit anemia. Kata Kunci: Analisis, tepung kulit kentang, zat besi Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008 JURNAL NERS Research & Learning in Nursing Science http://journal.universitaspahlawan.ac.id/index.php/ners METODE Metode penelitian ini adalah penelitian kualitatif dan kuantitatif dengan pendekatan eksperimen laboratorium. Penelitian dengan jenis kualitatif bertujuan untuk mengetahui adanya kandungan zat besi (Fe) pada tepung kulit kentang. Sedangkan jenis penelitian kuantitatif memiliki tujuan untuk menganalisis dari nilai kadar zat besi atau Fe yang terdapat pada tepung kulit kentang. Penelitian ini dilakukan di Laboratorium Sucofindo Surabaya pada bulan Maret- Juli 2023. Alat yang digunakan pada penelitian ini diantaranya corong gelas, gelas kimia, pipet tes, labu ukur, kertas saring, kaca arloji, dan spektrofotometer Serapan Atom (SSA). Sedangkan bahan yang dipakai adalah aquadest, sampel buah pepaya, tablet zat besi (multivitamin). Selanjutnya peneliti melakukan preparasi sampel, analisis kualitatif dan analisis kuantitatif. Kulit kentang bila tidak diolah maka akan menjadi sampah dan tidak bisa dikonsumsi. Akan tetapi, bila kulit kentang diolah dan dibuat menjadi bahan makanan, hal ini dapat dikonsumsi. Salah satunya dapat dibuat menjadi tepung kulit kentang. Tepung ini dapat dijadikan bahan membuat makanan yang dapat divariasikan menjadi makanan yang lagi tren saat ini (Fajiarningsih, 2013). Kentang adalah sejenis buah yang dapat dengan mudah diperoleh dan tidak mahal. Buah ini juga dapat ditanam sendiri dipekarangan rumah. Sehingga setiap hari dapat mengkonsumsi dari buah ini. Meskipun kandungan zat besi yang terdapat pada buah ini tidak terlalu besar bila dibandingkan dengan buah lainnya, akan tetapi kulit kentang dapat memenuhi kebutuhan zat besi yang dibutuhkan oleh tubuh manusia. Abstract Potato is a fruit that grows in Indonesia. This fruit has good benefits for the body when consumed. Some parts of this fruit can also be used multiplied, one of which is the skin. Potato skins, which are usually thrown away and not used, contain iron which is needed by the body. The purpose of this study was to determine and analyze the iron content in potato skin flour. The method used in this research is carried out on quantitative and qualitative analysis tests. The tool used to analyze the levels of iron in potato skin flour is the Atomic Absorption Spectrophotometer (AAS) which was carried out at the Sucofindo Laboratory in Surabaya in March-July 2023. The results of this study indicate that there is an iron content in potato skin flour as much as 111.8 in 100 gr. The conclusion is that potato skin can be used as a food ingredient because it has a high iron content when compared to potato flesh, so it can increase hemoglobin levels in a person's body in order to prevent or as a treatment for anemia. Keywords: Analysis, potato skin flour, iron @Jurnal Ners Prodi Sarjana Keperawatan & Profesi Ners FIK UP 2023 @Jurnal Ners Prodi Sarjana Keperawatan & Profesi Ners FIK UP 2023 Corresponding author : Address : Situbondo Email : nurhasanah_ifa@yahoo.com Phone : 085335724774 Corresponding author : Address : Situbondo Email : nurhasanah_ifa@yahoo.com Phone : 085335724774 ISSN 2580-2194 (Media Online) Jurnal Ners Universitas Pahlawan PENDAHULUAN dilakukan oleh Soelarso tahun 1997 yang menyebutkan bahwa kulit kentang memiliki kandungan gizi lima kali lebih besar bila dibandingkan dengan dagingnya. Menurut peneliti yang sama yang mengatakan bahwa kandungan gizi dari kulit kentang diantaranya adalah kalori 115 kal, serat 5 gr, vitamin C 7,8 gr, asam folat 5,5 mg, kalsium 19,8 mg, zat besi 4,1 mg, potassium 322 mg, dan sodium 3,1 mg (Soelarso, 2012). Negara Indonesia merupakan salah satu negara penghasil kentang terbesar di Asia Tenggara. Hal ini membuat masyarakat Indonesia sering mengkonsumsi kentang setiap harinya (Soelarso, 2012). Kentang adalah tanaman yang dapat dimanfaatkan untuk dijadikan sayur yang dapat dikonsumsi oleh banyak orang (Rahma, 2017). Selain rasanya yang enak, kentang ini juga memiliki kandungan gizi yang baik dan dibutuhkan oleh tubuh. Kandungan gizi yang terdapat pada kulit kentang dapat menjadi daya tarik bagi konsumen untuk mengkonsumsi tidak hanya pada daging kentang, namun juga dapat memanfaatkan kulit kentang. Kulit kentang dapat dikonsumsi dengan dibersihkan terlenih dahulu dan dikeringkan sehingga dapat dihaluskan menjadi tepung. Tepung kulit kentang dapat menjadi bahan dasar seseorang membuat olahan makanan seperti kue, atau jajanan lain yang dapat dikonsumsi dan sesuai selera konsumen. Selain itu, tepung kulit kentang ini tidak memiliki efek samping setelah mengkonsumsinya (Fajiarningsih, 2013). Sehingga aman dikonsumsi bagi penderita anemia atau untuk mencegah dari penyakit ini, utamanya bagi remaja perempuan dan ibu hamil yang rentan terkena anemia (Rahma, 2017). Salah satu kandungan gizi dari kentang adalah zat besi (Fe). Zat besi merupakan salah satu zat gizi yang diperlukan tubuh untuk memproduksi hemoglobin dalam sel darah merah yang dapat mencegah dari penyakit anemia yang rentan terjadi pada remaja perempuan dan ibu hamil. Dengan adanya kentang, dapat membantu seseorang yang mengalami anemia untuk meningkatkan kadar hemoglobin dalam darah (Rahma, 2017). Kentang terdiri dari daging, biji dan kulit. Daging dan biji pada kentang seringkali dimanfaatkan bahkan dapat dikonsumsi setiap hari. Namun, pada kulit kentang sering dijadikan sampah atau dibuang dan tidak dimanfaatkan. Padahal beberapa penelitian meneybutkan bahwa kulit kentang memiliki kandungan gizi yang tidak kalah dengan buah kentang itu sendiri. Salah satu kandungan gizi yang terdapat pada kulit kentang adalah zat besi (Fe). Hal ini diperkuat dengan penelitian yang dilakukan oleh Rahma Widyastuti tahun 2017 yang mengatakan bahwa penelitian dengan in vivo menyebutkan kulit kentang dapat meningkatkan kadar hemoglobin pada mencit (Rahma, 2017). Tujuan penelitian ini adalah untuk mengetahui dan menganalisis kadar zat besi (Fe) pada tepung kulit kentang. Jurnal Ners Universitas Pahlawan Analisis kuantitaif Uji analisis kuantitatif pada tepung kulit kentang bertujuan untuk menganalisis nilai kadar zat besi atau fe pada sampel dengan menggunakan alat Spektrofotometer Serapan Atom (SSA). Adapun tahap-tahap dalam melakukan uji analisis ini sebagai berikut. Pengukuran kadar besi (Fe) Sampel tepung kulit kentang yang telah dipreparasi sebelumnya dimasukkan kedalam SSA dengan cara diinjeksi. Lalu, ukur serapannya dengan gelombang yang sama yaitu 283,3 nm dan catat hasil pengukuran untuk selanjutnya dilakukan analisis. 1007| ANALISIS KADAR ZAT BESI (FE) PADA TEPUNG KULIT KENTANG tepung kulit kentang yang telah dilakukan dengan menggunakan alat spektrofotometer serapan atom ditunjukkan pada tabel dibawah ini. Tabel 1. Hasil analisis kadar zat (Fe) pada tepung kulit kentang Sampel Unit Hasil Tepung kulit kentang PPM 111,8 tepung kulit kentang yang telah dilakukan dengan menggunakan alat spektrofotometer serapan atom ditunjukkan pada tabel dibawah ini. kentang dan mudah saat dibersihkan. Selanjutnya kentang dikupas dan dipisah antara kulit dan dagingnya. Saat melakukan pengkupasan pada kulit kentang, usahakan daging tidak ikut pada kulit kentang (dikupas tipis). Kemudian kulit kentang yang sudah terpisah dengan daging, dilakukan penjemuran dibawah sinar matahari dengan suhu 55˚-60˚C selama 6-7 jam. Setelah kering, kulit kentang di haluskan dengan menggunakan alat penghalus (blender) dan diayak. Tepung kulit kentang dilarutkan dan disaring, lalu dilakukan penimbangan sebanyak 10 gr. Filtrat dari sampel tersebut dimasukkan kedalam labu ukur sebanyak 100 ml. Selanjutnya, sampel ditambahkan dengan aqudest sebagai pelarut hingga tanda batas dan dihomogenkan. Tabel 1. Hasil analisis kadar zat (Fe) pada tepung kulit kentang g Sampel Unit Hasil Tepung kulit kentang PPM 111,8 g Sampel Unit Hasil Tepung kulit kentang PPM 111,8 Pada tabel 1. Menunjukkan hasil analisis kadar zat besi pada tepung kulit kentang sebesar 111,8 dalam 100 gr. Hal ini membuktikan bahwa tepung kulit kentang memiliki kandungan zat besi yang tinggi bila dibandingkan dengan daging kentangnya. Analisis kuantitatif Hasil analisis kuantitatif pada tepung kulit kentang menunjukkan adanya kandungan zat besi didalamnya. Tepung kulit kentang dapat dimanfaatkan untuk dikonsumsi karena tidak memiliki efek samping setelah memakannya dan justru memiliki manfaat yang baik bagi tubuh yaitu dapat meningkatkan kadar hemoglobin dalam darah karena tepung kulit kentang memiliki kandungan zat besi (Suryowibisono, 2020). Tepung kulit kentang dapat di jadikan bahan untuk membuat olahan makanan yang dapat dikonsumsi oleh masyarakat khususnya bagi yang rentan terkena anemia yaitu pada remaja putri dan ibu hamil. Namun, zat besi alami tidak hanya diperoleh dari kulit kentang, akan tetapi juga dapat memanfaatkan bahan makanan lainnya, seperti ikan gabus yang di ekstrak juga dapat meningkatkan kadar Hb (Azzalina & Galaupa, 2023). Penelitian lain juga menunjukkan bahwa jus jambu merah dan jeruk sunkis madu juga berpengaruh terhadap peningkatan kadar Hb (Safitri, 2019). Bila di telaah dari banyaknya penelitian yang berkaitan dengan peningkatan kadar Hb sangatlah banyak dan mudah untuk didapatkan. Kadar Hb seseorang dapat meningkat disebabkan karena zat besi dalam darah terpenuhi dengan baik. Banyaknya sumber makanan yang dapat diperoleh untuk kebutuhan zat besi bertujuan untuk menurunkan kejadian anemia terutama bagi ibu hamil dan remaja. Analisis kualitatif Langkah awal yang dilakukan untuk mengetahui adanya kandungan Fe pada tepung kulit kentang adalah sampel uji yang telah dipreparasi sebelumnya diteteskan kedalam tabung reaksi sebanyak 5 tetes. Kemudian, teteskan sebanyak 3 tetes larutan kaliun tiosianida (KSCN) pada tabung yang sama. Penilaian yang dilakukan adalah melihat dari perubahan warna yang terjadi pada tabung reaksi tersebut. Perubahan warna merah yang terjadi pada larutan sampel menyatakan bahwa terdapat kandungan Fe didalam sampel. Pengukuran kadar larutan standar logam besi (Fe) Masing-masing larutan baku Fe dimasukkan dengan cara menginjeksikan kedalam alat SSA yang dibuat dengan konsentrasi 0 ppm, 5 ppm, 10 ppm, 20 ppm dan 50 ppm. Kemudian ukur serapannya pada Panjang gelombang 283,3 nm. Selanjutnya, hasil pengukuran dicatat dan dibuat kurva kalibrasi untuk mendapatkan persamaan liniernya. Preparasi sampel Tepung kulit kentang adalah sampel untuk dilakukan uji analisis kadar Fe. Kentang yang dipilih menjadi sampel adalalah kentang yang bagus dan tidak busuk. Langkah awal pembuatan sampel dimulai dari kentang yang dicuci bersih sampai tidak ada kotoran yang menempel pada kulit kentang. Sebelumnya kentang direndam satu hari agar kotoran tanah tidak menempel pada Dengan adanya penelitian ini, dapat memberitahukan bahwa kulit pada kentang juga dapat dimanfaatkan menjadi olahan makanan yang dapat dikonsumsi. Sehingga kulit kentang tidak lagi menjadi sampah atau tidak dimanfaatkan dengan baik oleh para konsumen. Penelitian yang ISSN 2580-2194 (Media Online) Jurnal Ners Universitas Pahlawan Jurnal Ners Universitas Pahlawan Analisis kualitatif Uji analisis kualitatif bertujuan untuk mengetahui hasil kadar zat besi pada tepung kulit kentang. Bila melihat pada tabel 1 yang menunjukkan bahwa kadar zat besi pada tepung kulit kentang sebnayak 111,8 dalam 100 gr (Rahma, 2017). Kadar zat besi pada tepung kulit kentang sangat besar bila dibandingkan dengan daging kentang itu sendiri. Hal ini dapat menambah informasi bagi masyarakat khususnya ibu yang sering memasak buah ini untuk tidak menjadikan kulit kentang sebagai sampah dan pada akhirnya dibuang. Namun, kulit kentang ini dapat dimanfaatkan dengan baik dan diolah secara benar sesuai dengan selera makanan yang diinginkan agar DAFTAR PUSTAKA memenuhi zat besi pada tubuh (Anova, W, & S, 2014). memenuhi zat besi pada tubuh (Anova, W, & S, 2014). Anova, T., W, H., & S, S. (2014). Substitusi Tepung Terigu dengan Tepung Kentang (Solanum Sp) pada Pembuatan Cookis Kentang. J Litbang Ind, 123-131 Vol. 4, No. 2 . Kebutuhan zat besi yang diperlukan oleh tubuh harus terpenuhi setiap harinya agar terhindar dari penyakit anemia. Beberapa data menunjukkan bahwa kelompok remaja putri dan ibu hamil memiliki jumlah penderita anemia yang lebih besar bila dibandingkan dengan kelompok umur lainnya. Pada kelompok remaja putri beresiko terkena anemia, disebabkan karena mengalami menstruasi setiap bulan sehingga berdampak pada pengeluaran darah dan akhirnya mengalami kekurangan darah. Hal ini juga terjadi pada ibu hamil khususnya pada trimester akhir yang memerlukan asupan zat besi lebih banyak dibandingkan sebelum hamil. Sehingga dengan adanya kebutuhan zat besi yang banyak, dapat didapatkan dari kulit kentang yang dibisa di olah menjadi makanan atau minuman sehat dan dapat dikonsumsi setiap hari (Winarno, 2004). Azzalina, N., & Galaupa, R. (2023). Perbandingan Pemberian Ekstrak Ikan Gabus dan Smooty Bayam Terhadap Peningkatan Kadar Hemoglobin pada Ibu Hamil di Wilayah UPT Puskesmas Ciomas Tahun 2023. Jurnal Ners, 121-126 Vol. 7, No. 1. Broto, W., T, H., & H, H. (1996). Effect of Sodium Bisulphite Concentration ang Thickness of Potato Tuber on Inhibition of Enzymatic and Non-Enzymatic Browning of. 331-346. Cicilia, S., Basuki, E., Prarudiyanto, A., Alamsyah, & Handito, D. (2018). pengaruh substansiTepung Terigu dengan Tepung Kentang Hitam (Coleus Tuberosus) Terhadap Sifat Kimia dan Organoleptik Cookies . J Ilmu dan Teknol. Pangan , 304-310 Vol. 4, No. 1 . Kentang dapat diperoleh di beberapa tempat dan mudah untuk mendapatkan nya. Selain itu, buah satu ini juga memiliki harga yang cukup murah, dan bahkan dapat ditanam sendiri di pekarangan rumah sehingga lebih mudah untuk mendapatkan dan dikonsumsi setiap hari (Broto, T, & H, 1996). Kulit kentang yang akan diolah dapat dijadikan tepung terlebih dahulu, agara memudahkan untuk membuat olahan makanan yang lagi trend saat ini, utamanya bagi kelompok remaja yang sering mengikuti jajanan viral saat ini (Fajiarningsih, 2013). Fajiarningsih, H. (2013). Pengaruh Penggunaan Komposit Tepung Kentang (Solanum Tuberosum L.) Terhadap Kualitas Cookies. Noscov, S. (2013 ). Pottasium, Physical and Chemical Properties . Rahma, W. (2017). Bioaktivitas Kulit Kentang (Solanum Tuberosum L.) Terhadap Peningkatan Kadar Haemoglobin Secara In Vivo. Namun, saat mengolah makanan dari bahan tepung kulit kentang harus diperhatikan bahan bahan campuran lainnya. HASIL DAN PEMBAHASAN Hasil uji analisis kadar zat besi (Fe) pada Jurnal Ners Universitas Pahlawan ISSN 2580-2194 (Media Online) 1008| ANALISIS KADAR ZAT BESI (FE) PADA TEPUNG KULIT KENTANG DAFTAR PUSTAKA Usahakan kulit kentang yang telah dibuat tepung, dikomposisikan lebih banyak sehingga zat besi yang diperlukan tubuh dapat terpenuhi dari tepung kulit kentang tersebut (Cicilia, Basuki, Prarudiyanto, Alamsyah, & Handito, 2018). Buah kentang yang dipilih juga harus segar, karena akan berdampak pada kulit kentang tersebut. Sehingga dengan memilih buah kentang yang segar, maka akan mendapatkan kulit kentang baik dan bagus serta dapat dijadikan tepung (Noscov, 2013 ). Membuat makanan dengan olahan tepung kulit kentang ini juga harus memperhatikan cara memasaknya, sehingga tidak merusak kandungan zat besi yang ada dalam tepung kulit kentang. Safitri, Y. (2019). Pengaruh Pemberian Jus bayam Merah, Jeruk Sunkis, Madu Terhadap kadar Haemoglobin pada Ibu Hamil yang Mengalami Anemia di UPT Puskesmas Kampar. Jurnal Ners, 72-83 Vol. 3, No. 2. Soelarso, B. (2012). Budi Daya Kentang Bebas Penyakit . Yogyakarta : Kanisius . Suryowibisono. (2020). Pemanfaatan Limbah Kulit Kentang Sebagai Bahan Tambahan Pembuatan Puding. Universitas Telkom Bandung . Winarno, F. (2004). Kimia Pangan dan Gizi . Jakarta : PT Gramedia Pustaka Utama . SIMPULAN Pada penelitian ini dapat disimpulkan bahwa tepung kulit kentang memiliki kadar zat besi sebanyak 111,8 dalam 100 gr. Tepung kulit kentang dapat dimanfaatkan oleh masyakarat utamanya pada kelompok remaja putri dan ibu hamil yang rentan beresiko terkena anemia. Sehingga hal ini dapat meningkatkan kadar hemoglobin dalam tubuh. ISSN 2580-2194 (Media Online) Jurnal Ners Universitas Pahlawan
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Development of Gene Expression–Based Score to Predict Sensitivity of Multiple Myeloma Cells to DNA Methylation Inhibitors
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TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly differentially expressed between control and decitabine treated cells were identified using SAM supervised paired analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set yielding to a maximum variance across control and decitabine treated cells. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly differentially expressed between control and decitabine treated cells were identified using SAM supervised paired analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set yielding to a maximum variance across control and decitabine treated cells. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly differentially expressed between control and decitabine treated cells were identified using SAM supervised paired analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set yielding to a maximum variance across control and decitabine treated cells. Probeset Gene Ratio Banding Affymetrix description Intercellular communication and membrane proteins 202304_at FNDC3A 0.40 13q14.2 fibronectin type III domain containing 3A 209541_at IGF1 0.43 12q22-q23 insulin-like growth factor 1 (somatomedin C) 221658_s_at IL21R 0.50 16p11 interleukin 21 receptor 210587_at INHBE 0.55 12q13.3 inhibin; beta E 219702_at PLAC1 0.42 Xq26 placenta-specific 1 209606_at PSCDBP 0.48 2q11.2 pleckstrin homology; Sec7 and coiled-coil domains; binding protein 202375_at SEC24D 0.31 4q26 SEC24 related gene family; member D (S. cerevisiae) 222838_at SLAMF7 0.51 1q23.1-q24.1 SLAM family member 7 222690_s_at TMEM39A 0.46 3q13.33 transmembrane protein 39A Signal transduction 224451_x_at ARHGAP9 0.38 12q14 Rho GTPase activating protein 9 /// Rho GTPase activating protein 9 212954_at DYRK4 0.42 12p13.32 dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 4 228831_s_at GNG7 0.33 19p13.3 guanine nucleotide binding protein (G protein); gamma 7 209314_s_at HBS1L 0.53 6q23-q24 HBS1-like (S. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly differentially expressed between control and decitabine treated cells were identified using SAM supervised paired analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set yielding to a maximum variance across control and decitabine treated cells. cerevisiae) 219188_s_at LRP16 0.36 11q11 LRP16 protein 229549_at OPN1SW 0.40 7q31.3-q32 Opsin 1 (cone pigments); short-wave-sensitive (color blindness; tritan) Cytoskeleton 213500_at COPB2 0.58 3q23 Coatomer protein complex; subunit beta 2 (beta prime) 204411_at KIF21B 0.53 1pter-q31.3 kinesin family member 21B 226438_at SNTB1 0.36 8q23-q24 Syntrophin; beta 1 (dystrophin-associated protein A1; 59kDa; basic component 1) 226181_at TUBE1 0.38 6q21 tubulin; epsilon 1 Cell cycle 214152_at CCPG1 0.44 15q21.1 cell cycle progression 1 223569_at PPAPDC1B 0.42 8p12 phosphatidic acid phosphatase type 2 domain containing 1B 223195_s_at SESN2 0.47 1p35.3 sestrin 2 Metabolism 231202_at ALDH1L2 0.45 12q23.3 aldehyde dehydrogenase 1 family; member L2 219572_at CADPS2 0.37 --- Ca2+-dependent activator protein for secretion 2 212816_s_at CBS 0.41 21q22.3 cystathionine-beta-synthase 218923_at CTBS 0.42 1p22 chitobiase; di-N-acetyl- 201791_s_at DHCR7 0.45 11q13.2-q13.57-dehydrocholesterol reductase 204646_at DPYD 0.48 1p22 dihydropyrimidine dehydrogenase 203397_s_at GALNT3 0.65 2q24-q31 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 203157_s_at GLS 0.42 2q32-q34 glutaminase 226183_at GSK3B 0.49 3q13.3 Glycogen synthase kinase 3 beta 1555037_a_atIDH1 0.53 2q33.3 isocitrate dehydrogenase 1 (NADP+); soluble 201625_s_at INSIG1 0.34 7q36 insulin induced gene 1 221760_at MAN1A1 0.43 6q22 Mannosidase; alpha; class 1A; member 1 222805_at MANEA 0.51 6q16.1 mannosidase; endo-alpha 232488_at MGC15875 0.55 5q35.3 hypothetical protein MGC15875 225520_at MTHFD1L 0.41 6q25.1 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like 202847_at PCK2 0.47 14q11.2 phosphoenolpyruvate carboxykinase 2 (mitochondrial) 221788_at PGM3 0.49 6q14.1-q15 Phosphoglucomutase 3 201397_at PHGDH 0.36 1p12 phosphoglycerate dehydrogenase 205194_at PSPH 0.38 7p15.2-p15.1 phosphoserine phosphatase 200831_s_at SCD 0.39 10q23-q24 stearoyl-CoA desaturase (delta-9-desaturase) 209610_s_at SLC1A4 0.54 2p15-p13 solute carrier family 1 (glutamate/neutral amino acid transporter); member 4 208916_at SLC1A5 0.40 19q13.3 solute carrier family 1 (neutral amino acid transporter); member 5 209921_at SLC7A11 0.41 4q28-q32 solute carrier family 7; (cationic amino acid transporter; y+ system) member 1 Protein binding 215930_s_at CTAGE5 0.30 14q13.3 CTAGE family; member 5 1554462_a_atDNAJB9 0.42 7q31|14q24.2-q24.3 DnaJ (Hsp40) homolog; subfamily B; member 9 222294_s_at EIF2C2 0.52 8q24 Eukaryotic translation initiation factor 2C; 2 235745_at ERN1 0.35 17q24.2 endoplasmic reticulum to nucleus signalling 1 219118_at FKBP11 0.38 12q13.12 FK506 binding protein 11; 19 kDa 218361_at GOLPH3L 0.45 1q21.2 golgi phosphoprotein 3-like 224763_at RPL37 0.35 5p13 ribosomal protein L37 201915_at SEC63 0.51 6q21 SEC63-like (S. cerevisiae) 202061_s_at SEL1L 0.56 14q24.3-q31 sel-1 suppressor of lin-12-like (C. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly differentially expressed between control and decitabine treated cells were identified using SAM supervised paired analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set yielding to a maximum variance across control and decitabine treated cells. elegans) 217790_s_at SSR3 0.40 3q25.31 signal sequence receptor; gamma (translocon-associated protein gamma) 202558_s_at STCH 0.47 21q11.1|21q11stress 70 protein chaperone; microsome-associated; 60kDa 222116_s_at TBC1D16 0.70 17q25.3 TBC1 domain family; member 16 218145_at TRIB3 0.41 20p13-p12.2 tribbles homolog 3 (Drosophila) Nuclear proteins and transcription factors 227558_at CBX4 0.49 17q25.3 chromobox homolog 4 (Pc class homolog; Drosophila) 219551_at EAF2 0.46 3q13.33 ELL associated factor 2 226982_at ELL2 0.45 5q15 elongation factor; RNA polymerase II; 2 202146_at IFRD1 0.51 7q22-q31 interferon-related developmental regulator 1 214179_s_at NFE2L1 0.59 17q21.3 nuclear factor (erythroid-derived 2)-like 1 213878_at RECQL 0.44 12p12 RecQ protein-like (DNA helicase Q1-like) 208763_s_at TSC22D3 0.53 Xq22.3 TSC22 domain family; member 3 225382_at ZNF275 0.35 Xq28 zinc finger protein 275 227132_at ZNF706 0.49 8q22.3 zinc finger protein 706 Others 227755_at --- 0.53 --- CDNA FLJ42435 fis; clone BLADE2006849 208725_at --- 0.46 --- Full-length cDNA clone CS0DD001YD20 of Neuroblastoma Cot 50-normalized of 244623_at --- 0.39 --- Transcribed locus 226719_at --- 0.53 --- CDNA FLJ34899 fis; clone NT2NE2018594 230570_at --- 0.37 --- Transcribed locus 229969_at --- 0.41 --- Transcribed locus; moderately similar to XP_508230.1 PREDICTED: zinc finger protein 195 [Pan troglodytes] 223136_at AIG1 0.41 6q24.2 androgen-induced 1 222545_s_at C10orf57 0.56 10q22.3 chromosome 10 open reading frame 57 220755_s_at C6orf48 0.49 6p21.3 chromosome 6 open reading frame 48 219802_at FLJ22028 0.44 12p12.1 hypothetical protein FLJ22028 212633_at KIAA0776 0.49 6q16.1 KIAA0776 229090_at LOC220930 0.45 10p11.23 hypothetical protein LOC220930 230570_at --- 0.37 --- Transcribed locus 229969_at --- 0.41 --- Transcribed locus; moderately similar to XP_508230.1 PREDICTED: zinc finger protein 195 [Pan troglodytes] 223136_at AIG1 0.41 6q24.2 androgen-induced 1 222545_s_at C10orf57 0.56 10q22.3 chromosome 10 open reading frame 57 220755_s_at C6orf48 0.49 6p21.3 chromosome 6 open reading frame 48 219802_at FLJ22028 0.44 12p12.1 hypothetical protein FLJ22028 212633_at KIAA0776 0.49 6q16.1 KIAA0776 229090_at LOC220930 0.45 10p11.23 hypothetical protein LOC220930
https://openalex.org/W4252808335
https://journal.tarbiyahiainib.ac.id/index.php/attalim/article/download/281/235
English
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High Touch Approach and Its Relationship with Students’ Learning Outcomes at IAIN Bukittinggi
Al-Ta'lim/Jurnal Al-Ta'lim
2,017
cc-by-sa
4,325
AL-TA’LIM JOURNAL, 24 (2), 2017, (165-173) (Print ISSN 1410-7546 Online ISSN 2355-7893) Available online at http://journal.tarbiyahiainib.ac.id/index.php/attalim Darul Ilmi Darul Ilmi Institut Agama Islam Negeri Bukittinggi, Indonesia. E-mail: ilmid10@yahoo.com Institut Agama Islam Negeri Bukittinggi, Indonesia. E-mail: ilmid10@yahoo.com Keywords: High touch, student-centered and learning outcomes Keywords: High touch, student-centered and learning outcomes How to Cite: Ilmi, D. (2017). High touch approach and its relationship with students’ learning outcomes at IAIN Bukittinggi. Al-Ta Lim Journal, 24 (2). doi:http://dx.doi.org/10.15548/jt.v24i2.281 How to Cite: Ilmi, D. (2017). High touch approach and its relationship with students’ learning outcomes at IAIN Bukittinggi. Al-Ta Lim Journal, 24 (2). doi:http://dx.doi.org/10.15548/jt.v24i2.281 High Touch Approach and Its Relationship with Students’ Learning Outcomes at IAIN Bukittinggi Received: 06th July 2017; Revised: 13th July 2017; Accepted: 17th July 2017 Permalink/DOI: http://dx.doi.org/10.15548/jt.v24i2.281 Abstract: Learning without students’ center tends to create less passionate students in following the lecture. They tend to ignore their lecturer. They have less attention, and try to create ineffective conditions, therefore, the learning atmosphere will become less effective to achieve the intended goals. The purpose of the research is to determine the process of learning, the implementation of High Touch approach, and factors that influence its implementation. Classroom action research approach was used at department of mathematics which involve students who enroll at academic year 2016/2017 IAIN Bukittinggi. The research was done in two cycles in which one cycle conducted three meetings. The instruments used were observation, essay questions, and tasks given to students. The results show that there is an improvement in students’ participation and activeness in learning when their lecturer builds learning experience through reinforcement, affection, guide, directive action, and good modeling by student–center andself- learning activities, and independent learning skill orientations showed in cycle I and II. It implies that implementing high touch may lead students to be more active, creative, and fun in learning. arul Ilmi, High Touch Approach and Its Relationship with Students’ Learning Outcomes… arul Ilmi, High Touch Approach and Its Relationship with Students’ Learning Outcomes… © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) INTRODUCTION resources. Educational investment is human resources used to develop human potentials in which includes both benefit and social returns (Collins & Clark, 2003; Davidsson & Honig, 2003; Weinberger, 1998). Education is a strategic investment to improve rivalry among countries in politics, laws, cultures, technologies, and defenses all over the world. In accordance to this condition, the developed countries simultaneously improve their education focusing on human capital and human investment. This is the reason to improve the capability to compete among nations by having high quality The quality of education output is affected by learning quality (Moore & Kearsley, 2011; Nightingale & O’Neil, 2012; Sims, Dobbs, & Hand, 2002). The quality is determined by prerequisite conditions of 165 © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 166 students’ mastery, learning skills, facilities, and physical and socio-emotional environment. Thus, learning process as operationalization of educational practice done by lecturer should contain high-tech and high-touch. The high- touch (the term used “authority”) is “educational tool” used by educator to touch students’ selves in educational relationship which directs to high-touch condition, in the meaning that educator’s treatment positively, constructively, and comprehensively touch students’ humanity aspects (Anderson, 2004; Gainor, Goins, & Miller, 2004; Kulchitsky, 2008; Noddings, 2013). atmosphere should be built by lecturer’s authority through engaging students in learning processes. Light, Calkins, & Cox (2009) furthermore, said that education phenomenon occurs when it grows and develops through authority actualization reflected in the way lecturer teaches in the classroom. Through this process, the lecturer can assure the development of learning situation. Based on the preliminary survey in the learning process of Mathematics education students in even semester in academic year 2015/2016 at IAIN Bukittinggi, the researcher found that the lecturer tended to show bad attitudes such as talkative, anger, and unfair, while they prefer good, friendly, smart, and kind attitudes. The results also show incompatible relation between lecturer and students because the lecturer tended to make the students inferior and passive. Moreover, students were less passionate to learn, reluctant to participate in discussion, and used to compare between one lecturer and another, thus, efficient learning environment is difficult to achieve. High-touch implementation or authority used to develop students’ personals includes acknowledgment, affection, reinforcement, guidance, directive actions, and modeling. Indicators of Success The indicators of success are presented as follow: The indicators of success are presented as follow: 90 - 100 = Very good 80 - 89 = Good 65 - 79 = Adequate 55 - 64 = Quite Good 0 - 54 = Not Good The learning process is success when students: 1. Solve the problems well 2. Ask many questions 3. Share their ideas 4. Done the task well 5. Present the result of task well 6. Pay attention, listen to, and give opinion to their peers’ work 7 Participate in giving opinion This research describes the improvement of learning process through high touch approach in Islamic education science subject and its relationship with learning outcomes of Mathematics education department students in even semester in academic year 2016/2017 at IAIN Bukittinggi in which the data are quantitative data. 7. Participate in giving opinion Setting of the Research The research was done at IAIN Bukittinggi toward Mathematics education department students in second semester in academic year 2016/2017. The samples were 35 students consisting of 8 males and 27 females. The steps in this research is named cycle in which one cycle consisted of three meetings. The researcher observed the planning, implementation, observation, and reflection activities. The reflection in the first cycle was used to take further action in the next cycle. Technique of Data Collection The students were coming from many different backgrounds including educational, socio-economic, and cultural backgrounds gathering in Islamic education science course class. To collect the data, the researcher used both direct observation and observation sheet in each cycle. The observation was done to observe the class atmosphere, learning process, students’ activities, and the results were written in observation sheet. Tests were also used at the beginning and the end of meeting. Test is a series of questions used to measure students’ competence. The tests were INTRODUCTION On the other hand, high-tech (education) is “educational tool” used by educator for realization of learning goals achievement directed to the use of high quality technology. It includes curriculum, learning methods, learning equipment, learning environment, and learning assessment. Learning process is done not only by using appropriate materials and methods, but also in line with students’ affective and social developments to achieve purposed goals. High tech and authority should be in harmony to improve the learning process and outcome. Regarding to the phenomena above, an analysis of learning process especially the implementation of authority and its relationship with learning outcomes is needed. The research was done towards Mathematics education department students in even semester in academic year 2016/2017 at IAIN Bukittinggi. The lecturer is expected to implement authority and education in learning process based on educational science principles which are related to the learning outcomes. Accordingly, the problems are identified as follow: The study done by Loughran (2013) showed that the application of authority and educational in learning process was not done well as expected as many problems emerged in relation with mastering difficulty, low of learning skill, insufficient facilities, students’ selfhood and physical and socio-emotional environment. Accordingly, efforts to enable learning process improve through implementing authority and education in learning process (Black, Harrison, Lee, Marshall, & Wiliam, 2004; Gould, 2009; Hord, 2009; Vescio, Ross, & Adams, 2008). 1. The lecturer’s implementation of authority (high touch) in learning process g g p 2. Learning activities tended to be passive 3. Learning outcomes 4. The relationship between authority and learning outcomes Kriz (2003) stated that in order to create effective learning environment, the classroom 5. Students’ problems include less passionate, less motivated, and passive. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Darul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… 167 METHOD science course was presented in even semester in academic year 2016/2017. This research is classified into Action Research in which describes facts, conditions of learning process, or phenomena systematically, factually, and actually Arikunto, (2002); Gay & Airasian (2000) stated that descriptive research is a research done to collect the information about a phenomenon as it is. The analysis used was descriptive analysis. According to Best (1982) descriptive analysis is an analysis done to know the relationship between independent and dependent variables. The independent variable in this research was high touch, while dependent variable was learning outcomes. Cycle I In the first meeting, the writer allowed the students to study and analyze the materials by using books, accessing internet and map and identify the material to be explained, the steps of learning process: 2. Observation sheet was used to measure students’ activities during the learning process. The activities to be observed included 3. The students who asked questions both from lecturer or peers 1. The students sat in group and were allowed to learn the materials to be presented in discussion in the classroom. 4. The students who shared their ideas 5. The students who discussed with their peers 2. The lecturer controlled the discussion; the group presenting was commented by three other groups. Later general comments were allowed to be given by all students. 6. Understanding and mapping the materials 7. Presenting the results in group 8. Paying attention to peers’ presentation 9. Participating in every discussion 3. The lecturer observed the discussion, the students who actively participated in giving comment. RESULT AND DISCUSSION in essay forms in which the answer is in the form of discussion, reasoning, and exploring ideas (Arikunto, 2002). The research was done in Mathematics Education Department in even semester in academic year 2016/2017 IAIN Bukittinggi in which the focus is on improving the students’ role in learning. The research, in general included students’ activities in learning and learning outcomes. Time of Research The research was done in four months, one semester, in which the Islamic education © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 168 Instrument of the Research The instruments used in the research: 1. Students’ activities sheet containing questions and structured tasks based on the sub-chapters of Educational Bases course. The structured tasks were given both in group and individual. Procedure of the Research 4. The lecturer and students concluded the materials discussed. The research was done in two cycles in which each cycle consisted of three meetings, the steps in each cycle: 5. The lecturer re-asked the conclusion made in order to check the students’ understanding. 1. Planning 1. Planning 2. Prepare learning equipment such as syllabus and SAP 6. The lecturer delivered the next materials and resources to be used. 3. Prepare the task to be done, questions to be answered, and observation sheet In the second meeting, the class discussed educational bases materials in which the procedure was quiet the same as the first meeting. The students were asked to bring visual aids in the form of carton related to the materials, then, it is presented by groups, the steps as follow: 4. Action 4. Action 4. Action The actions done by students were studying, understanding, and analyzing the materials based on the syllabus, and presenting group works through discussion. In this step, the approach was focused on students’ center learning in order to improve learning process quality and gain better outcomes. Thus, the learning scenario was constructed in the form of SAP. 1. The students sat in group and were allowed to learn the materials to be presented in discussion in the classroom. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Darul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… 169 2. The lecturer controlled the discussion; the group presenting was commented by three other groups. Later general comments were allowed to be given by all students. 1. The lecturer controlled the discussion; the group presenting was commented by three other groups. Later general comments were allowed to be given by all students. 2. The lecturer observed the discussion, the students who actively participated in giving comment. 3. The lecturer observed the discussion, the students who actively participated in giving comment. 3. The lecturer and students concluded the materials discussed. 4. The lecturer and students concluded the materials discussed. 4. The lecturer re-asked the conclusion made in order to check the students’ understanding. 5. The lecturer re-asked the conclusion made in order to check the students’ understanding. 6. The lecturer delivered the next materials and resources to be used 5. The lecturer delivered the next materials and resources to be used 6. At the end of the third meeting, the lecturer re-asked the materials learned from the first until the third meetings and the students were asked to collect them. In the third meeting, the class discussed educational bases materials in which the procedure was quiet the same as the first meeting. The students were asked to bring visual aids in the form of carton related to the materials, then, it is presented by groups, the steps as follow: 7. Observation The observation of learning activities was done during learning process. The observer completed the observation sheet and the data collected was analyzed descriptively to show students’ activities as described in the table below: 7. Observation The observation of learning activities was done during learning process. 4. Action The observer completed the observation sheet and the data collected was analyzed descriptively to show students’ activities as described in the table below: The students sat in group and were allowed to learn the materials to be presented in discussion in the classroom. Table 3 No Learning Activities First Meeting Second Meeting Third Meeting Average 1 The students completed the task given 60 70 80 70 2 The students shared their opinion 30 35 40 35 3. The students discussed each other 40 50 60 50 4. The students presented their works 20 30 40 30 5 The students paid attention to their friends’ presentation 50 60 70 60 6 The students who did not participate 80 60 50 65 7 The students who did not care 50 60 70 60 8 The students who were not discipline 30 20 20 25 9 The students who were chatting during learning 10 15 10 12,5 Table 3 not discipline was 25 %, there was an improvement, chatting during learning was 12,5 %, there was an improvement. The data above shows that there is an improvement from the first until the third meetings in preparing the task given by the lecturer by average was 70 %, giving opinion was 35 %, discussing with other students was 50 %, presenting the works was 30 %, paying attention to others’ presentation was 60%, not participating was 65 %, not caring was 60 %, In general, there were improvements in cycle I including doing tasks, giving opinion, the way of discussion, and the way of presenting the works, but there were still many © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 170 5. The lecturer re-asked the conclusion made in order to check the students’ understanding. students did not participate during learning such as not caring and not discipline as much as 60%. g 6. The lecturer delivered the next materials and resources to be used. In specific, various strategies implemented during educational bases course affected students’ ways in learning process. These results were gotten through implementing high touch approach including affection, mildness, guidance, reinforcement, directive actions, good modeling, and student center development oriented to students’ activities. In the second meeting, the class discussed educational bases materials in which the procedure was quiet the same as the first meeting. 4. Action The students were asked to bring visual aids in the form of carton related to the materials, then, they were presented by groups, the steps as follow: The results of cycle I describe that there was an improvement in the students’ learning process although the results were not satisfying. Only some students were active and wanted to present their works. These phenomena might be caused by great number of groups and lack of preparation. 1. The students sat in group and were allowed to learn the materials to be presented in discussion in the classroom. 2. The lecturer controlled the discussion; the group presenting was commented by three other groups. Later general comments were allowed to be given by all students. 3. The lecturer observed the discussion, the students who actively participated in giving comment. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Cycle II Accordingly, the writer minimized the number of groups in cycle II in which four or five students per group. Each student was given the materials’ summary and each group mapped the concept in simple visual aid to be presented, the steps are described as follow: 4. The lecturer and students concluded the materials discussed. 5. The lecturer re-asked the conclusion made in order to check the students’ understanding. 6. The lecturer delivered the next materials and resources to be used In the first meeting, the writer allowed the students to study and analyze the materials by using books, accessing internet and map and identify educational bases to be explained, the steps of learning process: In the third meeting: 1. The students sat in group and were allowed to learn the materials to be presented in discussion in the classroom. 1. The students sat in group and were allowed to learn the materials to be presented in discussion in the classroom. 2. The lecturer controlled the discussion; the group presenting was commented by three other groups. Later general comments were allowed to be given by all students. 2. The lecturer controlled the discussion, the group presenting was commented by three other groups. Later general comments were allowed to be given by all students. 3. The lecturer observed the discussion, the students who actively participated in giving comment. 3. The lecturer observed the discussion, the students who actively participated in giving comment. 4. The lecturer and students concluded the materials discussed. 4. The lecturer and students concluded the materials discussed. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) arul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… Darul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… 171 5. The lecturer re-asked the conclusion made in order to check the students’ understanding. 6. The lecturer delivered the next materials and resources to be used At the end of the third meeting, the lecturer re-asked the materials learned from the first until the third meetings and the students were asked to collect them. Table 4. The results of the observation in cycle II are described in the table below: No Learning Activities First Meeting Second Meeting Third Meeting Average 1 The students completed the task given 70 80 90 80 2 The students shared their opinion 40 45 50 45 3. Cycle II The students discussed each other 50 65 80 70 4. The students presented their works 30 40 50 40 5 The students paid attention to their friends’ presentation 60 70 85 72,5 6 The students who did not participate 60 40 40 45 7 The students who did not care 60 70 80 70 8 The students who were not discipline 20 15 15 17,5 9 The students who were chatting during learning 10 15 10 12,5 At the end of the third meeting, the lecturer re-asked the materials learned from the first until the third meetings and the students were asked to collect them. Table 4. The results of the observation in cycle II are described in the table below: No Learning Activities First Meeting Second Meeting Third Meeting Average 1 The students completed the task given 70 80 90 80 2 The students shared their opinion 40 45 50 45 3. The students discussed each other 50 65 80 70 4. The students presented their works 30 40 50 40 5 The students paid attention to their friends’ presentation 60 70 85 72,5 6 The students who did not participate 60 40 40 45 7 The students who did not care 60 70 80 70 8 The students who were not discipline 20 15 15 17,5 9 The students who were chatting during learning 10 15 10 12,5 6. The results of the first, second, and third meetings show that less than 50% students still not participating in discussion even some other never participate from cycle I until cycle II. 7. In the first, second, and third meeting there was an improvement in not caring, less motivated due to the students’ social and cultural background. 2. In the results of the first, second, and third meetings there was an improvement in giving opinion as much as 10 % inasmuch as the lecturer stimulated and respected the students’ opinion. 8. In cycle II, there were still some students came late to the class and take much time when the lecturer allowed them to go out of the class. 3. In presenting their works, still the same students in cycle I presented the materials in cycle II. The materials were presented well because they were directed by course book provided by the lecturer, but there were lack of reasoning and concrete examples. 9. Cycle II There still some students chatted during the learning process, but later they were able to follow the process. In general, cycle II covered improvement rather in cycle I. In other words, high touch approach implementation enables students to learn better, but when it is related to learning outcomes in form of essay examination, the results are not sufficient. They still have difficulty to do reasoning or to give operational and contextual examples. They may have lack preparation to follow examination such as not used to have autonomous learning or lack of reading outside the class. 4. In participating in discussion, still the students who were active in cycle I dominated the discussion, but some students who did not participate in cycle I were able to give their opinion in cycle II. 5. In the first, second, and third meeting, the students respected their classmates who presented the materials because the lecturer directed and gave them the reward. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 172 CONCLUSION AND RECOMMENDATION Gainor, S. J., Goins, R. T., & Miller, L. A. (2004). Using online modules in a multi- modality teaching system: A high-touch, high-tech approach to geriatric education. Gerontology & Geriatrics Education, 24(4), 45–59. Regarding to the findings of the research, it is concluded that the high touch approach implemented by teacher through affection, mildness, guidance, directive actions, and good modeling influenced students’ attitude in learning. The lecturer allows students to develop their creativity in independent learning skill, self-learning activities and student center, learning involved reward and punishment, respects students, and forms the class in U letter form, and gives tasks for students although the maximum achievement is not yet gained. Gay, L. R., & Airasian, P. (2000). Educational research: Competencies for. Analysis and Application,(6th Edition). Ohio: Prentice Hall. Gould, J. M. (2009). Understanding organizations as learning systems. Strategic Learning in a Knowledge Economy, 119–140. Hord, S. M. (2009). Professional learning communities. Journal of Staff Development, 30(1), 40–43. Based on the findings, the writer expects that the lecturer varies the learning through high touch approach. Although this research cannot be generalized, if the case and phenomenon is quiet the same, the approach may help lecturer during teaching learning process. Kriz, W. C. (2003). Creating effective learning environments and learning organizations through gaming simulation design. Simulation & Gaming, 34(4), 495–511. For policy makers, they need to complete learning medium and facilities to enable lecturers to improve their potentials. For the next researcher, further research is needed to improve learning process quality in order to help students to gain better learning outcomes. Kulchitsky, J. D. (2008). High-tech versus high-touch education: Perceptions of risk in distance learning. International Journal of Educational Management, 22(2), 151–167. Light, G., Calkins, S., & Cox, R. (2009). Learning and teaching in higher education: The reflective professional. Sage. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) REFERENCES Anderson, T. (2004). Towards a theory of online learning. Theory and practice of online learning, 2, 109–119. Loughran, J. (2013). Developing a pedagogy of teacher education: Understanding teaching & learning about teaching. Routledge. Arikunto, S. (2002). Prosedur suatu penelitian: Pendekatan praktek. edisi revisi kelima. Jakarta: Rineka Cipta. Moore, M. G., & Kearsley, G. (2011). Distance education: A systems view of online learning. Cengage Learning. Black, P., Harrison, C., Lee, C., Marshall, B., & Wiliam, D. (2004). Working inside the black box: Assessment for learning in the classroom. Phi Delta Kappan, 86(1), 8– 21. Nightingale, P., & O’Neil, M. (2012). Achieving quality learning in higher education. Routledge. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) arul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… 173 Noddings, N. (2013). Caring: A relational approach to ethics and moral education. Univ of California Press. Vescio, V., Ross, D., & Adams, A. (2008). A review of research on the impact of professional learning communities on teaching practice and student learning. Teaching and Teacher Education, 24(1), 80–91. Sims, R., Dobbs, G., & Hand, T. (2002). Enhancing quality in online learning: Scaffolding planning and design through proactive evaluation. Distance Education, 23(2), 135–148.
https://openalex.org/W3045031169
https://www.frontiersin.org/articles/10.3389/feduc.2020.00125/pdf
English
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Perceived Teacher Support and Communication in Strategizing Possible Selves
Frontiers in education
2,020
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Perceived Teacher Support and Communication in Strategizing Possible Selves Rebeca Mireles-Rios1* and Shadi Roshandel2 1 University of California, Santa Barbara, Santa Barbara, CA, United States, 2 University of California, East Bay, Hayward, CA, United States 1 University of California, Santa Barbara, Santa Barbara, CA, United States, 2 University of California, East Bay, Hayward, CA, United States During high school, adolescents develop future plans for their lives and profession and teachers are generally believed to play an important role. At present, however, there is limited evidence about exactly how this support affects students. This study uses possible-selves theory to explore the link between perceived teacher support and communication about the future to adolescents’ own hoped-for and feared possible selves. Surveys and structured interviews were used to gather views of 85 Latina/o and White students (Mage = 15.5 years). Results indicate that greater perceived support and better communication from teachers were both associated with adolescents expressing more concrete strategies for achieving all types of possible selves. This study offers initial evidence about the important role teachers may play in supporting and shaping how adolescents construct an action plan that will lead to their desired future selves. Specialty section: Specialty section: This article was submitted to Educational Psychology, a section of the journal Frontiers in Education Received: 10 January 2020 Accepted: 25 June 2020 Published: 21 July 2020 Edited by: Michael S. Dempsey, Boston University, United States Reviewed by: Patrick J. Carroll, The Ohio State University, United States Doug Hamman, Texas Tech University, United States Keywords: possible selves, perceived teacher support, motivation, goal intention, action plan, adolescence, teacher communication INTRODUCTION Throughout adolescence, there is a dramatic increase in cognitive skills, allowing students to think more concretely about their future (Piaget, 1964), and focus on who they are and who they want to become (Erikson, 1968). The construct of “possible selves” has been used to help understand how adolescents think about their future (Markus and Nurius, 1986; Roshandel and Hudley, 2018). Specifically, possible selves are components of the self-concept and are defined as both positive and negative mental representations of the self in the future which are constructed from their past and current perceptions of themselves (Markus and Nurius, 1986). They may include hoped-for selves (what one hopes to be), expected selves (what one expects to be), and feared selves (what one wants to avoid being) (Markus and Nurius, 1986). Each of these has been shown useful to help motivate adolescents to achieve a goal or avoid a fear they may have for their future (Markus and Nurius, 1986; Zhu and Tse, 2015). Possible selves are more proximal in nature and may serve as stronger behavioral motivators for attaining future selves than more ambiguous, distal goals for oneself (Oyserman et al., 2011). *Correspondence: Rebeca Mireles-Rios rebeca.rios@ucsb.edu ORIGINAL RESEARCH published: 21 July 2020 doi: 10.3389/feduc.2020.00125 Citation: Gollwitzer (1993, 1999) discusses the value of understanding implementation intentions of goals, which they define as the intentions to form a plan to meet a goal. Research has found that the connections created by implementation intentions facilitate goal attainment (Gollwitzer et al., 2004; Duckworth et al., 2011; Gollwitzer and Oettingen, 2011). Thus, by including strategies to the possible selves concept, we can further understand why some individuals successfully achieve desired selves and avoid feared selves, whereas others with similar expectations and fears do not manage the same outcome (Clinkinbeard and Murray, 2012). However, empirical research has yet to fully explore the contexts that facilitate strategy development. As high school represents an essential time in transitioning into adulthood, it is an optimal environment to foster the development of these critical strategies. Current research on possible selves, has linked the content of one’s identities to the behaviors or strategies individuals develop in order to achieve a desired expected self or avoid a feared possible self (Zhu and Tse, 2015). Possible selves have been associated with improved educational attainment (Destin and Oyserman, 2009), more gainful employment (Lee and Oyserman, 2009), and reduced riskiness in sexual behavior (Clark et al., 2007). However, there is limited research on how adolescents develop these strategies in order to influence their current behaviors to reach a desired outcome. During high school, adolescents have the potential to develop effective strategies that will help them achieve a goal, change their behavior, or take action to avoid a fear as they are further developing future-oriented thoughts (Markus and Nurius, 1986; Clinkinbeard and Murray, 2012; Zhu and Tse, 2015). High school is also a critical time as adolescents are making plans about their future and transitioning into their autonomous selves (National Research Council, 2013). In order to help adolescents develop effective strategies for changing their current behavior to achieve their possible selves, it is important to understand the contextual factors, such as support and interactions with teachers, which contribute to such processes. If students perceive support from supportive social contexts (e.g., teacher) during uncertain situations (e.g., starting a new internship or planning for their future after high school), they are more likely to explore new opportunities and feel less anxious (Gamboa et al., 2013). Citation: Mireles-Rios R and Roshandel S (2020) Perceived Teacher Support and Communication in Strategizing Possible Selves. Front. Educ. 5:125. doi: 10.3389/feduc.2020.00125 Research shows that some patterns of possible selves are universal, while others are sensitive to context and culture, resulting in individual differences in the processes related to possible selves (Cross and Markus, 1991; Knox et al., 1998). Oyserman et al. (2011) found that children in more disadvantaged neighborhoods were more likely to have school-focused possible identities than July 2020 | Volume 5 | Article 125 1 Frontiers in Education | www.frontiersin.org Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel children in less disadvantaged neighborhoods. More recently, Roshandel and Hudley (2018), identified gender differences in high school adolescents’ college-going possible selves, with females who perceived their teachers as more supportive being more likely to aspire and expect to attend a 4-year college compared to males. Turcios-Cotto and Milan (2013) found that Latin/o youth were less likely to picture themselves attending college, when compared to Black and White youth. Taken together, these studies highlight the importance of accounting for individual differences, such as gender and ethnicity, which may contribute to one’s possible selves. allowing one to have many possible selves (Oyserman et al., 2015). Being able to distinguish between expectations and fears for the future is critical in the development of possible selves as there is a belief that the current self is malleable, facilitating a confidence that change is possible (Oyserman et al., 2004). For the purpose of this study, we will use the term possible selves to refer to any future orientation, but expected selves and feared selves to distinguish between positive and negative future possible selves. According to research, adolescents with the most developed possible selves understand the necessary relationship between future expectations and fears, and have identified strategies in place to facilitate the achievement–avoidance of those selves (Oyserman and Markus, 1990; Oyserman et al., 2004). Strategies that are associated with expected and feared possible selves may be the most critical piece of future planning and the element that is most closely linked to actual behavior change (Oyserman et al., 2006; Clinkinbeard and Murray, 2012). Ajzen (1985) asserts that the type of goal chosen and the commitment to that goal are important determinants in whether an individual carries out the behaviors necessary for goal attainment. Perceived Teacher Support and Communication Possible selves research indicates that individuals often look to others as models for the development of their future selves (Oyserman and Fryberg, 2006; Anders and Olmstead, 2018). Communicating positive feedback from others fosters hope and has the potential to increase an individual’s self-efficacy and self- esteem, whereas negative feedback is associated with negative affect and lowered expectations that may stifle attempts to change or further develop (Ruvolo and Markus, 1992; Cross and Markus, 1994). One of the primary sources of this type of communication during high school are teachers (Clinkinbeard and Murray, 2012). Given the connections between feedback and behavior, it is theoretically possible that how teachers support and communicate with students can serve as a potential social resource to the development of strategies that guide students to achieving their expected selves and/or avoiding their feared selves. Citation: Research highlighting the predictors of effective planning can ultimately guide teachers in facilitating and designing curriculum and programs that enhance adolescents’ effective future-oriented thinking and the production of strategies (Oyserman et al., 2004; Dunkel and Kerpelman, 2006; Zhu and Tse, 2015). Yet, thus far, there is limited insight into how perceived teacher support and communication may be related to the development of adolescents’ strategies to achieve a hoped for and expected possible selves while avoiding their feared possible selves. This study explores the relationship between adolescents’ perceptions of their teachers support and communication and their possible selves. We examine not only their types of expectations and fears, but also the strategies they develop to meet the desired outcomes. Strategies Findings show that having strategies represents an “action plan” to any possible selves (Oyserman et al., 2004). Clinkinbeard and Murray (2012) make a distinction between “concrete” and “abstract” strategies by defining a “concrete strategy” as one that could be repeated by another person. One example of a concrete strategy to deal with an adolescent’s fear of dropping out of school would be to “take detailed notes in my math class,” as compared to an abstract strategy of “change my habits.” If this “action plan” is more concrete, adolescents have specific next steps in figuring out how to achieve their goal (Clinkinbeard and Zohra, 2012; Anders and Olmstead, 2018). These strategies thus represent the motivational resource most closely tied to changing behavior (Oyserman et al., 2004; Anders and Olmstead, 2018). Gollwitzer et al. (2004) discuss that the first step for implementation intention is to identify the goal and then plan actions that make the goal achievable. If we are more cognizant of adolescents’ possible selves, then teachers can provide them with the support of developing these concrete strategies that will increase the likelihood of adapting current behaviors to achieve the desired future goals. Thus, it is imperative to explore the role of perceived teacher support and communication in providing opportunities to develop the skillset of forming specific strategies related to possible selves. In order to provide an innovative approach to understanding the complex research agendas, this study used a mixed-methods approach to explore adolescents’ perceptions of support, by integrating both numerical data (e.g., survey) and text data (e.g., interviews) (Creswell, 2014). Specifically, an explanatory sequential design was chosen, where we initially collected and analyzed survey data, followed by qualitative data to help us better understand the quantitative findings (Mertens, 2007; Creswell, 2014; Creswell and Clark, 2017). The Present Study The present study explores how teachers may serve as a social resource to support the cognitive development of concrete strategies to help adolescents address their possible selves. First, we want to understand the types of possible selves and strategies high school students report. In terms of the strategies we also want to explore if these strategies are categorized as concrete or abstract. Secondly, we want to understand how perceptions of teacher support may be associated with the aforementioned strategies. We hypothesize when adolescents perceive more teacher support, they are more likely to have concrete strategies to deal with their possible selves. Specifically, we hypothesize that greater perceived teacher communication about the future will predict greater use of concrete over abstract strategies for both expected and feared possible selves. MATERIALS AND METHODS Participants and Procedures A total of 85 (41 males and 44 females) adolescents between the ages of 14 to 17 (M = 15.5, SD = 0.885) were recruited to participate in a study focusing on their thoughts about their future. Twenty-seven percent of the participants were in 9th grade, 42% were in the 10th grade, and 31% were in the 11th grade. For the purpose of this study, only results from participants who identified as White and Latin/o students were analyzed, as only a few participants identified as “other.” This ethnic breakdown of the students matched the overall school site demographics (45% White and 55% Latin/o). The school site has a 40% population of free or reduced lunch. Distinction Between Expected and Feared Possible Selves Teachers also create learning environments that provide students with resources and the space to explore how their current behaviors may impact their future goals that they Unlike general and ambiguous goals and fears, possible selves exist in any future domain (e.g., academic, social, and health), July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 2 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel may not receive otherwise (Strati et al., 2017; Roshandel and Hudley, 2018). Studies show that when adolescents perceive their teachers as supportive, they report higher levels of interest and engagement in their school work (Watson et al., 2016), more positive academic attitudes and values (Fredricks et al., 2019), and more academic engagement (Wentzel et al., 2016). In addition, from an adolescent perspective, it is not only interacting with their teachers that is vital but rather what the teachers are communicating (Elhay and Hershkovitz, 2019). Thus, these studies together confirm the significant impact perceived teacher support and communication has on adolescents’ academic engagement and motivation. experiences. Martinez and Castellanos (2018) found that students who were more engaged in school activities had greater opportunity to develop relationships with teachers who shared their own academic experiences with students, helping the students to carve out their own college trajectories. This finding supports prior research which argues that aspirations to attend college are not solely based on motivation, but can also be driven by school context and relationships with teachers, which can give students the confidence they need to make their aspirations a reality (Gutman and Akerman, 2008; Martinez and Castellanos, 2018). In addition, such future aspirations, can play a significant role in impacting current academic behavior and achievement. Given that teachers may influence students’ academic achievement and school engagement (Wentzel et al., 2016), it is theoretically possible to expect a relationship between perceived teacher support and adolescents’ strategies for achieving or avoiding their possible selves. The more we understand how teachers can influence students’ development of strategies through communication and support, the better we can prepare teachers to create the optimal learning environment that focuses on academics as well as development of the whole adolescent. Frontiers in Education | www.frontiersin.org Types of Strategies for Possible Selves Instructions and questionnaire matrices were adopted from Oyserman and James (2011). Participants were asked to write in up to four strategies for each expected possible selves (“What are doing to be that way in the future?”) and four strategies for each feared possible selves (“What are you doing to avoid being that way in the future?”). Academic Achievement Weighted grade point averages (GPA) were collected from official school records. Survey Measures Demographics Students were asked the following questions: Do your teachers talk about your future? What are the ways that teachers support you in the classroom to help you prepare for the future? Can you give concrete examples? The researchers told students they could answer the questions based on any teacher. The authors developed semi-structured interviews in order to focus on themes that would emerge rather than elicit specific answers about perceived teacher support and communication. Participants completed a questionnaire that included their age, grade level, and race/ethnicity. Students wrote in their age, checked offtheir grade (9th–12th), gender (male or female) and were asked their race/ethnicity (Latin/o, White, Asian, Black, and Other). Each category had the option to specify. For example, many students that checked Latin/o, wrote in Mexican-American. After students took the survey, they were asked if they were willing to participate in a 30-min interview. Thirty students (approximately 50% female, 50% male, 50% White, and 50% Latina/o) were interviewed at a separate time and location for the second phase of the study. The interviews took place either afterschool or at lunch, in a quiet and uninterrupted location at the school site. Parent consent and adolescent assent was received. Two graduate student researchers, who were trained and familiar with the study, facilitated the interviews. Again, participants were reminded that their answers would remain confidential. Students did not receive incentives to participate in any stage of the research. Types of Possible Selves Instructions and questionnaire matrices were adopted from Oyserman and James (2011). Participants were asked to write in up to four expected (“What are your expectations for the future?”) and four feared possible selves (“What are your fears for the future?”). Analytical Procedures Using categories identified from previous studies (e.g., Oyserman et al., 2004), the authors coded participants’ responses to the possible selves questionnaire into six categories: achievement (school or work related activities), relationships (social interactions with others), individual (personality), health (physical wellness), lifestyle (material possessions), and risky behavior (antisocial activity). Since students were able to mark more than one response, the authors used counts (ranging from 0 to 4) to measure each category. The authors of the paper coded the first 20 participants’ responses of possible selves and strategies together and then separately each coded the remainder 65 entries. They then compared coding and had an overall inter-rater agreement rate across the six categories of types of possible selves and strategies, ranging between 92 and 97% (Saldana, 2009). Any discrepancies were resolved through discussion. School Engagement Adolescents reported on an adapted version of a 3-item School Engagement Scale (Gonzales et al., 2014). Participants responded to the following items using a 4-point Likert scale ranging from “I strongly disagree” to “I strongly agree:” “I like to do well in school,” “It is very important to finish high school,” and “It is very important to go to college.” The ratings were summed to create a student engagement score. The scale had an alpha score of 0.87. School Engagement Researchers have also explored the relationship between perceived teacher support and educational outcomes such as academic engagement, effort, expectations, motivation, and achievement. Findings consistently indicate a positive relationship between teacher support and a myriad of advantageous outcomes, such as academic achievement, motivational outcomes, and educational aspirations (Wentzel, 1997, 1998; Wentzel et al., 2016; Roshandel and Hudley, 2018). For example, teachers can foster confidence in students by serving as role models by sharing their own academic Four general education, social studies classrooms representing the school demographics were identified and selected by the school administration to participate in the study. The overall teacher demographics of the school included 85% White, 12% Latin/o, 2% Asian, and 1% Black, with 56% female. For this study, Frontiers in Education | www.frontiersin.org July 2020 | Volume 5 | Article 125 3 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel Perceived Teacher Support and Communication We measured perceived informational support and perceived instrumental support (Malecki and Demaray, 2002; Roshandel and Hudley, 2018). A composite was created for the four-item measure of instrumental support (e.g., “My teachers explain things that I don’t understand”) and the four-item measure of informational support (e.g., “My teacher helps me solve problems well by giving me information”). We added the single-item question, “My teacher talks to me about my future plans.” Each item was analyzed separately in order to capture the various types of support teachers may provide and communicate. the demographics included three female teachers (two White, one Latina) and one male teacher (White). Students were given consent and assent forms, and those who returned the completed forms, received the survey the next class period. Two graduate student researchers and three undergraduate research assistants, who were trained and familiar with the study, administered the open-ended questionnaire during class time. All students were given instructions about the questionnaire and were reminded that their answers would remain confidential. Similar to previous studies in this field, students took approximately 30 min to complete the questionnaire (Elmore and Oyserman, 2012). The University’s Institutional Review Board approved this study. Frontiers in Education | www.frontiersin.org Types of Possible Selves Reported All participants identified at least one expected and one feared possible selves. Participants produced a total of 165 expected possible selves and 243 feared possible selves. At least one of each of the expected and feared possible selves focused on the achievement category, comprising of 52% of the total expected possible selves and 35% of the total feared possible selves. For example, an achievement expected possible selves was “I will go to college.” An achievement feared possible selves was “I am afraid I won’t get into a good university.” For the other categories of expected and feared possible selves and examples, see Table 2. Concrete and Abstract Strategies Measured variables Overall Male Female White Latina/o M (SD) M (SD) M (SD) M (SD) M (SD) School engagement (scale 0–4) 2.8 (0.9) 2.8 (0.6) 2.9 (0.6) 3.0 (0.6) 2.7 (0.5) Teacher support: instrumental (scale 0–4) 3.1 (0.6) 3.1 (0.5) 3.1 (0.6) 3.1 (0.5) 3.1 (0.5) Teacher support: informational (scale 0–4) 2.9 (0.8) 2.8 (0.7) 3.2 (0.6) 3.1 (0.7) 2.9 (0.7) Teacher communication: future (scale 0–4) 2.5 (0.8) 2.6 (0.8) 2.7 (0.7) 2.6 (0.8) 2.6 (0.7) Expected PS types Achievement 2.3 (0.8) 2.4 (0.7) 2.1 (0.8) 2.1 (0.8) 2.4 (0.8) Relationship 0.25 (0.5) 0.20 (0.4) 0.29 (0.5) 0.36 (0.5) 0.20 (0.2) Individual 0.29 (0.6) 0.13 (0.4) 0.41 (0.7) 0.36 (0.7) 0.20 (0.5) Health 0.18 (0.4) 0.03 (0.2) 0.02 (0.2) 0.0 (0) 0.04 (0.2) Lifestyle 0.51 (0.6) 0.53 (0.7) 0.48 (0.6) 0.49 (0.6) 0.54 (0.6) Risky behavior 0.02 (0.5) 0.0 (0) 0.05 (0.2) 0.05 (0.2) 0.0 (0) Feared PS types Achievement 0.89 (0.8) 0.85 (0.8) 0.82 (0.8) 0.88 (0.9) 0.84 (0.8) Relationship 0.03 (0.6) 0.23 (0.5) 0.36 (0.6) 0.26 (0.5) 0.33 (0.6) Individual 0.22 (0.6) 0.21 (0.6) 0.25 (0.5) 0.13 (0.4) 0.31 (0.7) Health 0.26 (0.5) 0.23 (0.5) 0.25 (0.5) 0.31 (0.6) 0.18 (0.4) Lifestyle 0.55 (0.6) 0.59 (0.8) 0.48 (0.5) 0.46 (0.6) 0.60 (0.7) Risky behavior 1.3 (0.9) 1.2 (0.9) 1.3 (0.9) 1.3 (0.8) 1.2 (0.9) Frequency expected PS strategies Achievement 2.0 (1) 2.2 (0.9) 1.9 (0.9) 1.9 (0.8) 2.1 (1) Relationship 0.29 (0.5) 0.20 (0.5) 0.34 (0.6) 0.33 (0.5) 0.22 (0.4) Individual 0.21 (0.5) 0.18 (0.4) 0.25 (0.6) 0.23 (0.5) 0.19 (0.5) Health 0.26 (0.5) 0.28 (0.5) 0.21 (0.5) 0.26 (0.4) 0.24 (0.5) Lifestyle 0.17 (0.4) 0.20 (0.4) 0.16 (0.4) 0.28 (0.5) 0.08 (0.3) Risky behavior 0.03 (0.2) 0.03 (0.2) 0.05 (0.2) 0.05 (0.2) 0.02 (0.1) Frequency feared PS strategies Achievement 1.0 (0.9) 1.1 (0.8) 0.91 (0.9) 0.89 (0.9) 1.1 (0.9) Relationship 0.20 (0.4) 0.13 (0.3) 0.27 (0.5) 0.28 (0.5) 0.13 (0.3) Individual 0.30 (0.4) 0.20 (0.5) 0.36 (0.6) 0.28 (0.6) 0.28 (0.5) Health 0.30 (0.5) 0.38 (0.6) 0.23 (0.5) 0.38 (0.6) 0.22 (0.4) Lifestyle 0.16 (0.4) 0.15 (0.4) 0.14 (0.3) 0.05 (0.2) 0.22 (0.5) Risky behavior 0.92 (0.8) 1.0 (0.8) 0.91 (1) 0.85 (0.8) 1.1 (0.9) Frequency of expected concrete strategies 0.9 (0.8) 1.1 (0.8) 0.7 (0.9) 0.95 (0.9) 0.87 (0.8) Frequency of expected abstract strategies 2.1 (0.1) 1.9 (1) 2.2 (1) 2.1 (1) 2.1 (1) Frequency of feared concrete strategies 1.3 (0.1) 1.4 (1) 1.2 (1) 1.0 (0.8) 1.5 (1) Frequency of feared abstract 1.6 (0.1) 1.6 (1) 1.5 (1) 1.7 (1) 1.4 (1) TABLE 1 | Means (SD) associated with measured variables. Concrete and Abstract Strategies conducting content analysis, created a category scheme by coding five transcripts together. We used an inductive procedure, adapting the category scheme to the content of the data. About 5% of the coding was discrepant, meaning that we agreed on 95% of the thought units in the text overall. We resolved the discrepancies through discussion. All transcripts including the training interviews were included in the analysis. Measured variables School engagement (scale 0–4) Teacher support: instrumental (scale 0–4) Teacher support: informational (scale 0–4) Teacher communication: future (scale 0–4) Expected PS types Achievement Relationship Individual Health Lifestyle Risky behavior Feared PS types Achievement Relationship Individual Health Lifestyle Risky behavior Frequency expected PS strategies Achievement Relationship Individual Health Lifestyle Risky behavior Frequency feared PS strategies Achievement Relationship Individual Health Lifestyle Risky behavior Frequency of expected concrete strategies Frequency of expected abstract strategies Frequency of feared concrete strategies Frequency of feared abstract strategies Finally, in order to gain a better understanding of adolescents’ possible selves, descriptive statistics were analyzed to identify the types of possible selves and the strategy types identified. Correlations and t-tests were run to distinguish any associations and group differences between variables. The means and standard deviations associated with academic motivation, perceived teacher support and communication, types of possible selves and strategies, and concrete and abstract strategies are presented in Table 1. Types of Strategies for Possible Selves Reported 2.0 (1) 2.2 (0.9) 1.9 (0.9) 1.9 (0.8) 2.1 (1) 0.29 (0.5) 0.20 (0.5) 0.34 (0.6) 0.33 (0.5) 0.22 (0.4) 0.21 (0.5) 0.18 (0.4) 0.25 (0.6) 0.23 (0.5) 0.19 (0.5) 0.26 (0.5) 0.28 (0.5) 0.21 (0.5) 0.26 (0.4) 0.24 (0.5) 0.17 (0.4) 0.20 (0.4) 0.16 (0.4) 0.28 (0.5) 0.08 (0.3) 0.03 (0.2) 0.03 (0.2) 0.05 (0.2) 0.05 (0.2) 0.02 (0.1) p Similar to the types of possible selves, the strategies identified by participants were categorized. The majority of participants (95%) provided at least one strategy for each type of possible selves. Achievement strategies were identified for the majority of the possible selves, consisting of 54% of strategies for expected selves and 32% for feared selves. Many students used achievement strategies to address their various types of fears. For example, a student with a risk behavior fear (getting pregnant in high school or getting involved in a gang, ID #77) reported an achievement strategy (focus on school, ID #77). In addition, fears of being homeless, living with parents, and living in poverty were also addressed with strategies to stay in school, further their education, graduate and go to college. For the other categories and examples of strategies identified for expected and feared possible selves, see Table 3. Concrete and Abstract Strategies We used Clinkinbeard and Murray (2012) definition of concrete strategies to categorize how students would address their given expectation or fear. A concrete strategy was one that could be replicated by another person. For example, “taking detailed notes” is a concrete response that others could repeat. All other responses were coded as abstract (e.g., “do better in school”). These codes were dichotomously coded. All audiotaped interviews were transcribed and a content analysis was used to discover patterns in the text related to the frequency of occurrence of particular themes (Krippendorff, 2018). The authors who had previous experience July 2020 | Volume 5 | Article 125 4 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel TABLE 1 | Means (SD) associated with measured variables. Concrete vs Abstract Strategies Concrete vs Abstract Strategies We also examined whether the strategy produced was concrete or abstract. The majority (60%) of strategies identified by participants for expected selves were abstract, whereas for feared selves, the strategies identified were evenly distributed among concrete (49%) and concrete (51%). See Table 3 for specific examples. July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 5 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel TABLE 2 | Types of possible selves by categories. Category type Expected selves Feared selves Percentage Examples Percentage Examples Achievement 52 “I expect to join the Marine Corp Reserves” (ID #63) 35 “I am afraid of dropping out of school” (ID# 73) Relationships 12 “I expect to be maintaining a great relationship with my family and friends” (ID #6) 9 “I am afraid of becoming too serious/detached from those I love” (ID# 68) Individual 11 “I expect to be happy and fulfilled” (ID #11) 5 “I am afraid of being cocky or arrogant” (ID# 32) Health 1.5 “I expect to stay active and healthy, stay in shape and eat properly” (ID #37) 7 “I am afraid of being overweight” (ID# 9) Lifestyle 22 “I want to be traveling the world” (ID #85) 16 “I am getting sucked into social media” (ID# 10) Risky Behavior 1.5 “I expect to be drug-free” (ID #49) 28 “I am afraid of getting pregnant in high school” (ID# 39) TABLE 3 | Types and examples of strategies for possible selves by categories. Concrete vs Abstract Strategies Category type Expected selves Feared selves Total percentage Concrete Examples Abstract Examples Total Percentage Concrete Examples Abstract examples Achievement 54 “I’m enrolling in as many computer science electives as possible” (ID# 14) “I’m keeping good grades” (ID# 67) 32 “I currently have a job so I can build my resume” (ID# 91) “I am never going to give up” (ID# 81) Relationships 13 “I’m going to college to and joining a business club to network with people” (ID# 19) “I’m planning on keeping in touch with people” (ID# 50) 9 “By spending a lot of time with my family and keeping a strong bond” (ID# 68) “I will try to not hang out with bad friends” (ID# 27) Individual 9 N/A “I’m working hard to stay happy and positive.” (ID# 48) 11 “I do my best not to underestimate people or come to conclusions based on appearances” (ID# 32) “I will stay true and pure to myself” (ID# 23) Health 13 “I’m going to gym and exercise every day.” (ID# 6) “I’m going to be healthy and fit.” (ID# 2) 12 “I work out every day” (ID# 9) “I will stay aware” (ID# 64) Lifestyle 9 “I’m going to open a bank and save $10 a week.” (ID# 55) “I’m living life each moment at a time.” (ID# 43) 6 “I read a lot of books” (ID# 10) “Do everything in my power to not fail in life” (ID# 69) Risky behavior 2 N/A “I’m not going to do drugs.” (ID# 78) 30 “I make sure to focus on school and not boys” (ID# 39) “Don’t do anything bad” (ID# 88) TABLE 2 | Types of possible selves by categories. Correlations Among the Measured Variables also varied significantly with the number of concrete feared strategies produced, t(82) = 2.23, p = 0.02, d = 0.19. Results also indicated a significant difference between those who reported at least one concrete strategy and those who did not. Those who reported a concrete strategy reported more school engagement compared to individuals who did not report a concrete strategy (M = 2.6, SD = 0.62), t(83) = −1.83, p = 0.05, d = 0.49. Finally, results of whether teachers did discuss their future or did not significantly varied between those who reported at least one concrete strategy and those who did not (M = 2.28, SD = 0.94), t(83) = −2.37, p = 0.05, d = 0.37. In other words, the more teachers communicated about the future, the more students’ reported concrete strategies. No other significant differences were found between groups and concrete and abstract strategies. Correlations were analyzed among the demographic variables, concrete and abstract strategies, and perceived teacher support and communication variables (see Table 4). T-tests were also conducted to examine group differences among significant variables. Demographic Variables Results indicated a significant difference in GPA between White students (M = 3.62, SD = 0.89) and Latina/o students (M = 2.96, SD = 0.8), t(83) = 3.61, p = 0.001, d = 0.79. No other significant differences were found between demographic variables. Concrete and Abstract Strategies Perceived Teacher Support and Communication Results indicated significant differences in those who perceived their teachers as providing more informational support between males (M = 2.83, SD = 0.71) and females (M = 3.19, SD = 0.63), Perceptions of Teachers’ Communication About the Future Perceptions of Teachers’ Communication About the Future One of the most common themes that emerged was how teachers communicate about students’ future in terms of going to college. Findings from the interviews indicated that most of their teachers did not discuss adolescents’ future. One participant stated “We don’t really talk about other things. Like the future doesn’t come up. Sometimes she’ll mention college, but then we never talk about it.” Other participants also discussed the college going process, and most participants interviewed felt that one of the reasons their teachers did not discuss their future was that they did not necessarily know much about the process of getting into college. For example, one participant said, “I think they just focus on their class, what they’re teaching. I don’t think they really know the college steps, just know what they’re going to teach and that’s their subject.” Similarly, another participant said, “I think she [the teacher] only knows science. We never talk about anything else.” I think it’s just that, like, like, he really does help me and I can come in at lunch and, like, he always says that we’re welcomed in the classroom. Like he’ll actually be there for us, I think it’s just that reassurance that he will actually help us, and, like, he’ll actually be there for us, is what helps. (Interview #7) The teacher will say, “oh, you missed this quiz. You can take it, like, on this day or during lunch or after school,” like, she gave me options. And then, like, if I miss an assignment or something, she’ll, like, tell me what I missed or check my syllabus and kind of, like, explain it. (Interview #5) Students also reported that it was during outside of class time that they would get more support and communication about ways to strategize and problem solve. For example, one student mentioned, Results also indicated that while across the board, students did not express detailed communication about the future, only the White students (9 out of 15) reported that their teachers encouraged them to go to college. I, um, really struggled with coming to class sometimes, and when we sat down at lunch he asked me to really think about, um, why I have been slacking. Perceptions of Teachers’ Communication About the Future Once I admitted that I didn’t understand the work, we came up, um, we talked about some ways I could ask questions to help me do the, um, the work and stuff. (Interview #22) Perceived Teacher Support and Communication She always answers questions and provides examples for each lesson that she gives us and we write notes but she writes them down while we write them, too and what to study. And sometimes she gives us, um, booklets that we can write down little notes like equations and stufffor the test. Um, that helps a lot, and he answers questions very well and always gives, like, real life examples. (Interview#4) Perceived Teacher Support and Communication The frequency of feared concrete strategies were significantly different between White (M = 1.0, SD = 0.79) and Latina/o (M = 1.52, SD = 1.13) students, t(83) = −2.42, p = 0.02, d = 0.54. Males (M = 1.41, SD = 0.98) and females (M = 1.23, SD = 0.97) July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 6 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel TABLE 4 | Correlations among measured variables (n = 85). 1 2 3 4 5 6 7 8 9 10 11 1. Grade level 2. Gender 0.05 3. Race 0.07 −0.12 4. GPA −0.03 0.07 −0.37** 5. Abstract strategies for expected selves −0.05 0.15 −0.04 0.01 6. Concrete strategies for expected selves −0.08 −0.25* −0.5 −0.01 −0.42** 7. Abstract strategies for feared selves −0.05 −0.02 −0.16 −0.03 −0.04 0.8 8. Concrete strategies for feared selves −0.02 −0.07 0.26* −0.07 0.29** 0.06 −0.38** 9. Academic engagement −0.37** 0.05 −0.11 0.28** −0.06 0.20 −0.05 0.03 10. Teacher support: instrumental −0.36** −0.12 0.07 0.07 0.14 0.12 −0.21 0.25** 0.24* 11. Teacher support: information −0.16 0.26* 0.17 0.06 0.02 0.11 −0.14 0.08 0.25* 0.37** 12. Teacher support: future −0.13 −0.63 0.14 −0.19 0.08 0.24* −0.07 0.26* 0.14 0.22* 0.29** *p < 0.05, **p < 0.01, and ***p < 0.001. They explain things well, um, they’ll write things in the board, and they’ll make sure you have your notes out and make sure you’re taking down your notes, um, they’ll be free to, like, e-mail with you if you have questions or be in at lunch to see if you need any help. I think there are some teachers like that here on campus. (Interview #12) t(81) = −2.46, p = 0.02, d = 0.54. That is, female students were more likely than their male counterparts to perceive that their teachers provided them with informational support. These quantitative results for this study give us a glimpse into adolescents’ possible selves and the types of strategies they believe will help them achieve an expected selves and avoid a feared selves. We explicitly get to see students use of abstract and concrete strategies. Lastly, we see how specific types of perceived support from teachers is associated with the various components of students’ possible selves. Interview Results The following themes emerged from the interviews. In addition, other participants mentioned how important it was for them to have additional time with teachers. To many students, offering time beyond regular class time signals a form of caring. Through this time in the classroom, four students found this space to help with academics. Two students mentioned, Various Forms of Support Another common trend that emerged was the different types of support students perceived from teachers. For example, July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 7 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel strategies significant for possible selves irrespective of being expected or feared. Finally, having high expectations also emerged as an important way that students perceived their teachers as supportive. One student said, Further supporting past research, we found that adolescents have a variety of possible selves regarding their future selves. Similar to previous studies, the types of possible selves produced reaffirm the variety of goals and identities adolescents are focusing on during this time. Participants from our study identified more feared possible selves than expected possible selves. This speaks to the uncertainty of adolescence during a transitional period of becoming an autonomous self. In addition, findings indicated that the majority of expected selves were related to achievement goals, which support how possible selves are more proximal in nature (e.g., students are currently in school) (Oyserman et al., 2011). Sometimes I used to think he was just out to get us like the other teachers. But then I realized, he really wanted us to do well. He would push us, um, to think, and um, do things we’ve never tried. (Interview#20) Similarly, another student talked about the importance of providing the expectation of a safe space for students to learn. He stated, When some kid tell them that they want to really learn and they don’t kick out the students, or you know, actually talk to the students that aren’t listening, you’re not supporting your other students that actually want to learn. (Interview #6) We also found that regardless of the expectation or fear, the majority of types of strategies students reported were achievement focused. Although it may seem obvious to list achievement strategies given that data was collected in a school setting, it is important to note that even students, who were not as academically engaged as their peers, still identified education as a protective factor for achieving some non-academic possible selves. This is important in our understanding of how to best support students in the classroom, as adolescents recognize that school may play a significant role in guiding students toward creating plans to meet their future goals or avoid their fears. Various Forms of Support School may serve as the blueprint to action plans adolescents develop (Oyserman et al., 2004, 2006). Based on these findings, it is apparent that students felt when they teachers gave them specific ways of doing something (take notes or ask questions) they felt they were not only supported but also that they teacher cared and knew what they were doing. Relevancy and Relatability The final theme that emerged was how students reported the importance of having teachers that can be relatable. For example, She’ll, um, just make sure everybody is doing their notes and she we’ll make class fun, like, if kids are talking she’ll just pretend to join in the conversation and be, like, “oh, that’s cool, what happened next?” Like, she makes it really fun and enjoyable. (Interview #14) Findings also indicated the significance of concrete strategies versus abstract ones. Specifically, we found that adolescents who produced at least one concrete strategy were more engaged and perceived their teachers as more supportive. This supports previous studies that more specific action plans serve as a motivational resource that are closely associated with changing behaviors in the present that may impact adolescents’ futures (Gollwitzer et al., 2004; Oyserman et al., 2004; Duckworth et al., 2011; Gollwitzer and Oettingen, 2011; Anders and Olmstead, 2018). Therefore, if adolescents have specific action plans (i.e., concrete strategies) to meet their expected possible selves or avoid their feared possible selves, they are more likely to have the next steps to achieve their goals (Clinkinbeard and Zohra, 2012; Anders and Olmstead, 2018). She teaches real life, I would say. Everything has an application, like especially in the virtual enterprise or other classes I’ve taken from her, like business applications and computer business. It’s just they all have real-life applications, like from writing checks, to making a balance sheet, to typing up a profit and loss statement. I feel like I can really use this information when I get out of here. (Interview #11) Such findings provide a glimpse into how high school students perceive support from their teachers and the way they communicate can impact their future goals. Furthermore, one contributing finding of this study is that students who perceived their teachers as supportive by communicating about their futures, provided more concrete strategies to deal with their possible selves, indicating that teachers may play a role in adolescents’ possible selves-strategy elaboration. That is, teachers have the potential to guide adolescents with the support of developing these concrete strategies that will increase the likelihood of adapting current behaviors to achieve the desired future goals. Our study found that when teachers talked to students about their future, the more concrete strategies they had to address their range of possible selves. Frontiers in Education | www.frontiersin.org DISCUSSION that when teachers themselves provided more concrete forms of support (e.g., accessible after class, teaching specific methods, providing relevant examples) versus abstract forms of support (e.g., available if needed, general comments), adolescents reported they felt more supported and trusted the expertise of their teachers. These results exemplify the types of support that are necessary for teachers to provide in order to support adolescents in the classroom and with their future goals. For example, having a teacher available during lunch to work with a student on specific methods to take notes to help them study for a test is more beneficial than a teacher saying they are available if a student needs it. These types of support model how teachers can show students the importance of creating concrete versus abstract strategies. The association between perceptions of teacher communication about the future and concrete strategies opens up future research to recognize and explore this relationship. That is, when students know how to come up with achievement strategies that are concrete, they are likely to address not only their achievement expectations and fears but also their risk behavior, health, individual, relationship and lifestyle possible selves. This may be particularly useful given that all students reported at least one risk behavior fear which was associated with more abstract solutions. If teachers can help guide students into developing concrete plans of action, they can indirectly address these types of fears that may not emerge in the classroom setting. Given that concrete strategies associated with future-oriented expectations and fears may be the most important pieces of future planning and are likely linked to actual behavior change (Gollwitzer et al., 2004; Oyserman et al., 2006; Clinkinbeard and Murray, 2012), our findings that perceived teacher support was associated with students concrete and abstract strategies further upholds previous research that students’ perceptions of teacher communication styles influences their motivation (Lin et al., 2016). Previous work on the achievement and opportunity gap have identified differences across gender and ethnicity in regards to student achievement (Boykin and Noguera, 2011; Carter and Welner, 2013). Our findings indicated that the only significant difference with GPA was between Whites and Latinas/os. DISCUSSION While it was expected that school engagement would be linked to GPA, our findings indicated no differences between GPA and the other variables and regardless of academic performance, adolescents recognized that their current behavior in school plays a vital role in their future goals. Therefore, teachers could use the context of possible selves as a motivator to engage all students by making content relatable and relevant to adolescents’ lives. Adolescents can then be motivated to engage with their teachers and in turn develop concrete strategies through communication and support. Even though previous research shows the importance and benefits of having concrete strategies, the majority of students identified abstract solutions for their expected selves. Given that there were significant group differences in GPA between White and Latina/o students, a possible solution for addressing this apparent achievement gap might be to focus on fostering teacher communication about the future and helping students to develop more concrete plans for their future. Interview data suggested that adolescents could benefit from their teachers sharing concrete examples from their own experiences as a way of providing more specific forms of support. Interestingly, when it came to feared possible selves, adolescents reported approximately 50% abstract and 50% concrete strategies to deal with their fears. Perhaps adolescents’ motivation to avoid their fears facilitates the development of more concrete strategies. These results also suggest that students in this study recognized that their current behavior in school plays a vital role in their future goals. Our research also indicates that youth are reporting that doing well in school as a form of resisting and coping with their fears, suggesting that they value education as a tool for creating a stable future. This is particularly significant because adolescents are able to move from concrete to abstract thinking and planning, making it possible to think about their futures (Piaget, 1964). In addition, as they are getting older they are developing critical thinking skills. It has been argued that critical thinking is a higher order mental function influenced by social factors and performed within social contexts which sometimes makes transferring what happens in a classroom setting to a real world setting challenging (Lee, 2018). By teaching students about concrete strategies, teachers could be providing relevant context for developing critical thinking skills that may help bridge this gap. DISCUSSION This study contributes to the possible selves research by exploring adolescents’ possible selves and the role of perceived teacher support as it is related to helping adolescents build strategies for achieving their expected selves and/or avoiding their feared selves. Based on our hypotheses, we found that when adolescents perceived more teacher support, they were more likely to have concrete strategies to deal with their possible selves. Although we hypothesized that greater perceived teacher communication about the future will predict greater use of concrete over abstract strategies for both expected and feared possible selves, we found significant results for concrete strategies in relation to possible selves more broadly. That is, we found the use of concrete Interview data provided us with deeper insight on how these perceived teacher support variables were related to how adolescents think about their future as well as the strategies they develop. Specifically, results from the interviews showed July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 8 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel times less likely to be encouraged by their teachers than their White counterparts (Witenko et al., 2016). These findings may be attribute to the mismatch demographics between students and teachers, given that the majority of teachers were White (Sleeter, 2001; Picower, 2009). Additionally, compared to females, males had more concrete strategies. For example, females perceived more informational support from teachers, compared to males. Previous studies show that teachers spend more time providing girls with more academic and informational support or that girls value this type of support more than males (Roshandel and Hudley, 2018). Teachers may also have different approaches to different types of students, as previous studies have indicated that teachers may have their own biases for students’ future paths (Boykin and Noguera, 2011). Together these findings indicate that based on individual differences, students may have different needs for the types of support they desire as well as different types of support they need in terms of reaching a desired outcome (Roshandel and Hudley, 2018). Horowitz et al. (2020) suggest that in some school contexts, teachers are able to provide the guidance needed for students while other teachers are not. This calls for further exploration around differences in race/ethnicity and gender in the development of possible selves and perceptions of teacher support. Frontiers in Education | www.frontiersin.org Implications for Teacher Communication With Adolescents Many studies focusing on possible selves recommend the need to understand the relationship between support systems and the development of concrete strategies (Oyserman et al., 2004; Oyserman et al., 2006; Clinkinbeard and Murray, 2012; Clinkinbeard and Zohra, 2012; Zhu and Tse, 2015). This study examines the associations with teachers, offering a new lens in viewing teacher communication and the classroom context as a space for “intervening” in the development of strategies for adolescents to develop their future selves. For example, by making teachers aware that achievement strategies are an important coping mechanism for adolescents when dealing with their fears, teachers can be more mindful about teaching students how to set goals and create “action plans” even within content area instruction. Hearing from the students provided insight into how they viewed their teachers’ knowledge about the future and ways that teachers can help support students in their development of strategies applicable to their possible selves. Despite needing a college degree to be a teacher, many students did not think that teachers know the route to college. One suggestion would be for teachers to share their journey to the profession with their students so they can help understand the steps needed for different types of careers. This could help students develop their own future road maps. Additionally, students mentioned the significance of teachers making time at lunch and afterschool to help them with their work. This space lends itself for opportunities for teachers to connect with students. Strategies associated with future-oriented expectations and fears may be the most important pieces of future oriented visualization and planning that are linked to actual behavior change (Ajzen, 1985; Oyserman and Markus, 1990; Gollwitzer and Oettingen, 2011). This may be because of a personal connection and relevancy to adolescents’ lives. Therefore, the findings from this study begin to shed light on the role teacher’s play in supporting and shaping how all adolescents can construct an action plan that will lead to the desired outcomes. Given that preservice and in service teacher research focuses on the development of 21st century skills (Urbani et al., 2017; Roshandel and Hudley, 2018), our findings might suggest to teachers that the use of possible selves and development of strategies could be a way of teaching adolescents higher order cognitive skills, such as critical thinking and creativity. DISCUSSION Interview data also revealed that Latina/o students were less likely to discuss being encouraged by their teachers to attend college than their White counterparts. Compared to White students, Latina/o students had more concrete strategies to address their possible selves. Similarly, a recent study examining who encourages students to enroll in advanced classes, even when controlling for a GPA, Latina/o students were over 5 By communicating with adolescents to think about their future, teachers have the opportunity to help with the July 2020 | Volume 5 | Article 125 9 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel development of concrete strategies to create tangible solutions to deal with their fears and help develop their future expectations. This is particularly telling, as much of the research on teacher support has focused on teachers working with younger students, often because high school teachers specialize in content and not necessarily the whole child (Noddings, 2012). Gehlbach et al. (2012) also discuss that early adolescence is a critical period for the teacher-student relationship; however, in comparison to elementary school, the interactions are less personal and frequent (Eccles et al., 1993). Pianta and Allen (2008) argue that as adolescents look to non-parental adults for social connections, these relationships are perhaps the most important factor for promoting positive youth development. Expanding the research around possible selves to the student-teacher relationship in secondary school may help students not only with their academic fears but in other arenas in their lives. development but also the possible enhancing of student self- efficacy in their own ability to generate an action plan. CONCLUSION Within possible selves literature, there is limited research exploring the changes of possible selves within one life stage. As high school is a time of dramatic developmental changes, in particular with the ability to plan and visualize their futures (Boyd and Bee, 2012), this may serve as an optimal time for adolescents’ possible selves-strategy elaboration. This current study expands research on the significant role that teachers can play to aid adolescents in the development of concrete solutions. Through instructional practices that can span across all content areas to the development of personal “action plans” we can provide teachers with practical ways to help students. Findings provide insight into the way students perceive how their teachers communicate and provide academic and emotional support which is associated with their development of concrete strategies. Better understanding the relationship of teacher communication about the future to the provision of feedback for the future can have a significant effect on the development of their future identities and cognitive skills. Limitations and Suggestions for Future Research It is important to note the limitations of this study. Given the small sample size, we were only able to explore how these variables are associated. The brevity of the survey did not allow us to get an in depth understanding the specific ways perceived teacher support can aid in the development of concrete strategy building for adolescents. Hearing from teachers about their experiences around awareness and action taken around addressing adolescents’ fears and strategy building would advance ways to best support students and teachers. More research is necessary to comprehend the relationship between sources of support and the contexts in which strategies and behavior change develops in adolescence. 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Cogn. 10, 95–124. doi: 10.1521/soco.1992. 10.1.95 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Saldana, J. (ed.) (2009). “An introduction to codes and coding,” in The Coding Manual for Qualitative Researchers (Thousand Oaks, CA: SAGE Publications). Copyright © 2020 Mireles-Rios and Roshandel. 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. Sleeter, C. E. (2001). Preparing teachers for culturally diverse schools: research and the overwhelming presence of whiteness. J. Teach. Educ. 52, 94–106. doi: 10.1177/0022487101052002002 Copyright © 2020 Mireles-Rios and Roshandel. 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. Strati, A. D., Schmidt, J. A., and Maier, K. S. (2017). Perceived challenge, teacher support, and teacher obstruction as predictors of student engagement. J. Educ. 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A new insect trackway from the Upper Jurassic—Lower Cretaceous eolian sandstones of São Paulo State, Brazil: implications for reconstructing desert paleoecology
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How to cite this article Peixoto BCPM, Mángano MG, Minter NJ, dos Reis Fernandes LB, Fernandes MA. 2020. A new insect trackway from the Upper Jurassic—Lower Cretaceous eolian sandstones of São Paulo State, Brazil: implications for reconstructing desert paleoecol- ogy. PeerJ 8:e8880 http://doi.org/10.7717/peerj.8880 A new insect trackway from the Upper Jurassic—Lower Cretaceous eolian sandstones of São Paulo State, Brazil: implications for reconstructing desert paleoecology Bernardo de C.P. e M. Peixoto1,2, M. Gabriela Mángano3, Nicholas J. Minter4, Luciana Bueno dos Reis Fernandes1 and Marcelo Adorna Fernandes1,2 1 Laboratório de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil 2 Programa de Pós Graduacão em Ecologia e Recursos Naturais, Centro de Ciências Biológicas e da Saúde, 1 Laboratório de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva, Universida Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil 3 Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 3 Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 4 School of the Environment, Geography, and Geosciences, University of Portsmouth, Portsmouth, Hampshire, United Kingdom ABSTRACT The new ichnospecies Paleohelcura araraquarensis isp. nov. is described from the Upper Jurassic-Lower Cretaceous Botucatu Formation of Brazil. This formation records a gigantic eolian sand sea (erg), formed under an arid climate in the south-central part of Gondwana. This trackway is composed of two track rows, whose internal width is less than one-quarter of the external width, with alternating to staggered series, consisting of three elliptical tracks that can vary from slightly elongated to tapered or circular. The trackways were found in yellowish/reddish sandstone in a quarry in the Araraquara municipality, São Paulo State. Comparisons with neoichnological studies and morphological inferences indicate that the producer of Paleohelcura araraquarensis isp. nov. was most likely a pterygote insect, and so could have fulfilled one of the ecological roles that different species of this group are capable of performing in dune deserts. The producer could have had a herbivorous or carnivorous diet or been part of the fauna of omnivores, being able to adopt herbivorous, carnivorous, and saprophagous diets when necessary. In modern dune deserts, some species of pterygote insects are detritivores (like Tenebrionidae), relying on organic matter that accumulated among the sand grains of the dunes during dry periods with no plant growth. The presence of additional burrows suggests that the Botucatu paleodesert would have had a detritivorous fauna like this. Based on the interpretation of the ichnofossil producers, it was possible to reconstruct the food web of this paleodesert. All the omnivorous and herbivorous invertebrates and the herbivorous ornithopod dinosaurs made up the primary consumers. These animals were, in turn, the food source for bigger carnivorous or omnivorous animals unable to feed on detritus, like arachnids, possible predatory insects, mammaliaforms, and theropod dinosaurs. The highest trophic level was occupied by larger theropod dinosaurs and mammaliaforms, which, because of their size, could prey upon a wide range of animals. The producer of Paleohelcura araraquarensis isp. nov. could have been a primary consumer if it were an omnivorous Submitted 6 November 2019 Accepted 10 March 2020 Published 22 May 2020 Corresponding author Bernardo de C.P. e M. Peixoto, b.peixoto@protonmail.com Academic editor Claudia Marsicano Additional Information and Declarations can be found on page 26 DOI 10.7717/peerj.8880 Copyright 2020 Peixoto et al. Distributed under Creative Commons CC-BY 4.0 Submitted 6 November 2019 Accepted 10 March 2020 Published 22 May 2020 Corresponding author Bernardo de C.P. e M. 1 Laboratório de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil 2 Programa de Pós Gradua¸cão em Ecologia e Recursos Naturais, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil 3 Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 4 School of the Environment, Geography, and Geosciences, University of Portsmouth, Portsmouth, Hampshire, United Kingdom 2 Programa de Pós Gradua¸cão em Ecologia e Recursos Naturais, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil ABSTRACT Peixoto, b.peixoto@protonmail.com Academic editor Claudia Marsicano Additional Information and Declarations can be found on page 26 DOI 10.7717/peerj.8880 Copyright 2020 Peixoto et al. Copyright 2020 Peixoto et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS detritivore or a herbivore, or a secondary consumer if it were produced by a predatory insect or an omnivore relying on animal biomass. The description of this new trackway expands the knowledge on the faunal composition of the Botucatu paleodesert and provides insights into the ecological relationships in ancient deserts. The presence of these arthropod trackways in Mesozoic eolian deposits helps to trace a continuity between Paleozoic and post-Paleozoic desert ichnofaunas, further reinforcing a single Octopodichnus—Entradichnus Ichnofacies for eolian deposits. detritivore or a herbivore, or a secondary consumer if it were produced by a predatory insect or an omnivore relying on animal biomass. The description of this new trackway expands the knowledge on the faunal composition of the Botucatu paleodesert and provides insights into the ecological relationships in ancient deserts. The presence of these arthropod trackways in Mesozoic eolian deposits helps to trace a continuity between Paleozoic and post-Paleozoic desert ichnofaunas, further reinforcing a single Octopodichnus—Entradichnus Ichnofacies for eolian deposits. Subjects Biodiversity, Ecology, Ecosystem Science, Entomology, Paleontology Keywords Deserts, Erg, Palaeoecology, Trophic web, Gondwana, Ichnofacies, Botucatu Formation, Paraná Basin Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 INTRODUCTION The Botucatu Formation, a stratigraphic unit of the Paraná Basin, is the testament of a gigantic sand desert (erg) that existed from the Late Jurassic to the Early Cretaceous in the south-central part of the supercontinent Gondwana, totaling an area of 1.5 × 106 km2, encompassing parts of Brazil, Argentina, Uruguay, Paraguay, Namibia and South Africa (Scherer & Goldberg, 2007). Ichnofossils are the only evidence of animal life in this ancient desert because no animal body fossils have been found. Therefore, trace fossils play a central role in understanding animal diversity and ecological relationships in this ancient erg. Eolian deposits have been traditionally considered of minor interest from an ichnologic perspective. However, this situation has changed at an accelerated rate in recent years with the publication of several papers on the topic (e.g., Ekdale, Bromley & Loope, 2007; Ekdale & Bromley, 2012; Good & Ekdale, 2014; Krapovickas et al., 2016; Carmona, Ponce & Wetzel, 2018; Xing et al., 2018; Buatois & Echevarría, 2019; Marchetti et al., 2019a; Marchetti et al., 2019b). A recent review emphasized the complex pattern of trace-fossil distribution in eolian and related facies, the debate surrounding definition of an archetypal ichnofacies, and delineation of macroevolutionary trends in desert environments (Krapovickas et al., 2016). Documentation of trace fossils in desert successions is of paramount importance to provide support to these models, and to help clarify the diagnostic characteristics of the so-called Octopodichnus-Entradichnus Ichnofacies. Several trackways of tetrapods and arthropods have been recovered from eolian dune deposits of different ages (McKeever, 1991, table 1). There has been contention over the preservation potential of such trackways in ‘dry’ dune deposits and whether they had to have been produced subaqueously (Brand, 1979; Brand & Tang, 1991; McKeever, 1991; Brand, 1992; Lockley, 1992; Loope, 1992). Moisture (Mckee, 1947; Sadler, 1993) and the presence of clay minerals (Loope, 1986; McKeever, 1991) between sand grains have both been proposed to play a role in trackway stabilization and preservation potential. Experiments have demonstrated that the combination of the two can lead to enhanced survivorship of arthropod trackways over those made in dry sand or sand with just surface moisture or the presence of clay minerals alone (Davis, Minter & Braddy, 2007). Nevertheless, it has been Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 2/37 recently argued that special conditions are not necessarily needed to preserve such trace fossils (see Davies & Shillito, 2018). Previous work Most of the previous studies in the Botucatu Formation focused on tetrapod trackways, with only two publications dealing in detail with invertebrate trace fossils (Fernandes, Netto & Carvalho de, 1988; Fernandes, Carvalho & Netto, 1990). Before these studies, invertebrate trackways were only mentioned within the context of vertebrate ichnofaunas as the source of food for the presumed mammaliaform producer of Brasilichnium elusivum (Leonardi, 1981, p. 803). Subsequently, Leonardi (1984, p. 54) illustrated invertebrate trackways identical to those documented in this study, but whose repository is unknown. Arthropod trackways from São Bento Quarry (Araraquara city—São Paulo State) were later illustrated as well by Leonardi & Sarjeant (1986, p. 83), but no further information regarding a repository was provided. Leonardi, Carvalho de & Fernandes (2007) reported trace fossils of insects and arachnids in Araraquara (São Bento Quarry), but no illustrations were provided. Fernandes (2005) identified arachnid tracks in slabs from São Bento Quarry, and interpreted them as made by scorpions and spiders. Peixoto et al. (2016) documented new findings of Taenidium serpentinum and Skolithos linearis, probably produced by insects. The occurrences of invertebrate ichnofossils are summarized in Table 1. The only plant fossils from the Botucatu Formation are conifer trunks, found in the region of Araguari (Minas Gerais State), north of the Tringulo Mineiro, within the limits of the sandstone occurrence area of this unit (Pires et al., 2011; Malaquias, Riff & Riff, 2017). Those trunks exhibit xylophagous marks assigned to termites (Isoptera) and beetles (Coleoptera) (Riff, Kloster & Riff, 2017). With respect to the vertebrate trace-fossil record of the Botucatu Formation, there are two ichnospecies of Brasilichnium produced by small mammaliaform organisms: one demonstrating cursorial locomotion described as B. elusivum (Leonardi, 1981; Fernandes & Carvalho, 2008), and the other one in hopping locomotion (D’Orazi Porchetti, Bertini & Langer, 2017a), described as B. saltatorium (Buck et al., 2017b). There is also a record of a burrow compatible with the Brasilichnium elusivum producer (Manes, Da Silva & Scheffler, 2017). There is some controversy in describing new ichnotaxa based on differences in locomotion patterns instead of objective morphological attributes of the footprints alone (Lockley, 2007; Minter, Braddy & Davis, 2007). Nevertheless, the presence of a hopping behavior (e.g., B. saltatorium) is useful in indicating a biomechanical capability that constrains eligible clades of possible producers, together with when this biomechanical capability appeared. INTRODUCTION The aims of this study are to: (i) describe the new ichnospecies, Paleohelcura araraquarensis isp. nov., which consists of trackways produced by pterygote insects walking on sand dunes; (ii) discuss the implications of this record with respect to ecological relationships within the Botucatu paleodesert; and (iii) assess its importance for our understanding of eolian dune ichnofacies. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Notes. SP, São Paulo State (Brazil); MG, Minas Gerais State (Brazil). Ichnofossils descriptions were translated literally from the original sources. Modified from Leonardi, Carvalho de & Fernandes (2007). Ichnofossils descriptions were translated literally from the original sources. Modified from Leonardi, Carvalho de & Fernandes (2007) Previous work Trackways of mammaliaforms larger than the producer of Brasilichnium elusivum have been described independently as Brasilichnium anaitti (D’Orazi Porchetti, Bertini & Langer, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 3/37 3/37 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Table 1 Occurrence of invertebrate ichnofossils from the Botucatu Formation and the first references describing them. Locality Ichnofossil Description Reference Quarry 3–4 km from São Carlos (SP) (probably Migliato or Araújo quarry) ‘‘Worm Tunnels’’ Pacheco & De Amaral (1913) Sierra of Botucatu (SP) ‘‘Worm tubes.’’ Almeida (1954) Pacaembú neighborhood, São Carlos (SP) ‘‘Worm trails.’’ Bjornberg & Tolentino (1959) São Tomás Ranch quarry, Ibaté Municipality (SP) ‘‘Fossil tracks of conchostracans(?)’’ Paraguassu (1970) Quarry near Araraquara (SP) ‘‘Vermiform trails and tracks of arthropods.’’ Leonardi (1980) ‘‘Arthropods trackways’’ (Leonardi, 1984) ‘‘Arthropod trails’’ (Leonardi & Sarjeant, 1986) ‘‘Invertebrate trackways and burrows’’ (Leonardi & Godoy, 1980) (...)Ten rare forms of invertebrate trails, mainly attributable to arthropods (arachnids and insects, adults or larvae)(...) (Leonardi, Carvalho de & Fernandes, 2007) São Bento Quarry, Araraquara (SP) Insects, scorpions and spiders (Fernandes, 2005). Itagua¸cu Farm Quarry, São Carlos (SP) ‘‘Trails of vermiform invertebrates.’’ Leonardi & Godoy (1980) Campo Minado Cave, Sierra of Itaqueri, Ipeúna (SP) Taenidium serpentinum and Skolithos linearis Peixoto et al. (2016) Sobradinho Farm, Araguari (MG) Burrows of xylophagous termites and Coleoptera insects in conifer wood. Riff, Kloster & Riff (2017) Notes. SP, São Paulo State (Brazil); MG, Minas Gerais State (Brazil). Ichnofossils descriptions were translated literally from the original sources. Modified from Leonardi, Carvalho de & Fernandes (2007). 4/37 4/37 2017b) and as Aracoaraichnium leonardii (Buck et al., 2017a). These two ichnotaxa bear several morphological similarities and were described from slabs reposited in different scientific collections. In addition, theropod and ornithopod dinosaur trackways have been recorded (Leonardi, 1979; Leonardi, 1980; Leonardi & Godoy, 1980; Leonardi, 1981; Leonardi, 1984; Leonardi & Sarjeant, 1986; Leonardi, 1987; Leonardi et al., 2002; Fernandes, 2005; Leonardi, Carvalho de & Fernandes, 2007; Francischini et al., 2015). Also noteworthy is the rare occurrence of an urolite, a biogenic mark interpreted as the result of the liquid extrusion of urine from dinosaurs onto unconsolidated sediment (Fernandes, Fernandes & Souto, 2004). Geological setting The Botucatu Formation is exposed in the Brazilian states of Mato Grosso, Mato Grosso do Sul, Goiás, Minas Gerais, São Paulo, Paraná, Santa Catarina, and Rio Grande do Sul with the same sedimentary system extending into Argentina, Uruguay, Paraguay, Namibia and South Africa, covering an area over 1.5 × 106 km2 (Scherer & Goldberg, 2007). In São Paulo State, the Botucatu Formation outcrops as a northeast-southwest strip (Fig. 1), with monotonous deposits mostly consisting of yellowish to reddish, very fine- to coarse-grained sandstone, mainly quartz arenite and subordinately subarkose. The quartz arenite is texturally and mineralogically supermature, whereas the subarkose is texturally submature to mature and mineralogically mature (Wu & Caetano-chang, 1992). The consensus is that the Botucatu Formation represents a giant dry eolian depositional system (erg) based on the presence of large to medium-sized cross-stratified sandstones (Scherer & Goldberg, 2007) (Fig. 2), and on the basis of the mineralogical and textural maturity of the dominant deposits (Wu & Caetano-chang, 1992). The landscape was dominated by linear, crescentic and some star dunes, representing a hyperarid system, according the classification framework of Mountney (2004), with winds predominantly coming from the north in the northern part of the Paraná Basin (Scherer & Goldberg, 2007) where the study area is located (Araraquara City). Fluvial/eolian sandstone of the Pirambóia Formation occurs below the Botucatu Formation in the northern portion of the basin (State of São Paulo) (Milani et al., 2007, p. 287; Soares, Soares & Holz, 2008a, their fig2). The contact between these two formations is still controversial (Giannini et al., 2004, p. 282; Soares, Soares & Holz, 2008b, their fig. 2; p.126) (Fig. 3). The Botucatu Formation is overlain by the magmatic extrusive rocks of the Serra Geral Group (former Serra Geral Formation) (Milani et al., 2007; Fernandes et al., 2018) (Fig. 3). Lenses of eolian sandstone (paleodunes) in the Serra Geral Group indicate that the eolian depositional system was active during volcanism. The Botucatu Formation and the Serra Geral Group have a concordant contact because the flow of lava over the unconsolidated sand of the paleodunes created marks on the paleodune surfaces (e.g., striations, crescentic ridges), formed breccias (peperites), and also preserved the relief of the ancient dunes (Milani et al., 1998; Scherer, 2000; Scherer, 2002; Waichel et al., 2007; Holz, Soares & Soares, 2008; Waichel, Scherer & Frank, 2008). Geological setting U–Pb baddeleyite/zircon dating for the lowest sub-unit of the Serra Geral Group that makes concordant contact with Botucatu Formation in São Paulo State (Chapecó-type Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 5/37 Figure 1 The localization of Araraquara City, where the fossils were collected, and outcrop area of the Botucatu and Pirambóia formations in the State of São Paulo, Brazil. Modified from Fernandes, Fernan- des & Souto (2004). Full-size DOI: 10.7717/peerj.8880/fig-1 Figure 1 The localization of Araraquara City, where the fossils were collected, and outcrop area of the Botucatu and Pirambóia formations in the State of São Paulo, Brazil. Modified from Fernandes, Fernan- des & Souto (2004). Full-size DOI: 10.7717/peerj.8880/fig-1 Figure 1 The localization of Araraquara City, where the fossils were collected, and outcrop area of the Botucatu and Pirambóia formations in the State of São Paulo, Brazil. Modified from Fernandes, Fernan- des & Souto (2004). Full-size DOI: 10.7717/peerj.8880/fig-1 dacites) yields an age of approximately 134 Ma (Janasi, Freitas & Heaman, 2011). This radiometric date, together with the oldest paleomagnetic date of the Botucatu Formation from southern Brazil (Tamrat & Ernesto, 2006) indicates a Late Jurassic to Early Cretaceous age for this unit. dacites) yields an age of approximately 134 Ma (Janasi, Freitas & Heaman, 2011). This radiometric date, together with the oldest paleomagnetic date of the Botucatu Formation from southern Brazil (Tamrat & Ernesto, 2006) indicates a Late Jurassic to Early Cretaceous age for this unit. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 MATERIALS & METHODS Paleontological material analyzed Paleontological material analyzed The ichnofossils described here were collected between 1997 and 2005, at São Bento quarry (21◦49′07.6′′S48◦04′28.8′′W), in the municipality of Araraquara (São Paulo State). All the ichnofossils here described were found during the commercial exploitation of the successive layers of sandstone of the slipface of a single paleodune (that was more than 100 m long and 20 m high) of the Botucatu Formation (Fig. 2. The full-size figures are in Figs. S1 and S2). The paleodune slipface dips at 29◦in the S-SW direction. The São Bento quarry is currently inactive and is part of the Ouro Ichnofossiliferous Site, in Araraquara (Leonardi et al., 2002), a region with several abandoned ichnofossiliferous quarries. The sandstone slabs containing the ichnofossils here analyzed were collected from sites A, C and D (sensu Fernandes, 2005) of the São Bento quarry and are: LPP-IC-0028, LPP-IC-0029, LPP-IC-0030, LPP-IC-0031, LPP-IC-0032, LPP-IC-0033, LPP-IC-0034, LPP-IC-0035. Sandstone extraction at the São Bento quarry was done without scientific monitoring, and most of the fossils were in slabs ready to be cut, or already cut for commercialization. Therefore, no data is available regarding orientation of specimens with respect to the slopes. All of these slabs are deposited in the Paleoichnology Collection of the Laboratório de Paleoicnologia e Paleoecologia (LPP) of the Federal University of São Carlos (UFSCar), São Carlos campus. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 6/37 Figure 2 Representative drawing of the location of the work fronts of São Bento quarry. Photographs 1, 2, 3 and 4 are the work fronts rock outcrop in March 2004, during the commercial exploitation of the Botucatu sandstone and collection of all ichnofossils here described. (A), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), (L) are the relative location of the sites of occurrence of ichnofossils, as described by Fernan- des (2005). The invertebrate ichnofossils occur in sites A, C, and D, in the work front 1. At present, the quarry is inactive, and the outcrop looks different because of the further exploitation and weathering. Drawing is not in scale (modified from Fernandes, 2005). The full-size image is in the Supplemental Infor- mation. Full-size DOI: 10.7717/peerj.8880/fig- Figure 2 Representative drawing of the location of the work fronts of São Bento quarry. Photographs 1, 2, 3 and 4 are the work fronts rock outcrop in March 2004, during the commercial exploitation of the Botucatu sandstone and collection of all ichnofossils here described. Paleontological material analyzed (A), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), (L) are the relative location of the sites of occurrence of ichnofossils, as described by Fernan- des (2005). The invertebrate ichnofossils occur in sites A, C, and D, in the work front 1. At present, the quarry is inactive, and the outcrop looks different because of the further exploitation and weathering. Drawing is not in scale (modified from Fernandes, 2005). The full-size image is in the Supplemental Infor- mation. Full-size DOI: 10.7717/peerj.8880/fig-2 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Full-size DOI: 10.7717/peerj.8880/fig-2 The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 7/37 7/37 Figure 3 Simplified stratigraphic column showing the lithology, relative age and contact relationships between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size DOI: 10.7717/peerj.8880/fig-3 The LSID for this publication is: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B- 75856AEAEAF5. The LSID for the here described Paleohelcura araraquarensis isp. nov. is: urn:lsid:zoobank.org:act:7D4303AE-BB63-4474-B79C-AD39AB144917. The online Figure 3 Simplified stratigraphic column showing the lithology, relative age and contact relationships between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size DOI: 10.7717/peerj.8880/fig-3 Figure 3 Simplified stratigraphic column showing the lithology, relative age and contact relationships between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size DOI: 10.7717/peerj.8880/fig-3 Figure 3 Simplified stratigraphic column showing the lithology, relative age and contact relationships between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size DOI: 10.7717/peerj.8880/fig-3 The LSID for this publication is: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B- 75856AEAEAF5. The LSID for the here described Paleohelcura araraquarensis isp. nov. is: urn:lsid:zoobank.org:act:7D4303AE-BB63-4474-B79C-AD39AB144917. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS The LSID for this publication is: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B- 75856AEAEAF5. The LSID for the here described Paleohelcura araraquarensis isp. nov. is: urn:lsid:zoobank.org:act:7D4303AE-BB63-4474-B79C-AD39AB144917. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trackway measurements The methodology and terminology of Trewin (1994), Braddy (2001) and Minter, Braddy & Davis (2007) for arthropod trackway description have been adopted herein (Fig. 4). For the description of preservation, the classification proposed by Seilacher (1964) is followed. For measurements of Paleohelcura araraquarensis isp. nov., four series on each side of the trackway were selected on slabs LPP-IC-0028, LPP-IC-0029, LPP-IC-0032, LPP-IC-0035. For those slabs with more continuous trackways, eight series on slab LPP-IC-0030 and nine series on slab LPP-IC-0031 were measured. The measured series are indicated in Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 8/37 Track width Series Track length Internal width External width Track 1 Track 2 Track 3 Stride Pace Anterior Posterior Left Right Figure 4 Nomenclature and measurements used for the analysis of Paleohelcura araraquarensis isp. nov. Measurements of the trackway and track characteristics. Full-size DOI: 10.7717/peerj.8880/fig-4 Figure 4 Nomenclature and measurements used for the analysis of Paleohelcura araraquarensis isp. nov. Measurements of the trackway and track characteristics. Full-size DOI: 10.7717/peerj.8880/fig-4 the photographs in Figs. S3 and S4. The series measured were chosen on the basis of the quantity and quality of the tracks on either side of the trackway. We attempted to select the series to be measured with regular distances between them along the trackway. In the slabs that show part and counterpart (LPP-IC-0029 with LPP-IC-0030 and LPP-IC-0031 with LPP-IC-0032), the same series were measured in the two slabs (i.e., negative epirelief and positive hyporelief). LPP-IC-0029 and LPP-IC-0032 present shorter trackways than their counterparts; therefore, only partial measurements were obtained. Measurements of internal and external width were taken; as were the pace, stride, and lengths and widths of the individual tracks (Fig. 4). Trewin (1994, p. 813) proposed using the ratio between the external width and the internal width as a trackway parameter; however, such a relationship is not practical in the case of Paleohelcura araraquarensis isp. nov. since there are sections of the trackways where the internal width is zero, producing fractions with zero as divisor, and it is impossible to divide by zero (Kaplan, 2000). Therefore, we adopt the inverse of the relation proposed by Trewin (1994, p. 813), that is, internal width/external width (I/E), and we propose this as a standard for the description of trackways. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trackway measurements The ratio between the length and width (L/W) of the tracks was also used to see if the tracks are circular (values close to one), or elliptical/elongated (values greater than one). The measurements were taken using a Vernier caliper. All measurements are listed in Table S1. The line drawing illustration of the holotype of Paleohelcura araraquarensis isp. nov. was produced using the Inkscape vector drawing program 0.92, whose license is free and open source (General Public License 3), using a photograph as a model for the footprint contour. All the graphic elements were produced with the aforementioned program. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 9/37 RESULTS AND DISCUSSION Ichnotaxonomy. Ichnogenus: Paleohelcura Gilmore, 1926 Type Ichnospecies: Paleohelcura tridactyla Gilmore, 1926. Type Ichnospecies: Paleohelcura tridactyla Gilmore, 1926. Emended Diagnosis: Trackways with external width greater than 20 mm, comprising two parallel track rows with series of commonly three tracks, but there can be fewer or up to four tracks per series. Series have alternating to staggered symmetry. Tracks vary from slightly elliptical to tapered or circular and can be in a linear or triangular arrangement within series. A medial impression may be present. Remarks: Historically, many arthropod trackway ichnotaxa were inadequately described and illustrated, at times based on few and/or poorly preserved specimens (Trewin, 1994, p. 821). Several trackway ichnotaxa with series of at most four tracks have been described, resulting in potential junior synonyms. Analysis of trackways from the Permian of Germany and southwestern United States (Minter, Braddy & Voigt, 2007; Minter & Braddy, 2009) showed intergradations between several ichnotaxa, underscoring morphological and preservational variations, due to small variations in locomotion or characteristics of the substrate. Paleohelcura (Gilmore, 1926) was originally described from the lower Permian Coconino Sandstone of western United States and subsequently described in other studies dealing with the ichnology of this and other units (Toepelman & Rodeck, 1936; Brady, 1939; Brady, 1947; Brady, 1961; Alf, 1968; Sadler, 1993; Braddy, 1995; Lucas & Lerner, 2004; Morrissey & Braddy, 2004; Voigt, Small & Sanders, 2005; Batchelor & Garton, 2013; Stoller, Rowland & Jackson, 2013). Only Paleohelcura tridactyla is accepted as a valid ichnospecies, and four forms are regarded as junior synonyms: P. dunbari (Brady, 1961), P. delicatula (Fischer, 1978), P. badensis Kozur, Loffler & Sittig (1994), and P.? lyonsensis (Toepelman & Rodeck, 1936), which was provisionally included in Paleohelcura when described. Sadler (1993) noted intergradations between P. tridactyla and P. dunbari, but retained them as separate ichnospecies because she considered the two ichnotaxa as morphologically different. On the contrary, it has been argued that the morphologic differences between P. tridactyla and P. dunbari are minor, with intergradations between the two, and so P. dunbari should be regarded as a junior synonym of P. tridactyla (Minter, Braddy & Davis, 2007; Minter & Braddy, 2009). Paleohelcura delicatula is only known from a single specimen that consists of comma-shaped tracks, opposite symmetry, and small size compared with P. tridactyla (Fischer, 1978). Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 RESULTS AND DISCUSSION New ichnotaxa should not ideally be erected on the basis of single specimens and the characteristics presented are not reliable to erect a new ichnospecies. Therefore, we regard Paleohelcura delicatula as a junior synonym of Stiaria intermedia. Paleohelcura badensis is regarded as a junior synonym of Stiaria intermedia (Minter & Braddy, 2009). Paleohelcura? lyonsensis has been considered a junior synonym of P. tridactyla (Braddy, 1995, p. 221). Gilmore (1927) erected Triavestigia niningeri, and Kozur, Loffler & Sittig (1994) referred it to Paleohelcura as a distinct ichnospecies; however, the holotype consists of an incomplete trackway, and the arrangement of the tracks within a Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 10/37 Table 2 Arithmetic mean of the measurements of the trackway. (I/E) Internal Width/External Width. LPP-IC-0033 and LPP-IC-0034 were not measured because of their poor preservation. Specimen External width mean (mm) Internal width mean (mm) I/E Mean Pace mean (mm) Stride mean (mm) LPP-IC-0028 22,80 2,43 0,11 12,73 12,80 LPP-IC-0029 23,43 2,70 0,11 10,78 11,30 LPP-IC-0030 22,75 1,68 0,07 5,49 10,92 LPP-IC-0031 23,22 1,41 0,06 6,13 11,75 LPP-IC-0032 22,10 1,95 0,09 10,90 11,40 LPP-IC-0035 23,55 3,11 0,10 11,18 11,27 Mean (mm) 22,97 2,21 0,09 9,53 11,57 Standard de- viation/Mean 0,05 0,57 0,62 0,14 0,11 series suggests that it is a junior synonym of P. tridactyla (Braddy, 1995, p. 221; Minter & Braddy, 2009). P l h l id l i i il S i i i di S i h 1909 (W lk 1985 Mi Table 2 Arithmetic mean of the measurements of the trackway. (I/E) Internal Width/External Width. LPP-IC-0033 and LPP-IC-0034 were not measured because of their poor preservation. series suggests that it is a junior synonym of P. tridactyla (Braddy, 1995, p. 221; Minter & Braddy, 2009). Paleohelcura tridactyla is similar to Stiaria intermedia Smith, 1909 (Walker, 1985; Minter & Braddy, 2009). Stiaria was described from continental fine-grained sediment lenses within andesites (Walker, 1985; Phillips & Smith, 2008, p. 5) from the Lower Devonian Old Red Sandstone of Scotland, and was revised by Pollard & Walker (1984), and Walker (1985), with the latter paper erecting a neotype, lectotype and paratypes not previously assigned to this ichnotaxon. Both ichnogenera may possess a medial impression and linear series with two to four tracks (Brady, 1947; Walker, 1985; Minter & Braddy, 2009). In fact, Walker (1985) suggested that Paleohelcura should be regarded, at least in part, as a junior synonym of Stiaria. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 RESULTS AND DISCUSSION Full-size DOI: 10.7717/peerj.8880/fig-5 2018), and is endorsed here. Paleohelcura araraquarensis isp. nov. is placed in Paleohelcura instead of Stiaria because it exhibits an external width greater than 20 mm. Figure 5 Holotype of Paleohelcura araraquarensis. isp. nov. (LPP-IC-0028). (A) Photograph of LPP- IC-0028 slab showing the positive hyporelief of Paleohelcura araraquarensis isp. nov. (B) Representative scheme of the holotype of Paleohelcura araraquarensis isp. nov. Lowercase letters c and d indicate exam- ples of footprint orientation within the series. The producer walked from bottom to top. Full-size DOI: 10.7717/peerj.8880/fig-5 2018), and is endorsed here. Paleohelcura araraquarensis isp. nov. is placed in Paleohelcura instead of Stiaria because it exhibits an external width greater than 20 mm. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 RESULTS AND DISCUSSION Stiaria quadripedia is a similar ichnospecies and was also revised by Walker (1985), but differs from Paleohelcura by presenting bifid or trifid tracks, which are possible to delineate in finer-grained sediments. Stiaria intermedia consists of trackways with up to three circular tracks, similar to Paleohelcura. In contrast, Stiaria quadripedia may have four tracks, and is larger than S. intermedia. Size is not regarded as an appropriate ichnotaxobase (Bertling et al., 2006), but analysis of the external widths of specimens assigned to Stiaria intermedia and to Paleohelcura tridactyla has shown that they fall into two separate size classes (Minter & Braddy, 2009). Whilst not separated by an order of magnitude, a working model was proposed (Minter & Braddy, 2009) whereby Stiaria intermedia should be used for trackways with an external width of less than 20 mm, and Paleohelcura for those with an external width greater than 20 mm, like the trackways here described (see Table 2). The clarification of the ichnotaxonomic status of Stiaria intermedia and Paleohelcura tridactyla remains to be achieved through examination of their holotypes and neotypes (Minter & Braddy, 2009). In any case, Paleohelcura is a well-accepted ichnotaxon, which has been recorded extensively. The working model proposed by Minter & Braddy (2009) has been adopted in many papers (Lucas et al., 2005; Minter & Braddy, 2009; Fillmore, Lucas & Simpson, 2010; Poschmann & Braddy, 2010; Batchelor & Garton, 2013; Getty et al., 2013; Getty et al., 2017; Bernardi, Marchetti & Gobbi, 2018; Uchman, Gazdzicki & Blazejowski, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 11/37 Figure 5 Holotype of Paleohelcura araraquarensis. isp. nov. (LPP-IC-0028). (A) Photograph of LPP- IC-0028 slab showing the positive hyporelief of Paleohelcura araraquarensis isp. nov. (B) Representative scheme of the holotype of Paleohelcura araraquarensis isp. nov. Lowercase letters c and d indicate exam- ples of footprint orientation within the series. The producer walked from bottom to top. Full-size DOI: 10.7717/peerj.8880/fig-5 2018), and is endorsed here. Paleohelcura araraquarensis isp. nov. is placed in Paleohelcura instead of Stiaria because it exhibits an external width greater than 20 mm. Figure 5 Holotype of Paleohelcura araraquarensis. isp. nov. (LPP-IC-0028). (A) Photograph of LPP- IC-0028 slab showing the positive hyporelief of Paleohelcura araraquarensis isp. nov. (B) Representative scheme of the holotype of Paleohelcura araraquarensis isp. nov. Lowercase letters c and d indicate exam- ples of footprint orientation within the series. The producer walked from bottom to top. Figures 5 and 6A. g Horizon and type locality: São Bento Group, Botucatu Formation; Locality: Ouro; municipality: Araraquara; São Paulo State (Fig. 1); São Bento quarry (Corpedras company) (Fig. 2), geographical coordinates: 21◦49′07.6′′S 48◦04′28.8′′W, altitude: 670 m. Holotype: LPP-IC-0028: sandstone slab showing slightly curved trackway, preserved in positive hyporelief over a length of 34 cm. Reposited in the Paleoichnology collection of the Laboratório de Paleoecologia e Paleoicnologia (LPP) of the Federal University of São Carlos (UFSCar) campus São Carlos-SP. Horizon and type locality: São Bento Group, Botucatu Formation; Locality: Ouro; municipality: Araraquara; São Paulo State (Fig. 1); São Bento quarry (Corpedras company) (Fig. 2), geographical coordinates: 21◦49′07.6′′S 48◦04′28.8′′W, altitude: 670 m. Holotype: LPP-IC-0028: sandstone slab showing slightly curved trackway, preserved in positive hyporelief over a length of 34 cm. Reposited in the Paleoichnology collection of the Laboratório de Paleoecologia e Paleoicnologia (LPP) of the Federal University of São Carlos (UFSCar) campus São Carlos-SP. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 12/37 Figure 6 Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC- 0028 (holotype). (B) LPP-IC-0029 and (C) LPP-IC-0030. The producer walked from bottom to top. Scale bar: 10 cm. Full-size DOI: 10.7717/peerj.8880/fig-6 Figure 6 Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC- 0028 (holotype). (B) LPP-IC-0029 and (C) LPP-IC-0030. The producer walked from bottom to top. Scale bar: 10 cm. Full-size DOI: 10.7717/peerj.8880/fig-6 Full-size DOI: 10.7717/peerj.8880/fig-6 Paratypes: Sandstone slabs: LPP-IC-0029 (negative epirelief) and its counterpart LPP- IC-0030 (positive hyporelief); LPP-IC-0031 (negative epirelief) and its counterpart LPP- IC-0032 (positive hyporelief); LPP-IC-0033 (negative epirelief) and its counterpart LPP- IC-0034 (positive hyporelief); LPP-IC-0035 (negative epirelief) and with no apparent counterpart slab. All slabs are reposited in the Paleoichnology collection of the Laboratório de Paleoecologia e Paleoicnologia (LPP) of the Federal University of São Carlos (UFSCar) campus São Carlos-SP. Etymology: It is dedicated to the city of Araraquara, São Paulo State, where these trackways were found, along with most of the ichnofossils of the Botucatu Formation. Diagnosis: Trackways composed of two rows, whose internal width between the rows is less than one-quarter of the external width; with alternating to staggered series, consisting of up to three tracks with different sizes that may vary from slightly elongated to tapered or circular in shape. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figures 5 and 6A. Description: Due to the similarity between the size of the sandstone grains and the size of the locomotory appendages of the producer, the tracks of Paleohelcura araraquarensis isp. nov. in all the analyzed slabs have little definition. The following slabs have counterparts: LPP-IC-0029 (negative epirelief) and LPP-IC-0030 (positive hyporelief), Figs. 6B and 6C, respectively; LPP-IC-0031 (negative epirelief) and LPP-IC-0032 (positive hyporelief), Figs. 7A and 7B, respectively; LPP-IC-0033 (negative epirelief) and LPP-IC-0034 (positive hyporelief), Figs. 7C and 7D, respectively. The organization of the tracks in the series follows a pattern with two, usually smaller, tracks grouped anteriorly and often more externally, and a longer track more posteriorly and commonly internally positioned (Fig. 5C). In places, the series adopt a linear configuration (Fig. 5D) that, despite showing some recurrence, is Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 13/37 Figure 7 Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC- 0031. (B) LPP-IC-0032. (C) LPP-IC-0033. (D) LPP-IC-0034. (E) LPP-IC-0035. The producer walked from bottom to top. Scale bar: 10 cm. Full-size DOI: 10.7717/peerj.8880/fig-7 Table 3 Arithmetic mean of the measurements of each track in the series. (L/W) Length /Width. LPP-IC-0033 and LPP-IC-0034 were not mea- sured because of their poor preservation. Figure 7 Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC- 0031. (B) LPP-IC-0032. (C) LPP-IC-0033. (D) LPP-IC-0034. (E) LPP-IC-0035. The producer walked from bottom to top. Scale bar: 10 cm. Full-size DOI: 10.7717/peerj.8880/fig-7 Figure 7 Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC- 0031. (B) LPP-IC-0032. (C) LPP-IC-0033. (D) LPP-IC-0034. (E) LPP-IC-0035. The producer walked from bottom to top. Scale bar: 10 cm. Full-size DOI: 10.7717/peerj.8880/fig-7 Table 3 Arithmetic mean of the measurements of each track in the series. (L/W) Length /Width. LPP-IC-0033 and LPP-IC-0034 were not mea- sured because of their poor preservation. Figures 5 and 6A. Specimen Track 1 (Mean) Track 2 (Mean) Track 3 (Mean) Length (mm) Width (mm) L/W Length (mm) Width (mm) L/W Length (mm) Width (mm) L/W LPP-IC-0028 5,41 3,14 1,73 4,94 3,03 1,63 5,11 3,79 1,35 LPP-IC-0029 4,98 2,83 1,76 4,38 3,41 1,28 4,28 3,37 1,27 LPP-IC-0030 4,43 2,73 1,62 4,13 2,77 1,49 4,32 3,21 1,35 LPP-IC-0031 4,60 2,97 1,55 4,53 3,07 1,47 4,81 3,77 1,27 LPP-IC-0032 4,96 3,10 1,60 4,44 3,01 1,47 4,77 3,53 1,35 LPP-IC-0035 4,28 2,84 1,51 4,73 3,28 1,44 5,41 3,74 1,45 Mean (mm) 4,78 2,93 1,63 4,52 3,09 1,47 4,78 3,57 1,34 Standard de- viation/Mean 0,19 0,17 0,17 0,15 0,15 0,15 not an appropriate feature for the diagnosis because it is a variation in the more consistent triangular pattern shown in Fig. 6C. The arithmetic means of the measurements of the trackway parameters are summarized in Tables 2 and 3. All measurements are in the Table S1. The average external width of the Specimen Track 1 (Mean) Track 2 (Mean) Track 3 (Mean) Length (mm) Width (mm) L/W Length (mm) Width (mm) L/W Length (mm) Width (mm) L/W LPP-IC-0028 5,41 3,14 1,73 4,94 3,03 1,63 5,11 3,79 1,35 LPP-IC-0029 4,98 2,83 1,76 4,38 3,41 1,28 4,28 3,37 1,27 LPP-IC-0030 4,43 2,73 1,62 4,13 2,77 1,49 4,32 3,21 1,35 LPP-IC-0031 4,60 2,97 1,55 4,53 3,07 1,47 4,81 3,77 1,27 LPP-IC-0032 4,96 3,10 1,60 4,44 3,01 1,47 4,77 3,53 1,35 LPP-IC-0035 4,28 2,84 1,51 4,73 3,28 1,44 5,41 3,74 1,45 Mean (mm) 4,78 2,93 1,63 4,52 3,09 1,47 4,78 3,57 1,34 Standard de- viation/Mean 0,19 0,17 0,17 0,15 0,15 0,15 not an appropriate feature for the diagnosis because it is a variation in the more consistent triangular pattern shown in Fig. 6C. The arithmetic means of the measurements of the trackway parameters are summarized in Tables 2 and 3. All measurements are in the Table S1. The average external width of the trackways is 22.97 mm, and the average internal width is 2.21 mm, with absolute values of the latter varying from 3.80 mm to 0 mm (i.e., no internal separation between series). The internal trackway width is, on average, approximately one-tenth of the outer trackway width. The ratio between the length and width of the tracks (L/W) is always greater than one, which indicates that they are elongated. It is rare in Paleohelcura araraquarensis isp. nov. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figures 5 and 6A. for tracks to have lengths and widths with similar values, that is, with a circular shape. Specimen Track 1 (Mean) Track 2 (Mean) Track 3 (Mean) Length (mm) Width (mm) L/W Length (mm) Width (mm) L/W Length (mm) Width (mm) L/W LPP-IC-0028 5,41 3,14 1,73 4,94 3,03 1,63 5,11 3,79 1,35 LPP-IC-0029 4,98 2,83 1,76 4,38 3,41 1,28 4,28 3,37 1,27 LPP-IC-0030 4,43 2,73 1,62 4,13 2,77 1,49 4,32 3,21 1,35 LPP-IC-0031 4,60 2,97 1,55 4,53 3,07 1,47 4,81 3,77 1,27 LPP-IC-0032 4,96 3,10 1,60 4,44 3,01 1,47 4,77 3,53 1,35 LPP-IC-0035 4,28 2,84 1,51 4,73 3,28 1,44 5,41 3,74 1,45 Mean (mm) 4,78 2,93 1,63 4,52 3,09 1,47 4,78 3,57 1,34 Standard de- viation/Mean 0,19 0,17 0,17 0,15 0,15 0,15 not an appropriate feature for the diagnosis because it is a variation in the more consistent triangular pattern shown in Fig. 6C. The arithmetic means of the measurements of the trackway parameters are summarized in Tables 2 and 3. All measurements are in the Table S1. The average external width of the trackways is 22.97 mm, and the average internal width is 2.21 mm, with absolute values of the latter varying from 3.80 mm to 0 mm (i.e., no internal separation between series). The internal trackway width is, on average, approximately one-tenth of the outer trackway width. The ratio between the length and width of the tracks (L/W) is always greater than one, which indicates that they are elongated. It is rare in Paleohelcura araraquarensis isp. nov. for tracks to have lengths and widths with similar values, that is, with a circular shape. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 14/37 There were variations observed between track measurements in the negative epirelief and their corresponding counterpart slab. This may have two causes: (i) subjectivity may have caused variation in the measurement of tracks of different toponomy, one in negative epirelief and another one in positive hyporelief (i.e., methodological bias); or (ii) it would suggest that arthropod tracks are susceptible to another type of alteration, after the production of the footprint and the lithification of the substrate, generated by the splitting of the layers in to part and counterpart slabs (i.e., taphonomic bias). In this situation, tracks can lose parts, become smaller, or retain the sediment of the counterpart slab, thereby modifying their size. Figures 5 and 6A. As such, the measurements taken from an ichnofossil may not correspond precisely to the size of the tracks left by the animal when the substrate was unconsolidated. It was not possible to take accurate track measurements of the counterpart slabs LPP-IC-0033 and LPP-IC-0034 (Figs. 7C and 7D) due to the poor preservation of the tracks, but they are included as paratypes because they represent part of the variation that Paleohelcura araraquarensis isp. nov. can exhibit, whether due to preservation, taphonomic process or produced by the disaggregation of the layers. The paratypes (LPP-IC-0029, LPP-IC-0030, LPP-IC-0031, LPP-IC-0032, LPP-IC-0033, LPP-IC-0034) did not present significant differences in relation to the holotype (LPP-IC-0028). Specimens in LPP-IC-0028, LPP-IC-0029, LPP-IC-0030, LPP-IC-0031, LPP-IC-0032, LPP-IC-0035 show sediment displacement associated with the tracks (Fig. 8). In slabs with positive hyporelief (LPP-IC-0028, LPP-IC-0030, LPP-IC-0032), the displacement appears as a faint depression attached to the track. This displacement indicates the direction of movement, being located on the opposite side from the direction of movement of the animal, generated by the effort that the locomotory appendage applied to the unconsolidated substrate to generate propulsion. However, we cannot rule out the possibility that the displacement could have been generated by sliding of the animal caused by the slope of the dune; this interpretation is less likely because the orientation of the displacement is the same in all the specimens that exhibit it. Therefore, we consider that the displacement was more likely to have been generated by the propulsion of the animal over the sand. The commercial extraction of the sandstone at the São Bento quarry was undertaken without scientific monitoring, and most of the fossils were in slabs ready to be cut, or already cut for commercialization. Thus, the fossils were rescued and there was no record of the orientation of the slabs in relation to the slope or whether the holotype and the paratypes could be parts of the same large but fragmented individual trackway. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Comparisons The main difference between Paleohelcura araraquarensis isp. nov. and Paleohelcura tridactyla, and its junior synonyms (revised in Remarks section of the ichnogenus Paleohelcura) is the ratio of the internal width to the external width. Paleohelcura araraquarensis isp. nov. has an internal width equal to or less than one-quarter of the external width (on average one-tenth of the external width). The internal width in other ichnospecies of Paleohelcura is greater than one-quarter of the external width, even in Paleohelcura with narrow internal widths like some of from the lower Permian Robledo Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figure 8 Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit- ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco- motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the photo. Full-size DOI: 10.7717/peerj.8880/fig-8 g y y g p y y Series within Lithographus and P. araraquarensis isp. nov. share two usually smaller tracks that are grouped anteriorly and commonly more externally, and a longer track that is positioned more posteriorly and internally. We suggest that this characteristic may reflect the pterygote insect leg arrangement (see Trace-fossil producer section). The main morphological characteristic of Lithographus are its linear tracks with varied orientations, which differs from the rounded to elliptical tracks of Paleohelcura araraquarensis isp. nov., whose long-axes are subparallel to the midline of the trackway. It is, therefore, more reasonable to assign the trackways described here to Paleohelcura, and to establish a new ichnospecies for forms with a narrow internal width. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figure 8 Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit- ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco- motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the photo. Full-size DOI: 10.7717/peerj.8880/fig-8 Figure 8 Specimen LPP-IC-0035 of Paleohelcura ara ing deformation in the sediment by the effort of loco motion effort; White arrow: the direction of movemen photo. Figure 8 Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit- ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco- motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the photo. Full-size DOI: 10.7717/peerj.8880/fig-8 Figure 8 Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit- ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco- motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the photo. Full-size DOI: 10.7717/peerj.8880/fig-8 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Mountains and Coconino Sandstone of the USA that have external widths of similar or lower absolute values to P. araraquarensis isp. nov. (Minter & Braddy, 2009, their fig. 30; 31), and those from the Permian Lyons Sandstone of Colorado (USA) (Toepelman & Rodeck, 1936, their fig. 1). The triangular series arrangement and ellipsoidal tracks shown by Paleohelcura araraquarensis isp. nov. (Fig. 5C) slightly resemble Lithographus and some of its junior synonyms. Lithographus comprises trackways with series of up to three tracks with alternate to staggered symmetry but differs from Paleohelcura in that the tracks are linear and have varied orientations with respect to the midline of the trackway (Minter & Braddy, 2009). Several ichnogenera were synonymized with Lithographus by Minter & Braddy (2009). The triangular series arrangement and ellipsoidal tracks shown by Paleohelcura araraquarensis isp. nov. (Fig. 5C) slightly resemble Lithographus and some of its junior synonyms. Lithographus comprises trackways with series of up to three tracks with alternate to staggered symmetry but differs from Paleohelcura in that the tracks are linear and have varied orientations with respect to the midline of the trackway (Minter & Braddy, 2009). Several ichnogenera were synonymized with Lithographus by Minter & Braddy (2009). Series within Lithographus and P. araraquarensis isp. nov. share two usually smaller tracks that are grouped anteriorly and commonly more externally, and a longer track that is positioned more posteriorly and internally. We suggest that this characteristic may reflect the pterygote insect leg arrangement (see Trace-fossil producer section). Trace-fossil producer The photographs are frames of a video courtesy of R.E. Ritzmann showing a cockroach (Blaberus discoidalis) in ventral view walking on an oiled glass plate (Video S1). Full-size DOI: 10.7717/peerj.8880/fig-9 1º step 2º step 3º step 4º step 5º step 6º step Series { Footprint being marked Already marked footprint A C B Series { C Series { C 1º step 2º step 3º step 4º step 5º step 6º step Footprint being marked Already marked footprint A B A C B Figure 9 Illustration simulating the marks left by a cockroach while walking (ventral view). (A) The photographs show the advancement of a cockroach and the marks that would be left by its feet. The red footprints on the photograph indicate footprints that have just been produced; magenta footprints indi- cate already produced footprints. (B) Footprints produced in each step, broken down by color. (C) Asso- ciation between the footprints within the series and the pairs of feet that produced them. The photographs are frames of a video courtesy of R.E. Ritzmann showing a cockroach (Blaberus discoidalis) in ventral view walking on an oiled glass plate (Video S1). Full-size DOI: 10.7717/peerj.8880/fig-9 p. 2); therefore, neoichnological studies provide strong grounds to exclude scorpions as producers of P. araraquarensis isp. nov. Neoichnological studies with cockroaches produced trackways with elongated tracks because they walk on tarsal segments (tarsomeres) (Davis, Minter & Braddy, 2007). Therefore, segmented tarsi appear to be an important feature to generate tracks similar to those of the ichnogenus Lithographus and its junior synonyms. Within the Hexapoda, Protura, Diplura, Monura and Collembola possess undivided tarsi (Kristensen, 1998, p. 289; Bitsch & Bitsch, 2000, p. 140), probably producing tracks similar to those of an arachnid because they also do not have segmented tarsi. Only the true insects (Zygentoma, Archaeognatha, and Pterygota) have segmented tarsi (Kristensen, 1998, p. 289; Bitsch & Bitsch, 2000; Gorb & Beutel, 2001, p. 534). The neoichnology of representatives of Zygentoma and Archaeognatha revealed that they produced circular/elliptical to elongated tracks (Getty et al., 2013), which resemble those produced by arachnids. In this case, it is due to the low mobility of the tarsomeres, which lead to the digitigrade posture (on the pretarsus). Trace-fossil producer Although series arrangement and track shape are variable, the most common pattern identified is useful for making neoichnological comparisons. An alternating tripod gait is a relatively robust locomotion pattern for Hexapoda (Wöhrl, Reinhardt & Blickhan, 2017a). Arachnids, although possessing four pairs of locomotory appendages, can produce series with three tracks, either by adopting a hexapedal gait or for taphonomic reasons (Davis, Minter & Braddy, 2007; Schmerge, Riese & Hasiotis, 2013). Trackways comprising series with alternating symmetry on either side of the medial line, and with a maximum of three tracks per series, indicate that the animal maintained at least three feet on the ground while walking (Fig. 9). Therefore, we restrict the discussion of the producer to the Arachnida and Hexapoda. The trackways made by spiders do not resemble Paleohelcura araraquarensis isp. nov. because they have larger internal width, circular tracks, and a different series arrangement Davis, Minter & Braddy, 2007, their Fig. 9). When scorpions leave series with three tracks they resemble P. araraquarensis isp. nov., but with a different arrangement of tracks within the series. In P. araraquarensis isp. nov., there are two tracks anteriorly positioned, and a usually longer track more posteriorly and internally positioned. In scorpion trackways, there are two tracks more posteriorly positioned and one track that lays anteriorly, and the former are usually longer and commonly most internally positioned (Fig. 10; Davis, Minter & Braddy, 2007, their fig. 7–8). In addition, even small scorpions, with comparable size to the animal that made Paleohelcura araraquarensis isp. nov., leave trackways with a large internal width, differing from the narrow internal width of P. araraquarensis isp. nov. (Fig. 10). Modern scorpions are very similar to Paleozoic scorpions (Polis, 1990, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 17/37 1º step 2º step 3º step 4º step 5º step 6º step Series { Footprint being marked Already marked footprint A C B Figure 9 Illustration simulating the marks left by a cockroach while walking (ventral view). (A) The photographs show the advancement of a cockroach and the marks that would be left by its feet. The red footprints on the photograph indicate footprints that have just been produced; magenta footprints indi- cate already produced footprints. (B) Footprints produced in each step, broken down by color. (C) Asso- ciation between the footprints within the series and the pairs of feet that produced them. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trace-fossil producer The increased mobility of the tarsomeres in Pterygota is linked to the evolutionary pressure to climb and walk on a variety of new substrates due to their ability to fly and the necessity to hold onto leaves and plant stems (Gorb & Beutel, 2001, p. 533). Most Pterygota walk on tarsomeres (Manton, 1972; Zollikofer, 1994, p. 98; Frazier et al., 1999; Boggess et al., 2004; Davis, Minter & Braddy, 2007; Gladun & Gorb, 2007; Clemente & Federle, 2008; Wöhrl, Reinhardt & Blickhan, 2017b); cockroach video in Video S1 of this publication, courtesy of R.E. Ritzmann). As observed in neoichnological experiments on cockroaches (Davis, Minter & Braddy, 2007), they all probably produce elongated or elliptical tracks. Nevertheless, it is not possible to assume this for all pterygote insects without more neoichnological experiments because there are species that walk on a few distal tarsomeres and on their pretarsus (Niederegger & Gorb, 2003; Gladun & Gorb, 2007; Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 18/37 C B A Figure 10 Scorpion Tityus serrulatus. tracks in sand (A and B) and Paleohelcura araraquarensis isp. nov. holotype LPP-IC-0028 (C). Scale bar: 1 cm. The arrow indicates the direction of the animal move- ment. (A) and (B) photographs courtesy of Ravi Sampaio, 2015. Full-size DOI: 10.7717/peerj.8880/fig-10 B C A Figure 10 Scorpion Tityus serrulatus. tracks in sand (A and B) and Paleohelcura araraquarensis isp. nov. holotype LPP-IC-0028 (C). Scale bar: 1 cm. The arrow indicates the direction of the animal move- ment. (A) and (B) photographs courtesy of Ravi Sampaio, 2015. Full-size DOI: 10.7717/peerj.8880/fig-10 Endlein & Federle, 2015), thereby probably producing elongated tracks, but less so than those of full plantigrade insects. Trackways of some desert darkling beetles (Tenebrionidae) closely resemble Paleohelcura araraquarensis isp. nov. in that they comprise a narrow internal width, elliptical tracks, and series that usually exhibit the same arrangement of P. araraquarensis isp. nov. (Fig. 11). Despite being made by pterygote insects, they do not show strong linear tracks like the cockroaches used in experiments by Davis, Minter & Braddy (2007) that made trackways similar to Lithographus. Even with segmented tarsi, these Tenebrionidae leave elliptical tracks just like in P. araraquarensis isp. nov. This is probably due to the small size of the animal compared with the sand grain size, which diminishes the resolution of the tracks. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trace-fossil producer The cockroaches used by Davis, Minter & Braddy (2007) were relatively large compared to the grain size of the substrate, creating trackways of greater than 40 mm external width, and so it is expected that the tracks reflected more faithfully the morphology of the locomotory appendages. The series arrangement in the trackways of Tenebrionidae are also similar to those observed in Paleohelcura araraquarensis isp. nov. and cockroach trackways, with two usually smaller tracks grouped anteriorly and often more externally and a longer track more posteriorly and often internally positioned. This arrangement could be related to the Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 19/37 A B Figure 11 Darkling beetles (Tenebrionidae) and their tracks from Morocco dunes. (A) Oblique view of the trackway and the walking darkling beetle. (B) Perpendicular view of the trackway and the walk- ing darkling beetle. The photos were not taken with scales, but these beetles are around 2 cm long. Pho- tographs courtesy of Martin Harvey, 2004. Full-size DOI: 10.7717/peerj.8880/fig-11 A A Figure 11 Darkling beetles (Tenebrionidae) and their tracks from Morocco dunes. (A) Oblique view of the trackway and the walking darkling beetle. (B) Perpendicular view of the trackway and the walk- ing darkling beetle. The photos were not taken with scales, but these beetles are around 2 cm long. Pho- tographs courtesy of Martin Harvey, 2004. Full-size DOI: 10.7717/peerj.8880/fig-11 positions of the legs and the role of each leg in the gait of pterygote insects. Therefore, we consider that the producer of P. araraquarensis isp. nov. would probably be an insect from the Pterygota rather than an arachnid, and suggest Recent Tenebrionidae as a plausible analog, noteworthy for their high abundance and diversity in deserts, as a result of their striking ability to adapt to hyperarid settings (Cloudsley-Thompson, 2001). This contrasts with the interpretations of the producers of other ichnospecies of Paleohelcura, inferred to have been made by scorpions and spiders (Davis, Minter & Braddy, 2007). Although a beetle affinity is proposed, more neoichnological studies are necessary to discriminate other potential producers since the diversity of pterygote insects is high (Clapham et al., 2016). In addition, it will enable greater understanding of the effect on trackway morphology of the interaction among the size of an arthropod, the morphology of its limbs, the grain size, and the moisture in the substrate. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Paleoautoecologic implications sharing some morphological characteristics with trackways of some dune desert Tenebrionidae (see Trace-fossil producer section), it is not possible to establish a confident link between the fossil tracks and the group without eliminating other Pterygota as candidates through neoichnology or the discovery of associated body fossils. Nevertheless, the existence of Taenidium isp. and Skolithos linearis burrows (Fernandes, Netto & Carvalho de, 1988; Fernandes, Carvalho & Netto, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Paleoautoecologic implications Looking at modern deserts, among Pterygota reaching similar size, the Coleoptera are one of the most conspicuous and abundant animals in arid environments (Holm & Scholtz, 1980; Cloudsley-Thompson, 2001; Whitford, 2002). Some, like Tenebrionidae, possess several morphological, physiological, and mainly behavioral adaptations to deal with extremely dry and hot environments (ultra-psammophilous) (Cloudsley-Thompson, 2001). Beyond their remarkable incidence in deserts, these animals play an ecological role in dune deserts of consuming detritus among sand grains and being able to rely on this biomass even when there is no primary production during dry periods in hyperarid deserts (Seely & Louw, 1980; Southgate, Masters & Seely, 1996). Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 20/37 The producer of Paleohelcura araraquarensis isp. nov. being a pterygote insect could predominately have been a herbivore, carnivore, or an omnivore. Omnivory is important in desert food webs since primary productivity is limited by moisture availability (Polis, 1991). Such an animal would be able to feed on the organic matter that accumulated in the paleodunes, but also be capable of adopting herbivorous, saprophagous, or carnivorous diets when appropriate. Tenebrionidae are a notable example of this in modern dune deserts (Koch, 1961; Holm & Scholtz, 1980; Robinson & Seely, 1980; Seely & Louw, 1980; Cloudsley- Thompson, 2001). If it had been carnivorous, the producer of Paleohelcura araraquarensis isp. nov. could be a predatory insect, like some species of Carabidae (Coleoptera) living in dunes of the Negev desert (Filser & Prasse, 2008). Despite Paleohelcura araraquarensis isp. nov. sharing some morphological characteristics with trackways of some dune desert Tenebrionidae (see Trace-fossil producer section), it is not possible to establish a confident link between the fossil tracks and the group without eliminating other Pterygota as candidates through neoichnology or the discovery of associated body fossils. Nevertheless, the existence of Taenidium isp. and Skolithos linearis burrows (Fernandes, Netto & Carvalho de, 1988; Fernandes, Carvalho & Netto, 1990; Fernandes, 2005; Peixoto et al., 2016) shows that the Botucatu paleodesert played host to a community of omnivorous detritivorous insects just like modern dune deserts, where they comprise most of the animal biomass on the dune slip-face (e.g., Namib dune desert: Seely & Louw, 1980). P. araraquarensis isp. nov. could be produced by an insect of this community like omnivorous Tenebrionidae, which produces similar trackways in modern dune deserts. Despite Paleohelcura araraquarensis isp. nov. Paleoecology of the Botucatu desert The trace-fossil record of the Botucatu Formation may provide indirect evidence on the abundance and diversity of organisms that inhabited these eolian dunes. Through the interpretation of the phylogenetic affinity and probable nutrient source of the producers, it is possible to make broad inferences about the ecological relationships of these organisms in the context of the Late Jurassic—Early Cretaceous Botucatu desert. Modern desert food webs are complex due to a high frequency of omnivory, generating highly connected food webs, with species interacting with many predators and prey (Polis, 1991). Omnivorous insects comprise most of the animal biomass on the dune slip-face of modern deserts (e.g., Namib dune desert; Seely & Louw, 1980). A similar situation is envisaged for the Botucatu paleodesert. Taenidium isp. and Skolithos linearis were most likely produced by insects feeding on detritus among the sand grains (Figs. 12G to 12E), like Tenebrionidae in modern dune deserts. Those with compatible size could produce Paleohelcura araraquarensis isp. nov. when walking on the sand. Insects feeding on living plant material as omnivores or herbivores could also produce P. araraquarensis isp. nov. (Fig. 12H to 12E). Accordingly, relying on plant material, the detritivorous and herbivorous invertebrates are regarded as part of the primary consumers in the trophic web of the Botucatu paleodesert. The geomorphological characteristics of an erg create habitats with abiotic characteristics that determine the productivity, biomass and diversity found in the interdune, and the windward and slip-face subenvironments of the dune field (Seely & Louw, 1980; Southgate, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 21/37 Figure 12 Reconstruction of the Botucatu paleodesert food web based on the interpretation of the probable producers of the ichnofossil of Botucatu Formation. The dashed arrows represent the ecologi- cal relations and the flow of energy in the Botucatu paleodesert. (A) Large mammaliaform. (B) Theropod dinosaurs. (C) Small mammaliaform. (D) Arachnids and possible insects. (E) Insects. (F) Ornithopod di- nosaurs. (G) Detritus in the sand and blown by the wind. (H) Plants. On the left, the possible ecological roles that the Paleohelcura araraquarensis isp. nov. producer could have played in the Botucatu desert are shown. This scheme was made with modifications of artworks of different authors: Carivorous Insect from Rafael Pasini; Scorpion in (D) from Gareth Monger; (A) and (C) Mammaliaforms from Ceri Thomas; (B) Theropod dinosaur from Frederic Wierum; (F) Ornithopod dinosaur from Nobumichi Tamura. Paleoecology of the Botucatu desert All art- works are under the CC-BY-SA 4.0 (https://creativecommons.org/licenses/by/4.0/). Full-size DOI: 10.7717/peerj.8880/fig-12 Figure 12 Reconstruction of the Botucatu paleodesert food web based on the interpretation of the Figure 12 Reconstruction of the Botucatu paleodesert food web based on the interpretation of the probable producers of the ichnofossil of Botucatu Formation. The dashed arrows represent the ecologi- cal relations and the flow of energy in the Botucatu paleodesert. (A) Large mammaliaform. (B) Theropod dinosaurs. (C) Small mammaliaform. (D) Arachnids and possible insects. (E) Insects. (F) Ornithopod di- nosaurs. (G) Detritus in the sand and blown by the wind. (H) Plants. On the left, the possible ecological roles that the Paleohelcura araraquarensis isp. nov. producer could have played in the Botucatu desert are shown. This scheme was made with modifications of artworks of different authors: Carivorous Insect from Rafael Pasini; Scorpion in (D) from Gareth Monger; (A) and (C) Mammaliaforms from Ceri Thomas; (B) Theropod dinosaur from Frederic Wierum; (F) Ornithopod dinosaur from Nobumichi Tamura. All art- works are under the CC-BY-SA 4.0 (https://creativecommons.org/licenses/by/4.0/). Full-size DOI: 10.7717/peerj.8880/fig-12 Masters & Seely, 1996). Even though the slip-face occupies a small area of the erg, when compared to the windward or interdune areas (in the case of ergs with well-spaced dunes), and is a region of low plant growth, it has a high concentration of biomass per unit area in the form of detritus, concentrated by the wind mainly at the base of the dunes (Seely & Louw, 1980; Southgate, Masters & Seely, 1996). This detritus originates from adjacent subenvironments, more conducive to plant growth where there is moisture (Seely & Louw, 1980; Southgate, Masters & Seely, 1996), and could even be carried by the wind from distant locations, where the climatic regimes allow greater primary productivity (Robinson & Seely, 1980). During prolonged periods of drought, with low primary productivity, the dune slip-faces maintain high concentrations of biomass as detritus is deposited by the wind, providing food for detritivores and letting them survive until periods of increased availability of moisture and biomass, when they can proliferate (Southgate, Masters & Seely, 1996). Masters & Seely, 1996). Paleoecology of the Botucatu desert Therefore, we can infer that the Botucatu paleodesert landscape could have had some shrubs, similar to modern arid dune deserts (e.g., Namib Desert: Fig. 13). The presence of plants makes it reasonable to infer that some invertebrates in the area may not have relied exclusively on detritus to survive but could also have been herbivorous (Fig. 12: flowing energy from H to E). of the Botucatu Formation as ‘‘dwarf’’ when compared with the fauna of the older and paleoenvironmentally wetter Guará Formation. It is possible that this could be due to the lower primary productivity of the arid Botucatu paleodesert that would only support viable populations of smaller herbivorous dinosaurs. We infer that the presence of these small-sized herbivorous dinosaurs, less than 68 cm in height to the pelvic girdle, is evidence that there was localized plant growth at least for some period in the region (Fig. 12H to F). This is supported by the fact that the home range of modern herbivorous animals increases with increasing body size (e.g., ungulates: Ofstad et al., 2016), so these small ornithopods with limited home range should not have fed far from where their tracks were found. Therefore, we can infer that the Botucatu paleodesert landscape could have had some shrubs, similar to modern arid dune deserts (e.g., Namib Desert: Fig. 13). The presence of plants makes it reasonable to infer that some invertebrates in the area may not have relied exclusively on detritus to survive but could also have been herbivorous (Fig. 12: flowing energy from H to E). Contrasting with most ‘‘dwarf’’ trackway producers, a trackway of a large dinosaur (3.6 m high and 5 m long) interpreted as that an ornithopod has been documented as well (Fernandes, 2005; Fernandes & Carvalho, 2007; Francischini et al., 2015). Since the trackway of only one animal was found and there are no trackways of associated predators, it is likely to infer that there was no settled population of this animal in this location at the time. The large ornithopod dinosaur that produced this trackway probably had an extensive home range, encompassing habitats with different primary productivity to support their large body size, and this trackway of an anomalously large animal for the environment would be the record of an animal crossing a less suitable area, like modern desert-dwelling elephants (Viljoen, 1989) and giraffes (Fennessy, 2009; Flanagan et al., 2016). Peixoto et al. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Paleoecology of the Botucatu desert Even though the slip-face occupies a small area of the erg, when compared to the windward or interdune areas (in the case of ergs with well-spaced dunes), and is a region of low plant growth, it has a high concentration of biomass per unit area in the form of detritus, concentrated by the wind mainly at the base of the dunes (Seely & Louw, 1980; Southgate, Masters & Seely, 1996). This detritus originates from adjacent subenvironments, more conducive to plant growth where there is moisture (Seely & Louw, 1980; Southgate, Masters & Seely, 1996), and could even be carried by the wind from distant locations, where the climatic regimes allow greater primary productivity (Robinson & Seely, 1980). During prolonged periods of drought, with low primary productivity, the dune slip-faces maintain high concentrations of biomass as detritus is deposited by the wind, providing food for detritivores and letting them survive until periods of increased availability of moisture and biomass, when they can proliferate (Southgate, Masters & Seely, 1996). Ornithopods are known to be primary consumers in several Cretaceous ecosystems (Barrett, 2014), therefore, they likely assumed this role in the Botucatu paleodesert as well (Fig. 12F). Based on trackways, Francischini et al. (2015) interpreted the dinosaur fauna Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 22/37 Figure 13 Kahani Dunes in the arid Namib Desert showing some plant growth (Stipagrostis sabuli- cola). Photography courtesy of Oliver Halsey, 2016. Full-size DOI: 10.7717/peerj.8880/fig-13 Figure 13 Kahani Dunes in the arid Namib Desert showing some plant growth (Stipagrostis sabuli- cola). Photography courtesy of Oliver Halsey, 2016. Full-size DOI: 10.7717/peerj.8880/fig-13 of the Botucatu Formation as ‘‘dwarf’’ when compared with the fauna of the older and paleoenvironmentally wetter Guará Formation. It is possible that this could be due to the lower primary productivity of the arid Botucatu paleodesert that would only support viable populations of smaller herbivorous dinosaurs. We infer that the presence of these small-sized herbivorous dinosaurs, less than 68 cm in height to the pelvic girdle, is evidence that there was localized plant growth at least for some period in the region (Fig. 12H to F). This is supported by the fact that the home range of modern herbivorous animals increases with increasing body size (e.g., ungulates: Ofstad et al., 2016), so these small ornithopods with limited home range should not have fed far from where their tracks were found. Paleoecology of the Botucatu desert (2020), PeerJ, DOI 10.7717/peerj.8880 23/37 The next trophic level includes animals from various phylogenetic groups that feed on detritivores and herbivores, therefore, they are carnivores and omnivores (secondary consumers). In the Botucatu Formation, this would include arachnids such as scorpions and spiders (Fig. 12D), the producer of Paleohelcura araraquarensis isp. nov. if it had been a predator or an omnivore feeding on animal biomass, small mammaliaform organisms that produced Brasilichnium elusivum and B. saltatorium (Fig. 12C), and small theropod dinosaurs (Fig. 12B). Due to the high connectivity of the trophic web, there would be interactions among the representatives of this level. Trophic interactions would have been limited by the ability of an animal to prey upon another, which is linked to the size of the prey. Therefore, the animals grouped here probably preyed on smaller animals from the same trophic level or even eggs and juveniles of larger animals. The existence of predatory arachnids in the Botucatu paleodesert does not preclude the existence of predatory insects because this coexistence is observed in modern dune deserts (e.g., Negev: Filser & Prasse, 2008). Brasilichnium elusivum is the most common vertebrate trackway recorded in the Botucatu Formation (Leonardi, Carvalho de & Fernandes, 2007) and is a typical ichnotaxon in other Mesozoic paleodeserts around the world (Krapovickas et al., 2016; Xing et al., 2018). Lizards and snakes that could have occupied the same trophic level are common inhabitants of all modern hot deserts (Cloudsley-Thompson, 1991; Whitford, 2002, p. 129), but no records of these types of animals have been found in the Botucatu Formation. Snakes appeared during the Middle Jurassic (Caldwell et al., 2015), and may not have been widespread in desert ecosystems during the time of the Botucatu paleodesert (Late Jurassic—Early Cretaceous). There are a few records of lizard-like tracks in paleodeserts from the Permian (Loope, 1984; Haubold et al., 1995; Lockley & Hunt, 1995; Lockley et al., 1998). Tracks assigned to lizards (‘‘lacertoids’’) generally show digit impressions with an inturned ‘‘comb’’ of curved digits, and pes tracks that are longer than wide (Hunt & Lucas, 2006), however, these were not found in the Botucatu Formation. The top trophic level is hard to define due to the trophic generalism of desert animals. Paleoecology of the Botucatu desert This level is represented by animals that, because of their size, would be able to prey on non-detritivorous animals, like the producer of Brasilichnium elusivum, arachnids and small herbivorous dinosaurs (ornithopods), as well as the small detritivorous animals. Such organisms would include the larger theropod dinosaurs reported from trackways in the Botucatu Formation (Francischini et al., 2015) (Fig. 12B), and mammaliaform producers of Brasilichnium anaitti, which are larger than the producers of Brasilichnium elusivum and B. saltatorium (Fig. 12A). Implications for the definition of the Octopodichnus-Entradichnus Ichnofacies of eolian environments Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Implications for the definition of the Octopodichnus-Entradichnus Ichnofacies of eolian environments Two invertebrate eolian ichnofacies were defined independently, the Octopodichnus Ichnofacies of Hunt & Lucas (2007) and the Entradichnus Ichnofacies of Ekdale, Bromley & Loope (2007). Subsequently, they were both integrated as the Octopodichnus-Entradichnus Ichnofacies (Buatois & Mángano, 2011, p. 78; Krapovickas et al., 2016; Buatois & Echevarría, 2019). In particular, Krapovickas et al. (2016) suggested that this ichnofacies is characterized Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 by (1) low to rarely moderate trace fossil diversity, (2) dominance of simple sub-superficial dwelling traces produced mostly by coleopterans, orthopterans and arachnids, with horizontal (e.g., Palaeophycus) and/or vertically oriented dwelling burrows (e.g., Skolithos, Digitichnus); (3) superficial locomotion traces produced by arthropods, especially arachnids (e.g., Octopodichnus, Paleohelcura); and (4) subordinate simple (Planolites) and meniscate (e.g., Taenidium, Entradichnus) feeding burrows. The Chelichnus Ichnofacies of Hunt & Lucas (2007) is considered the archetypal vertebrate ichnofacies of eolian environments. The combined analysis of sedimentary facies and variations in the occurrence, abundance, and diversity of trace fossils may allow differentiation among hyper-arid, arid and semi-arid deserts (Krapovickas et al., 2016). Like ichnofacies from other continental environments, arid eolian environments display recurrence in the characteristics of their ichnocoenoses, which represent behavioral convergence as a response to abiotic features of habitat and substrate (Buatois & Mángano, 2011). There is also recurrence of certain tracemakers in arid eolian environments, such as scorpions and spiders, which can produce Paleohelcura and Octopodichnus (Brady, 1947; Davis, Minter & Braddy, 2007), both common ichnotaxa in arid deserts (Krapovickas et al., 2016). The Octopodichnus Ichnofacies is dominated by arthropod trackways and was based on the study of Permian ichnoassemblages, illustrated by the Coconino Sandstone (Hunt & Lucas, 2007). In contrast, the Entradichnus Ichnofacies is characterized as dominated by simple shallow vertical and horizontal burrows, as well as meniscate trace fossils, having been based on the study of Jurassic examples, in particular the Navajo Sandstone (Ekdale, Bromley & Loope, 2007). It has been noted that the diverging characterization of these ichnofacies was the result of the disparate databases (Buatois & Mángano, 2011; Krapovickas et al., 2016). Undoubtedly, the apparent contrasting nature of Paleozoic and post-Paleozoic eolian ichnofaunas has been detrimental to a unifying approach to ichnofacies definition. In this regard, the presence of arthropod trackways in Mesozoic eolian deposits (like P. araraquarensis isp. Implications for the definition of the Octopodichnus-Entradichnus Ichnofacies of eolian environments nov.) helps to trace a continuity between Paleozoic and post-Paleozoic desert ichnofaunas, further reinforcing the notion of a single Octopodichnus-Entradichnus Ichnofacies for eolian deposits. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 CONCLUSIONS Paleohelcura araraquarensis isp. nov. is characterized by elliptical tracks and a narrow internal width to the trackway. Despite being included in Paleohelcura, an ichnogenus usually attributed to arachnids, Paleohelcura araraquarensis isp. nov. was most likely produced by a pterygote insect on the basis of neoichnological observations. The producer of Paleohelcura araraquarensis isp. nov. could have occupied one of the ecological roles that different species of Pterygota are capable of performing in modern dune deserts. It could have been a herbivore, or a carnivore (like carnivorous Carabidae in some modern dune deserts) or been part of the fauna of omnivores, being able to adopt herbivorous, carnivorous, and saprophagous diets when opportune. As an omnivore, like the abundant Tenebrionidae beetles in some modern dune deserts, it could have been Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 capable of relying on organic particles that accumulated among the sand grains of the dunes during dry periods with no plant growth. The presence of Taenidium isp. and Skolithos linearis burrows suggests that the Botucatu paleodesert would have had a detritivorous fauna like modern dune deserts, with the producers of these burrows having a compatible size with the plausible producers of Paleohelcura araraquarensis isp. nov. Based on the interpretation of the ichnofossil producers, it was possible to reconstruct the food web of this paleodesert, in which the producer of Paleohelcura araraquarensis isp. nov. could have been a primary consumer if it were a herbivorous or an omnivorous detritivorous insect, or a secondary consumer if it had been produced by predatory insects or omnivores relying on animal biomass. To date, Paleohelcura araraquarensis isp. nov. is only known from the Botucatu Formation, but the presence of such types of arthropod trackways in Mesozoic eolian deposits helps to trace a continuity between Paleozoic and post-Paleozoic desert ichnofaunas, further reinforcing the notion of a single Octopodichnus-Entradichnus Ichnofacies for eolian deposits. ACKNOWLEDGEMENTS We thank R.E. Ritzmann for providing high-speed videos of cockroaches walking and authorizing their use (for Fig. 9 and in Video S1); Dr. Stanislav N. Gorb for helpful discussions regarding insect and arachnid foot morphology; Silvina de Valais for provided photos of holotype and other specimens of Hexapodichnus casamiquelai; Emma Rainforth for sharing her thesis; Martin Harvey, Oliver Halsey, Ravi Sampaio for sharing their photographs for use in this publication; Ceri Thomas, Frederic Wierum, Gareth Monger, Nobumichi Tamura, Rafael Pasini for letting us modify and publish their drawings in this paper under CC-BY-SA 4.0; Ana Maria Ribeiro for letting us modify and publish the Fig. 1 map published in Revista Brasileira de Paleontologia. We are also grateful to the PeerJ reviewers Verónica Krapovickas and Anthony Shillito, and the PeerJ editor Claudia A. Marsicano for their comments that helped to improve this paper. Funding This work was funded by the National Council for Scientific and Technological Development (CNPq) of Brazil with a Master Degree grant to Bernardo de Campos Pimenta e Marques Peixoto (process No. 133988/2015-5) and a Ph.D. Degree grant (process number 141220/2018-0) to Bernardo de Campos Pimenta e Marques Peixoto. PeerJ’s Article Processing Charge (APC) was funded by the PROAP-CAPES. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions • Bernardo de C.P.e M. Peixoto conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. • M. Gabriela Mángano and Nicholas J. Minter performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. • Luciana Bueno dos Reis Fernandes performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. • Marcelo Adorna Fernandes conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. Grant Disclosures The following grant information was disclosed by the authors: National Council for Scientific and Technological Development (CNPq). PROAP-CAPES. 26/37 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Competing Interests Competing Interests The authors declare there are no competing interests. Data Availability The following information was supplied regarding data availability: The full resolution photographs of the quarry and the work fronts are available in Figs. S1 and S2. Indications of the series measured are available in Figs. S3 and S4. All the measurements taken are available in Table S1. The cockroach video used to produce Fig. 9 is available in Video S1. All the slabs with the ichnofossils (LPP-IC-0028, LPP-IC-0029, LPP-IC-0030, LPP-IC- 0031, LPP-IC-0032, LPP-IC-0033, LPP-IC-0034, LPP-IC-0035) are housed in Laboratório de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil. New Species Registration The following information was supplied regarding the registration of a newly described species: PublicationLSID: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B-75856AEAEAF Paleohelcura araraquarensis isp. nov. LSID: urn:lsid:zoobank.org:act:7D4303AE-BB63- 4474-B79C-AD39AB144917 Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.8880#supplemental-information. 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Comparing adherence to MDR-TB treatment among patients on self-administered therapy and those on directly observed therapy: non-inferiority randomized controlled trial
Trials
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© 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Comparing adherence to MDR‑TB treatment among patients on self‑administered therapy and those on directly observed therapy: non‑inferiority randomized controlled trial Clara Wekesa1*   , Christine Sekaggya‑Wiltshire1, Stella Zawedde Muyanja1, Ivan Lume1, Maria Sarah Nabaggala1, Rosalind Parkes‑Ratanshi1,2 and Susan Adakun Akello3 Abstract Background  Adherence is key to the treatment success of multi-drug resistant tuberculosis (MDR-TB) and pre‑ vention of community transmission. Directly observed therapy (DOT) is the recommended approach for the man‑ agement of patients with MDR-TB. Uganda implements a health facility-based DOT approach where all patients diagnosed with MDR-TB report to the nearest private or public health facility for daily observation of ingesting their medicines by a health care provider. Directly observed therapy is very costly for both the patient and health care system. It follows the assumption that MDR TB patients have a history of poor adherence to TB treatment. But only 21% of MDR-TB patients notified globally and 1.4–12% notified in Uganda had been previously treated for TB. The shift to all oral treatment regimen for MDR-TB provides an opportunity for the exploration of self-administered therapy for this group of patients even with use of remotely operated adherence technology. We are conducting a non-inferiority open-label randomized controlled trial to compare adherence to MDR-TB treatment among patients on self-adminis‑ tered therapy (measured by Medication Events Monitoring System (MEMS) technology) with a control group on DOT. Methods  We plan to enrol 164 newly diagnosed MDR-TB patients aged ≥ 8 years from three regional hospitals based in rural and urban Uganda. Patients with conditions that affect their dexterity and ability to operate the MEMS-oper‑ ated medicine equipment will not be eligible to participate in the trial. Patients are randomized to either of the two study arms: self-administered therapy with adherence being monitored using MEMS technology (intervention arm) or health facility-based DOT (control arm) and will be followed up monthly. Adherence is measured by the number of days the medicine bottle is open to access medication as recorded by the MEMS software in the intervention arm and treatment complaint days as recorded in the TB treatment card in the control arm. The primary outcome is the comparison of adherence rates between the two study arms. Discussion  The evaluation of self-administered therapy for patients with MDR-TB is important to inform cost-effec‑ tive management strategies for these patients. The approval of all oral regimens for the treatment of MDR-TB provides an opportunity for innovations such as MEMS technology to support sustainable options for MDR-TB treatment adherence support in low-resource settings. Trials Trials Trials Wekesa et al. Trials (2023) 24:326 https://doi.org/10.1186/s13063-023-07314-z Open Access Abstract *Correspondence: Clara Wekesa clara.wekesa@gmail.com Full list of author information is available at the end of the article *Correspondence: Clara Wekesa clara.wekesa@gmail.com Full list of author information is available at the end of the article © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 10 Wekesa et al. Trials Wekesa et al. Trials (2023) 24:326 Trial registration  Pan African Clinical Trials Registry, Cochrane #PACTR202205876377808. Retrospectively registered on 13 May 2022 Keywords  Multi-drug-resistant tuberculosis, Medication events monitoring system technology, Directly observed therapy, Differentiated care, TB treatment adherence, TB treatment outcomes, Resource-limited settings, Sub-Saharan Africa, TB drug PK levels Trial registration  Pan African Clinical Trials Registry, Cochrane #PACTR202205876377808. Retrospectively registered on 13 May 2022 Keywords  Multi-drug-resistant tuberculosis, Medication events monitoring system technology, Directly observed therapy, Differentiated care, TB treatment adherence, TB treatment outcomes, Resource-limited settings, Sub-Saharan Africa, TB drug PK levels Keywords  Multi-drug-resistant tuberculosis, Medication events monitoring system technology, Directly observed therapy, Differentiated care, TB treatment adherence, TB treatment outcomes, Resource-limited settings, Sub-Saharan Africa, TB drug PK levels Administrative information Title Comparing adherence to MDR-TB treatment among patients on self- administered therapy and those on Directly Observed Therapy: Non Inferiority Randomized Controlled Trial (WISECAP Study) Trial registration Pan African Clinical Trials Registry, Cochrane #PACTR202205876377808 on ­13th May 2022 Protocol version 23.09.21 Version 1.2 Name and contact information for the trial sponsor This Trial is under funding by Janssen Global Public Health, a division of Janssen Pharmaceutica NV, under grant number 1550786 Turnhoutseweg 30, B-2340 Beerse, Belgium Names, affiliations, and roles of protocol contributors Clara ­Wekesa1, Christine Sekaggya ­Wiltshire1, Stella Zawedde ­Muyanja1, Ivan Lume 1, Maria Sarah ­Nabagalla1, Rosalind Parkes-Ratanshi1,2, Susan Adakun ­Akello3 1. Infectious Diseases Institute, Mak‑ erere University 2. Roles and responsibilities: committees Department of Public Health, Cambridge University 3. TB unit, Mulago National Referral Hospital SA, CS, SZ, RPR and CW conceived the study, participated in the pro‑ posal and protocol development. SN wrote up the statistical methods of the study and IL contributed to development of the study tools. All authors read and approved the final manuscript. R l f Th f di b d did k Administrative information Background and rationale Multi-drug-resistant tuberculosis (MDR-TB), which is resistance to both isoniazid and rifampicin, poses a chal- lenge to TB control programmes globally because of the risk of worse treatment outcomes including death com- pared to drug-sensitive TB [1]. In 2020, 71% (2.1 of 3 million) of patients found to have pulmonary TB tested positive for rifampicin resistance, a ten percent incre- ment from the previous year [2]. Treatment success for drug-resistant TB remains sub-optimal, below the World Health Organization (WHO) target of 90%, increasing risk for disease relapse and continued community trans- mission. In Uganda, for the period between July 2019 and June 2020, 513 patients tested positive for rifampicin resistance and or MDR-TB (RR/MDR-TB) and the treat- ment success rate in the same period was 77.9% [3]. Global mortality resulting from MDR-TB remains high, and more so among people co-infected with HIV/AIDS. Mortality rate among people living with HIV (PLHIV) is 20% in comparison to 15% among mono-infected MDR- TB patients on treatment [4]. Observational studies done in Uganda have reported a similar mortality rate of 16–18% among MDR-TB patients on treatment [4, 5].ff In an effort to counter the effects of non-adherence, including poor treatment outcomes and transmission of resistance strains, directly observed therapy (DOT) was adopted as a strategy, where patients are observed as they take their daily dose by either a community health care worker, family member or health care worker. The obser- vation may occur at a health facility when the patient reports for their daily dose or may entail the health pro- vider visiting the patient at their home premise. Uganda implements a health facility-based DOT approach where all patients diagnosed with MDR-TB report to the near- est private or public health facility for DOT. Although treatment success rates on patients treated using the DOT strategy in Uganda have gradually improved from 70.6% in 2018 to 77.9% in 2020, these rates are still below the 90% target set by the WHO. Factors that contribute to low treatment success rates with DOT include lack of financial resources to make daily clinic visits, poor road infrastructure, long distances to nearest health facility, insufficient and overworked health care workers, and overburdened facilities [6, 7]. Therefore, innovations that Role of sponsor The funding body did not take part in the conception of the experiment and will be independent of the study conduct, analysis and interpretation of the results. The funding body did not participate in the write-up of this manuscript. The Data Monitoring Board and the Trial Steering Committee will make recommendations concerning the conduct of the trial based on periodi‑ cally shared data progress reports and interim results. The independent Trial Monitor will make recommen‑ dations of the adherence to study protocol and operating procedures as well as ensuring participant safety during the course the study. Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 3 of 10 outcomes between MDR-TB patients on self-adminis- tered therapy and standard DOT can support remote monitoring of MDR-TB treatment should be explored to reduce time and financial costs. A systematic review on strategies to improve adherence to MDR-TB treatment demonstrated that community DOT had varied effectiveness and that barriers to achieving adherence are patient-based; there is therefore a need to develop personalized interventions [8].h Methods We used the SPIRIT reporting guidelines in reporting our study methods [11]. Trial designh This study will be conducted as a multi-centre open- label randomized clinical trial with two parallel arms and 1:1 enrolment ratio (82 patients in the intervention arm and 82 controls). We shall enrol a total of 164 par- ticipants newly diagnosed with pulmonary MDR-TB or rifampicin-resistant TB (RR-TB) from 3 TB tertiary care centres from 2 regions of the country. The Medication Events Monitoring System (MEMS) (AARDEX®) is a portable smart medication device designed to monitor treatment adherence by transmit- ting electronic information on the number of times the medicine cap is opened, specifying dates and times. This information is taken to correlate with actual times that patients ingest their medication and can be used to vali- date the accuracy of self-reported adherence. The device is operated by a long-lasting battery and is able to trans- mit software information in real time and store infor- mation for many months following battery outage. The information on patient non-adherence as captured by MEMS technology has been shown to accurately corre- late with treatment outcomes among people living with HIV [9]. In addition, the device has also shown to out- perform self-report or pill counts and is acceptable to patients who found it easy to open and carry it on their person [9, 10]. Explanation for choice of comparators For the purposes of this study, we shall be using patients on standard of care (DOT), as the comparator group. This comprises patients with MDR-TB who have to report daily to a DOTS facility to be observed as they ingest their medication. DOT is the standard of care for MDR-TB patients and presently the only alternative for patient follow-up. Category 1—A bedaquiline-based injection-shorter treatment regimen (STR) (9 to 12 months) reserved for MDR-TB/RR-TB patients who have not been pre- viously treated with second-line drugs and in whom resistance to fluoroquinolones and second-line injectable agents has been excluded. Drug options for shorter treatment regimen include an intensive phase of 4 months (extended to 6 months in case of delayed sputum smear conversion) containing high-dose gati- floxacin or moxifloxacin, kanamycin, prothionamide, clofazimine, high-dose isoniazid, pyrazinamide, and ethambutol followed by a continuation phase of 5 months containing gatifloxacin or moxifloxacin, clo- fazimine, ethambutol and pyrazinamide [12]. Diagnosis and management of MDR‑TB Diagnosis of MDR-TB is based on Gene Xpert® testing of sputum for identification of resistance to rifampicin (RR- TB) followed by culture and drug susceptibility testing (DST) for phenotypic characterization; however, treat- ment is started prior to the receipt of culture and sensitiv- ity results due to long turnaround time of 10–16 weeks. Treatment options for MDR-TB/RR-TB currently include: Study setting Th d This study will be conducted in Mulago National Referral Hospital in the central region and Lira regional referral hospital in the northern region. These hospitals regis- ter the highest number of MDR-TB patients in Uganda. Both these hospitals are public government hospitals not for profit. The hospitals house specialized TB wards and clinics, operating a weekly TB clinic to follow up patients with drug-sensitive and drug-resistant TB on therapy and to initiate therapy for newly diagnosed patients. Being referral hospitals, these facilities receive patients referred from health facilities within a radius of up to 30km and in some cases even further. We plan to conduct an open-label randomized non- inferiority clinical trial among patients receiving MDR- TB treatment in Uganda to compare adherence rates among those using self-administered therapy with MEMS technology vs DOT, which is the current standard of care in Uganda for MDR-TB. We will correlate adher- ence information with serum drug concentrations and compare determined treatment outcomes between the two groups. Specific objectivesh The primary objective of the study is to determine if adherence to MDR-TB treatment among patients on self-administered therapy (measured by MEMS technol- ogy) is non-inferior to that among patients on directly observed therapy (DOT). The secondary objectives of the study include determining the correlation between MDR- TB drug serum concentrations and adherence as meas- ured by MEMS technology and comparing treatment Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 4 of 10 Category 2—The longer treatment regimens (18 to 20 months) based on core backbone agents but indi- vidualized depending on age, fluoroquinolone resist- ance, and site of disease. patterns of missed medication and brainstorming for solutions with the patient (Table 1). Determination of TB treatment outcomes At the end of therapy treatment (9 months for partici- pants that qualify for shorter MDR-TB/RR-TB treatment regimen and 18 months for those selected for the longer MDR-TB/RR-TB treatment regimen), clinical and labora- tory information is routinely compiled by each MDR-TB centre and forwarded to the National TB Program (NTP) review committee for discussion on consensus on treat- ment outcome based on a specified criterion (Table  2). TB treatment outcomes for this study will be as deter- mined by the review committee. Adherence to interventions Participants on the intervention arm will be given monthly study visits in which the adherence software shall be read to determine information on use of the medicine cap and box. Based on the information dis- played, study personnel provide tailored adherence coun- selling to the participants. For participants below the age of 18 and under guardianship, their caretakers will be included in the adherence counseling sessions. Study outcomes O i Our primary outcome is the adherence rates among MDR-TB patients on self-administered therapy vs DOT. Our secondary outcomes include (1) drug con- centrations and adherence data for patients on self- administered therapy vs DOT; (2) proportion of patients with sputum conversion at 2, 8 and 11 months in each study arm; and (3) MDR-TB treatment out- comes (cure, treatment completion, loss to follow-up or death) by study arm. Intervention arm For this trial, we shall use two kinds of MEMS-oper- ated devices, the MEMS cap and the MEMS Electronic DosePak® (EDP). At enrolment, patients on the self- administering arm will be provided with both devices; the MEMS cap will be placed over a medicine bottle containing bedaquiline. The concomitant medication will be packaged in a convenient pack containing all tab- lets to be swallowed each day and placed into the medi- cation box with the EDP® technology Patients will be instructed on how to use the MEMS devices. Each time a patient opens up the cap over the bedaquiline bottle or the lid of the medicine box, the time and date of this event will be immediately captured. Adherence data from the MEMS devices will be downloaded by the study team every month during the routine visits. The devices will provide information on specific calendar dates and times when the devices were opened. Information on the cal- endar days with event markings to signify the date and time when medication containers were opened will be displayed on a computer. The software will contain two separate input calendars for both the MEMS cap and the EDP box for each participant. This same adherence data will be shared with primary clinician and used to tailor adherence counselling given to patients, exploring Interventions: modifications Participants may withdraw from the study at any time at their own request, or they may be withdrawn at any time at the discretion of the investigator or sponsor for safety or behavioural reasons, or the inability of the subject to comply with the protocol required schedule of study visits or procedures. Patients who are withdrawn at any time of the study may be replaced. According to standard of care, patients with MDR-TB are managed as in-patients at central MDR treatment facilities (which include Mulago and Lira hospitals) in the intensive phase of treatment after which they are discharged and required to report to the central MDR treatment facility monthly for review and drug refills. Following discharge, during the continuation phase, patients are required to report daily to the nearest private or public facility registered under DOT programme [13]. Monthly reviews during routine care include safety labo- ratory investigations including a full blood count, liver and renal function tests, sputum culture, DST and adher- ence counseling. Eligibility criteria We shall enrol consented patients with newly diagnosed pulmonary MDR-TB and RR-TB patients aged ≥8 years, initiated on oral MDR-TB therapy (category 1 or 2). Patients with any condition that inhibits the use of the MEMS device, e.g. dexterity or debilitating arthritis, will be excluded from participation. Control: DOT No evidence of clinical deterioration Treatment completed (success) 1. A patient who has culture converted and 2. Received treatment for a total duration of 9–11 months and 3. Has less than 3 consecutive negative TB cultures at least 30 days apart and 4. No evidence of clinical deterioration Loss to follow-up A patient with treatment interrupted for 2 consecutive months and more Treatment failure 1. A patient who failed to culture convert by month 5 (shorter regimen) or month 6 (longer regimen) or 2. In the initial 6 months of treatment more than 2 of 5 cultures are positive or 3. Treatment stopped due to adverse events 4. Permanent discontinuation of two Group A drugs (BDQ, LZD, or LFX) in the first 6 months of the modified shorter regimen or 5. More than 2 new drugs added because of poor clinical response or 6. Panel decision to terminate any further DR-TB treatment Died A patient who died for any reason during treatment Definition Time from initiation of treatment to the time point where the patient has two consecutive negative cultures 1. A patient who has culture converted and 2. Received treatment for a total duration of 9–11 months and 3. Has at least 3 consecutive negative TB cultures at least 30 days apart and 4. No evidence of clinical deterioration 1. A patient who has culture converted and 2. Received treatment for a total duration of 9–11 months and 3. Has less than 3 consecutive negative TB cultures at least 30 days apart and 4. No evidence of clinical deterioration A patient with treatment interrupted for 2 consecutive months and more 1. A patient who failed to culture convert by month 5 (shorter regimen) or month 6 (longer regimen) or 2. In the initial 6 months of treatment more than 2 of 5 cultures are positive or 3. Treatment stopped due to adverse events 4. Permanent discontinuation of two Group A drugs (BDQ, LZD, or LFX) in the first 6 months of the modified shorter regimen or 5. More than 2 new drugs added because of poor clinical response or 6. Panel decision to terminate any further DR-TB treatment A patient who died for any reason during treatment Time to culture conversion Treatment completed (success) Died A patient who died for any reason during treatment Recruitment In order to enrol the required sample size in the stipu- lated time frame of the study, we shall enrol from sites with high volume numbers of MDR-TB in the country, which include Mulago National Referral Hospital and Lira Regional Referral Hospital. We shall also endeav- our to enrol all eligible participants at each clinic day at both sites. Randomization and allocation of intervention for the patients using MEMS technology is ≥ 70%, with study power of 0.9, precision of 0.05, and 20% loss to follow-up rate. We therefore require a sample size of 164 patients (82 in each arm) to demonstrate a 19% incre- ment in MDR-TB treatment success. The intervention (MEMS technology) approach is considered non-inferior (19%) to the control (DOT) approach if the lower limit of the 95%CI of the difference between the two groups (i.e. MEMS group minus DOT group) is > −10%. Blinded randomization will be performed by the study team (medical officer or study nurse) at the time of enrol- ment using an automated randomization sequence within the Redcap data capture tool. The randomization will be done in real time on the Redcap tool as it will be Inter- net enabled. Participants will be randomized to either intervention arm (arm 1; self-administered therapy) or control arm (arm 2; DOT). Stratified randomization will be performed according to age group and gender. Due to the nature of the intervention and organization of patient care, it will not be possible to blind either participants or study staff to the study arm allocation Control: DOT Device: MEMS CAP without LCD display and EDP® technology on medicine box. Standard MEMS devices record the time and date when the bottle is opened The MEMS technology automatically complies drug dosing history information by electronically recording the date and time of each opening of the medication cap and or box Behavioural: Adherence counselling. Patients will receive adherence counselling as scheduled by their primary care health providers Patients report daily to the nearest health unit to be observed by a health care pro‑ vider as they ingest their medication Behavioural: Adherence counselling. Patients will receive adherence counsel‑ ling as scheduled by their primary care health providers Table 2  Description of MDR-TB treatment outcomes Outcome Definition Time to culture conversion Time from initiation of treatment to the time point where the patient has two consecutive negative cultures Cured 1. A patient who has culture converted and 2. Received treatment for a total duration of 9–11 months and 3. Has at least 3 consecutive negative TB cultures at least 30 days apart and 4. No evidence of clinical deterioration Treatment completed (success) 1. A patient who has culture converted and 2. Received treatment for a total duration of 9–11 months and 3. Has less than 3 consecutive negative TB cultures at least 30 days apart and 4. No evidence of clinical deterioration Loss to follow-up A patient with treatment interrupted for 2 consecutive months and more Treatment failure 1. A patient who failed to culture convert by month 5 (shorter regimen) or month 6 (longer regimen) or 2. In the initial 6 months of treatment more than 2 of 5 cultures are positive or 3. Treatment stopped due to adverse events 4. Permanent discontinuation of two Group A drugs (BDQ, LZD, or LFX) in the first 6 months of the modified shorter regimen or 5. More than 2 new drugs added because of poor clinical response or 6. Panel decision to terminate any further DR-TB treatment Died A patient who died for any reason during treatment Table 2  Description of MDR-TB treatment outcomes Outcome Definition Time to culture conversion Time from initiation of treatment to the time point where the patient has two consecutive negative cultures Cured 1. A patient who has culture converted and 2. Received treatment for a total duration of 9–11 months and 3. Has at least 3 consecutive negative TB cultures at least 30 days apart and 4. Sample size Based on records from the NTP, treatment success rate for MDR-TB is 77.9%; from clinical trials, adequate adherence has been defined as 76–80% adherence rate; and from a meta-analysis, it has shown that 70% of patients on MDR-TB therapy are adherent [14, 15]. Our sample size calculation assumed that the adherence rate Page 5 of 10 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Table 1  Description of study arms Allocation concealment mechanismh The designing of the randomization sequence within the Redcap tool was performed by a biostatistician who is blinded from the participant enrolment activities. The variables used to design the randomization sequence included gender and age group. At the stage of enrol- ment, the randomization is conducted in real time on the Internet-enabled Redcap tool by the member of the study team conducting the enrolment. The study team have no control over the randomization sequence. Control arm Patients randomized to the control arm will receive daily DOT provided by a peripheral health facility close to their residence. Under the DOT programme, patients Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 6 of 10 report to the nearest DOT provider facility daily and drug intake is observed. On each visit, the TB card is ticked off in correspondence with the number of days they made a visit to the health centre and this card is retained at the health facility. At the monthly visits, the TB card is taken to the central MDR treatment facility for review by the managing clinician. At this point, the study team will record the number of doses taken/missed according to the TB card. Participants will receive adherence counsel- ling on all routine visits at the central facility (Table 1). daily), it was not feasible to blind study participants, pri- mary care providers, the study team and or the data manager. report to the nearest DOT provider facility daily and drug intake is observed. On each visit, the TB card is ticked off in correspondence with the number of days they made a visit to the health centre and this card is retained at the health facility. At the monthly visits, the TB card is taken to the central MDR treatment facility for review by the managing clinician. At this point, the study team will record the number of doses taken/missed according to the TB card. Participants will receive adherence counsel- ling on all routine visits at the central facility (Table 1). Data collection plan All participants will be reviewed by the study team monthly during routine visits (Table  3). On the ini- tial visit, participants will be taken through a screen- ing questionnaire to assess eligibility. Patients found to be eligible will then be taken through the informed consent process in the language of their preference. Enrolled participants will provide demographic infor- mation including age and gender. At each monthly study visit, participants will be taken through an interviewer-led questionnaire to capture clinical his- tory (reported adverse events, treatment outcomes) and adherence information (adherence data down- loads from MEMS devices, adherence data from TB treatment cards, pills counts, and participant-reported adherence on days of treatment missed). Adherence data will be used to provide tailored counselling for each participant to encourage treatment completion and this data will be shared with the participant’s pri- mary clinician. Laboratory assessments Blood sampling to measure the concentrations of bedaquiline and linezolid will be conducted on months 2, 8 and 11 of treatment. Drug intake on the days for blood sampling will be performed by DOT and blood samples taken off prior to drug intake and 1-h post-drug intake. The blood samples will be placed in cooler boxes Allocation implementationhfi The study team (medical officer and or study nurse) hav- ing determined participant eligibility enter the partici- pant gender and age into the automated randomization sequence within the Redcap tool and click a button to determine study allocation and or study arm in real time. * Drug susceptibility testing: sputum culture and sputum DST will be provided as standard of care by the health facility Statistical: outcomes Adherence assessment Th i The study follow-up duration will be 24 months to cater for patients on both short- and long-term oral MDR- TB therapy. Treatment duration will be approximately 9 to 18 months: 9 months for participants that qualify for shorter MDR-TB/RR-TB treatment regimen and 18 months for those selected for the longer MDR-TB/RR-TB treatment regimen. The primary outcome is the overall adherence rates among MDR-TB patients on self-administered therapy and on DOT. For the participants on the intervention arm, opening of the devices will be equated to a patient swallowing their medication and adherence will be derived from the number of days the device was opened as a proportion of the total number of days in the entire period of their MDR-TB treatment. Patients on the intervention arm will be required to take no less than 70% of their medication to be considered as having good adherence. For participants on the control arm (DOT), adherence information will be obtained from their TB treatment cards. Adherence rates will be calculated from the number of days that they report to the DOTS facility as a proportion of the total number of treatment complaint days in the entire period of their MDR-TB treatment. Participants who take their medication at least 80% of the time (missing no more than 5 days each month) will be considered as having good adherence. To ensure data quality, study information is captured with an e-questionnaire in Redcap that has been designed to send real-time notifications for inputted variables that may be out of range. The tool also does not accept for the person collecting data (medical officer and or study nurse) to verify the tool, but is redirected to the study coordinator and Data Manager to do the final verification for correct and missing data. In addition, the entire study team was trained afore on the use of the study Redcap tool and on other study-related standard operating pro- cedure manuals including the collection of blood speci- mens for continued reference. In the event of a protocol violation, retraining on study protocol and procedures will be conducted. The primary analysis will be a comparison of overall adherence in each arm. Adherence by self-administra- tion (proportion of expected openings of the MEMS devices) and DOT (proportion of DOT visits) will also be compared at 2 months and 6 months in the interven- tion and control arms. Retention of study participants in the Trial For all persons approached to participate in the study, full information will be provided about the trial its aims, study procedures and duration. Participants will be given an opportunity to ask any questions they may have at the time of consent and any other time of the study. Data from participants that have withdrawn and or been withdrawn from the study will be retained for sub-analysis. All participants that have been withdrawn from study participation will be replaced to ensure the sample size is achieved. Data management Clinical data will be entered into a study-specific data- base by trained designated study staff on a regular basis from completed Case Record Forms (CRF) using Red- cap. Information will primarily be captured using Red- cap and CRFs will be printed out for filing and storage. Blinding (masking) Given the nature of the intervention (a visible medicine box and the requirement not to report to a DOT facility Table 3  Description of study procedures * Drug susceptibility testing: sputum culture and sputum DST will be provided as standard of care by the health facility Table 3  Description of study procedures Wekesa et al. Trials (2023) 24:326 Page 7 of 10 Case Record Forms and other source documents will be kept in locked cabinets. No participant-identifying information will be disclosed in any publication or at any conference activities arising from the study. The participant data captured in the Redcap tool will be accessed in real time by the data manager, who will provide data queries for the study team to address. In addition, the Redcap data collection tool has been fash- ioned to generate automated checks on entry of certain variables that are out of range. Data management pro- cedures are detailed in the standard operating manual. and transported to the laboratory on dry ice for samples from Lira Hospital and in a cooler box for samples from Mulago Hospital, processed and stored at −4 to −80 °C. Pharmacokinetic analysis will be performed at the Infectious Diseases Institution (IDI) translation labora- tory. Quantification of the drug concentrations will be performed in batches using validated high-performance liquid chromatography (HPLC) and mass spectrometry at the Translation Laboratory of the Infectious Diseases Institute, Makerere University using MS. Sputum smears and cultures will be performed as part of routine care and testing conducted at the government reference laboratory.h Statistical: outcomes Adherence assessment Th i The chi-square test and 1-way analysis of variance models will be used to compare out- comes by randomization arm. We shall use independ- ent samples t-test to test for statistical difference mean adherence rates in the patient in the intervention and control arms. We will use an intent-to-treat approach (analysed all participants regardless of drug refills) to compare adherence outcomes by randomization arm using a generalized estimating equations model using repeated measures with identity link, exchangeable cor- relation structure and robust variance estimates. Data monitoring: formal committeeh Data monitoring: formal committeeh Harms All observed or volunteered adverse events (AEs) regard- less of treatment group or suspected causal relationship will be reported. During each study visit, the study doc- tors will assess AEs that may have occurred since the pre- vious visit. The severity of adverse events will be graded according to the National Institute of Health Division of AIDS (DAIDS) classification system for reporting adverse experiences in adults [16]. Additional analyses and handling of missing data y g g Interim data analysis will be conducted once 50% of the overall sample size has been achieved with the purpose of guiding the data monitoring board on recommendations for the study. Missing data will be dealt with as missing covariate and outcome data. As a generalized estimating equations model will be used, missing outcome data will be dealt with using a missing at random assumption. If > 3% of data for covariate data is missing, multiple impu- tation will be used for the primary analysis. Given the way the adherence rate is computed, we anticipate that there may be a number of ways that the outcome meas- ure could be missing. These include loss to follow-up and or partial information collected at anyone study visit. A further analysis will be performed in which missing pri- mary outcome data will be imputed. Multiple imputation will be also performed on missing covariate and outcome data for all secondary clinical analyses in a similar way to that performed for the primary analysis. Auditing d An independent study monitor will be assigned to the study. The study monitor will perform at least two study audits annually to assess adherence to study protocol, SOPs, observe for study deviations and integrity of con- duct of the study. The study monitor will provide a writ- ten report of their findings and recommendations after each study visit. Secondary outcomes To assess correlation of specific MDR-TB drug serum concentrations with observed adherence, bedaquiline Wekesa et al. Trials (2023) 24:326 Page 8 of 10 Page 8 of 10 Steering Committee. The DMB is chaired by Dr. Simon Kasasa Senior Lecturer, in the Department of Epidemiol- ogy & Biostatistics at Makerere University School of Public Health in the College of Health Sciences, and other mem- bers include Dr. Suzanne Kiwanuka, a review Coordinator for Centre for Systematic Reviews on Human Resources for Health; Dr Paul Otuba, National MDR-TB Coordina- tor at the NTP; and Dr. Dathan Byonanebye, a lecturer in the Department of Community Health and Behavioural Sciences, Makerere University. The DMB is independent from the sponsor and has no competing interests. and linezolid pre-dose concentrations ­(Ctrough) will be used. To cater for those who may be adherent but have low bedaquiline or linezolid concentrations, we will use of cut-off value of bedaquiline ­Ctrough from previous stud- ies, below which a participant will be classified as non- adherence. Linezolid will be used to assess more recent adherence than bedaquiline. We will compare time to sputum culture conversion in the intervention and control arm using time to event analysis. We shall also use chi-square testing to com- pare treatment success rates in the intervention and control arms. Treatment success will be evaluated using a composite outcome that includes TB cure (for micro- biologically diagnosed patients) and TB treatment com- pletion (for clinically diagnosed TB). We will undertake per-protocol and intention to treat analyses to compare TB cure rates in the two study arms. We will determine the proportions of patients retained in care of retention in care at 2 and 6 months in the two study arms using chi-square tests. Study outcomes by randomization arm will be compared using a generalized estimating equa- tions (GEE) model using repeated measures with iden- tity link, exchangeable correlation structure and robust variance estimates. Adjusted coefficients along with their corresponding 95% CIs will be generated. Statistical sig- nificance will be set at the P < .05 level and statistical analyses performed using Stata version 14 (StataCorp, College Station, TX, USA). Data monitoring: Interim analysishi The first interim analysis will be conducted when 50% of the participants complete the study medication. Trial monitoring is conducted independent of the investiga- tors by internal monitors at the Infectious Diseases Insti- tute. The Trial Steering Committee is composed of Stavia Turyahabwe, program manager at the NTP in Uganda; Angom Esther, Nurse in charge at Lira Regional Referral Hospital; Dr Agnes Kiragga, a senior statistician with expe- rience in HIV clinical trials and cohort studies; and Eliza- beth Tindyebwa, a lay representative from the Friends’ Council (patient group) at the Infectious Disease Insti- tute, Uganda. Since this trial is unblinded, the DMB will have access to intervention assignment. They will ensure that we protect the safety of the participants while assur- ing study integrity. The DMB will periodically (every 6 months) review the data, including interim analyses, and communicate any concerns to the study team and TSC. Data access Access to the database will be given to authorized per- sonnel only (study investigators, project coordinator and data manager) and a log of authorized personnel will be stored in the trial master file. Dissemination plans Dissemination workshops will be held with study par- ticipants, funders and other stakeholders to commu- nicate the results of the study. The study results will be presented to officials in charge of the National TB Pro- gram in Uganda and at other national and international conferences. We shall also aim to publish in high-impact, peer-reviewed journals with a focus on open access. Full anonymity of participants’ details will be maintained throughout. We shall adhere to the International Com- mittee of Medical Journal Editor (ICMJE) recommenda- tions on the definition and roles of authorship. We shall not procure the services of professional writers. Declaration of interestsh The authors have no competing interests to declare. Ethical considerationsh The independent Data Monitoring Board (DMB) will make recommendations concerning the study to the Trial The study has received approvals from the Mulago Hospital Research and Ethics Committee (Ref No: Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 9 of 10 MHREC2125), the Uganda National Council for Science and Technology (Ref No: HS1796ES) and the National Drug Authority (RefNo:476/NDA/DPS/08/2021). The informed consent/assent document are in compliance with GCP and local regulatory authorities. Participants are taken through the consent process by the study team (Medical Officer or Study Nurse). Assent is requested from children between 8 and 17 years, and in addition, consent is sought from their parents or legally authorized representative. Informed consent is sought from partici- pants aged 18 years and above and emancipated minors. Approval for protocol amendments shall be submitted to the Mulago Hospital Research and Ethics Committee for approval and then communicated to the National Drug Authority and The Uganda National Council for Science and Technology before implementation. Participants will be free to withdraw at any point of the study without any compromise to receiving their standard therapy. Children aged 8–17 years that do not provide assent but whose guardians have signed consent or vice versa will not be included in the study. Discussionh The introduction of new strategies like the use of shorter course regimen still does not address the operational limi- tations of DOT, including the unguaranteed accuracy of patient adherence information. In as much as MEMS technology may not be absolutely accurate in estimating patient adherence since opening the device may not imply drug ingestion, it does provide a patient-centred solution, relieving the burden on patients to meet an obligation of frequent clinic visits and maintaining patient autonomy [17]. MEMS technology also has the potential to meaning- fully engage both caregivers and patients via its interactive functions such as LCDs on drug balances, reminders and real-time electronic display of patient adherence informa- tion. The adherence data abstracted from the MEMS tech- nology enables caregivers to understand their patients’ adherence patterns and using this information, tailor coun- selling information, a better approach to improving adher- ence. The limitations of the MEMS technology include its inability to capture patient records on taking more than the required dosing and pill dumping [18]. The MEMS technology may also overestimate adherence by being unable to differentiate other instances in which the medi- cine devices are opened simply out of curiosity and altered behaviour as a result of being observed. The device may also be associated with inconvenience (on person carriage) and device fatigue which can negatively impact adherence [9]. Despite these limitations, this is one of the few studies that may address some of the facility and patient-based fac- tors associated with non-adherence to MDR-TB therapy. It may also provide a cheaper and sustainable alternative for effective long-term follow-up of patients with MDR-TB with favourable outcomes including completion of therapy. The addition of pharmacokinetic data further strengthens adherence assessment in addition to the devices, patient self-reports and pill counts. We believe this study will dem- onstrate how MEMS technology may be used to remotely assess and therefore strengthen adherence in patients with tuberculosis for favourable treatment outcomes. Confidentialityf All study staff will ensure the protection of participant personal data and will not include participant names on any forms, reports, publications, or in any other disclosures. Protocol Version: 1.2 23.09.2021 MRHEC Ref No: MHREC2125 UNCST Ref No: HS1796ES NDA Ref No: 476/NDA/DPS/08/2021 Start date: 15.11.2021 Protocol Version: 1.2 23.09.2021 MRHEC Ref No: MHREC2125 UNCST Ref No: HS1796ES NDA Ref No: 476/NDA/DPS/08/2021 Start date: 15.11.2021 Protocol Version: 1.2 23.09.2021 MRHEC Ref No: MHREC2125 UNCST Ref No: HS1796ES NDA Ref No: 476/NDA/DPS/08/2021 Start date: 15.11.2021 Approximate date for completion of recruitment: 31.01.2023 Participant enrolment is at 70% and expected completion is January of 2023. Page 10 of 10 Page 10 of 10 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Abbreviations BDQ Bedaquiline CRF Case Report Form DAIDS National Institutes of Health Division of AIDS DMB Data Monitoring Board DOT Directly observed therapy DR-TB Drug-resistant tuberculosis DST Drug susceptibility testing EDP Electronic DosePak® GCP Good Clinical Practice GEE Generalized estimating equations HF-DOT Health facility-based directly observed therapy HIV Human immunodeficiency virus HPLC High-performance liquid chromatography IDI Infectious Diseases Institute LCD Liquid crystal display LFX Levofloxacin LZD Linezolid MDR-TB Multi-drug-resistant tuberculosis MEMS Medications Events Monitoring System MHREC Mulago Hospital Research Ethics Committee NTLP National Tuberculosis and Leprosy Program PK Pharmacokinetics PLHIV People living with HIV RR-TB Rifampicin-resistant tuberculosis SAE Severe adverse event SMS Short Messaging Service WHO World Health Organization 5. Baluku JB, Nakazibwe B, Naloka J, Nabwana M, Mwanja S, Mulwana R, et al. Treatment outcomes of drug resistant tuberculosis patients with multiple poor prognostic indicators in Uganda: a countrywide 5-year retrospective study. J Clin Tuberc Other Mycobact Dis. 2021;23:100221. g g y y retrospective study. J Clin Tuberc Other Mycobact Dis. 2021;23:1002 6. Fiseha D, Demissie M. Assessment of Directly Observed Therapy (DOT) following tuberculosis regimen change in Addis Ababa, Ethiopia: a quali‑ tative study. BMC Infectious Diseases. 2015;15(1):405. 7. Queiroz EM, De-La-Torre-Ugarte-Guanilo MC, Ferreira KR, Bertolozzi MR. Tuberculosis: limitations and strengths of directly observed treatment short-course. Revista latino-americana de enfermagem. 2012;20(2):369–77. 8. Pradipta IS, Houtsma D, van Boven JFM, Alffenaar JC, Hak E. Interventions to improve medication adherence in tuberculosis patients: a systematic review of randomized controlled studies. NPJ Prim Care Respir Med. 2020;30(1):21. 9. Shellmer DA, Zelikovsky N. The challenges of using medication event monitoring technology with pediatric transplant patients. Pediatr Trans‑ plant. 2007;11(4):422–8. 10. Schoenthaler A, Ogedegbe G. Patients’ perceptions of electronic monitoring devices affect medication adherence in hypertensive African Americans. Ann Pharmacother. 2008;42(5):647–52. 11. Chan AW, Tetzlaff JM, Gøtzsche PC, Altman DG, Mann H, Berlin JA, et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clini‑ cal trials. BMJ (Clinical research ed). 2013;346: e7586. 12. Competing interests The authors declare that they have no competing interests. Received: 9 December 2022 Accepted: 17 April 2023 Received: 9 December 2022 Accepted: 17 April 2023 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Authors’ contributions 17. Alipanah N, Jarlsberg L, Miller C, Linh NN, Falzon D, Jaramillo E, et al. Adherence interventions and outcomes of tuberculosis treatment: a systematic review and meta-analysis of trials and observational studies. PLoS medicine. 2018;15(7): e1002595. 17. Alipanah N, Jarlsberg L, Miller C, Linh NN, Falzon D, Jaramillo E, et al. Adherence interventions and outcomes of tuberculosis treatment: a systematic review and meta-analysis of trials and observational studies. PLoS medicine. 2018;15(7): e1002595. 18. Lyimo RA, van den Boogaard J, Msoka E, Hospers HJ, van der Ven A, Mushi D, et al. Measuring adherence to antiretroviral therapy in northern Tanzania: feasibility and acceptability of the Medication Event Monitoring System. BMC Public Health. 2011;11(1):92. Protocol Version: 1.2 23.09.2021 MRHEC Ref No: MHREC2125 UNCST Ref No: HS1796ES NDA Ref No: 476/NDA/DPS/08/2021 Start date: 15.11.2021 Prasad R, Gupta N, Banka A. Shorter & cheaper regimen to treat multidrug-resistant tuberculosis: a new hope. Indian J Med Res. 2017;146(3):301–3. 13. WHO consolidated guidelines on drug-resistant tuberculosis treatment. Geneva; World Health Organisation; 2019. 13. WHO consolidated guidelines on drug-resistant tuberculosis treatment. Geneva; World Health Organisation; 2019. 14. Vernon A, Fielding K, Savic R, Dodd L, Nahid P. The importance of adher‑ ence in tuberculosis treatment clinical trials and its relevance in explana‑ tory and pragmatic trials. PLoS Med. 2019;16(12):e1002884. 14. Vernon A, Fielding K, Savic R, Dodd L, Nahid P. The importance of adher‑ ence in tuberculosis treatment clinical trials and its relevance in explana‑ tory and pragmatic trials. PLoS Med. 2019;16(12):e1002884. Supplementary Information 15. Nellums LB, Rustage K, Hargreaves S, Friedland JS. Multidrug-resistant tuberculosis treatment adherence in migrants: a systematic review and meta-analysis. BMC medicine. 2018;16(1):27. 15. Nellums LB, Rustage K, Hargreaves S, Friedland JS. Multidrug-resistant tuberculosis treatment adherence in migrants: a systematic review and meta-analysis. BMC medicine. 2018;16(1):27. 16. Wandeler G, Mulenga L, Vinikoor MJ, Kovari H, Battegay M, Calmy A, et al. Liver fibrosis in treatment-naive HIV-infected and HIV/HBV co- infected patients: Zambia and Switzerland compared. Int J Infect Dis. 2016;51:97–102. Author details 1 Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 1 Infectious Diseases Institute, Makerere University, Kampala, Uganda. 2 Depart‑ ment of Public Health, Cambridge University, Cambridge, UK. 3 TB unit, Mulago National Referral Hospital, Kampala, Uganda. Received: 9 December 2022 Accepted: 17 April 2023 References 1. Lange C, Dheda K, Chesov D, Mandalakas AM, Udwadia Z, Hors‑ burgh CR Jr. Management of drug-resistant tuberculosis. The Lancet. 2019;394(10202):953–66. 2. Organisation WH. Global Tuberculosis Report. World Health Organisation; 2018. 3. Uganda National TB and Leprosy Program Jul 2019 – June 2020 Report. Republic of Uganda Ministry of Health; 2020. 4. Kizito E, Musaazi J, Mutesasira K, Twinomugisha F, Namwanje H, Kiyemba T, et al. Risk factors for mortality among patients diagnosed with multi- drug resistant tuberculosis in Uganda- a case-control study. BMC Infec‑ tious Diseases. 2021;21(1):292. 1. Lange C, Dheda K, Chesov D, Mandalakas AM, Udwadia Z, Hors‑ burgh CR Jr. Management of drug-resistant tuberculosis. The Lancet. 2019;394(10202):953–66. 2. Organisation WH. Global Tuberculosis Report. World Health Organisation; 2018. 3. Uganda National TB and Leprosy Program Jul 2019 – June 2020 Report. Republic of Uganda Ministry of Health; 2020. 4. Kizito E, Musaazi J, Mutesasira K, Twinomugisha F, Namwanje H, Kiyemba T, et al. Risk factors for mortality among patients diagnosed with multi- drug resistant tuberculosis in Uganda- a case-control study. BMC Infec‑ tious Diseases. 2021;21(1):292.
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The Association of Palmitoylethanolamide with Luteolin Decreases Autophagy in Spinal Cord Injury
Molecular neurobiology
2,015
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7,331
2 Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 South Grand Blvd, St Louis, MO 63104, USA 1 Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres, 31, 98166 Messina, Italy Rosalba Siracusa1 & Irene Paterniti1 & Giuseppe Bruschetta1 & Marika Cordaro1 & Daniela Impellizzeri1 & Rosalia Crupi1 & Salvatore Cuzzocrea1,2 & Emanuela Esposito1 Received: 9 March 2015 /Accepted: 26 June 2015 /Published online: 5 July 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Spinal cord injury (SCI) is a devastating condition of the central nervous system (CNS) often resulting in severe functional impairment and for which there are not yet restor- ative therapies. In the present study, we performed a widely used model of SCI to determine the neuroprotective propriety of palmitoylethanolamide (PEA) and the antioxidant effect of a flavonoid luteolin (Lut), given as a co-ultramicronized com- pound co-ultraPEALut. In particular, by western blot analysis and immunofluorescence staining, we investigated whether this compound (at the dose of 1 mg/kg) was able to modulate autophagy. Our results showed that treatment with co- ultraPEALut after SCI reduced the expression of proteins pro- moter of autophagy such as Beclin-1 and microtubule- associated protein 1A/1B-light chain 3 (MAP-LC3). In con- trast, this compound decreased the levels of mammalian target of rapamycin (mTOR), p-Akt, and p-70S6K which are pro- teins that inhibit autophagy. These data confirmed that the protective role of co-ultraPEALut is associated with inhibition of excessive autophagy and regulation of protein degradation. Therefore, treatment with co-ultraPEALut could be consid- ered as a possible therapeutic approach in an acute traumatic lesion like SCI. TRACTED Keywords Neuroprotection . Anti-inflammatory . Anti-oxidant . Autophagy . Spinal compression Introduction D ART The Association of Palmitoylethanolamide with Luteolin Decreases Autophagy in Spinal Cord Injury & sito1 otection . Anti-inflammatory . phagy Spinal compression RTICLE & Marika Cordaro1 & 1,2 & Emanuela Esposito1 2015 rlink.com Keywords Neuroprotection . Anti-inflammatory . Anti-oxidant . Autophagy . Spinal compression Introduction D ARTICLE Rosalba Siracusa1 & Irene Paterniti1 & Giuseppe Bruschetta1 & Marika Cordaro1 & Daniela Impellizzeri1 & Rosalia Crupi1 & Salvatore Cuzzocrea1,2 & Emanuela Esposito1 Mol Neurobiol (2016) 53:3783–3792 DOI 10.1007/s12035-015-9328-6 Mol Neurobiol (2016) 53:3783–3792 DOI 10.1007/s12035-015-9328-6 Co-ultramicronization Process of PEA and Lut E The co-ultramicronization process was performed using jet mill equipment (Sturtevant, Inc., 348 Circuit Street Hanover, MA, USA) with a chamber of 300 mm in diameter, operated with Bspiral technology^ and driven by compressed air at 10 to 12 bars. Crashing was determined by the high number of collisions that occurred among particles as a result of the high level of kinetic (not mechanical) energy. This process is effec- tive not only in reducing product particle size but also in modifying crystalline structure. Scanning electron microscopy showed an intimate intermixing of PEA and Lut, while anal- ysis by differential scanning calorimetry and X-ray diffraction indicated transformation into a crystalline form different from the original two, definable as Ba higher energy content form.^ The composite showed the following particle size distribution: 96 % <10 μm, 80 % <5 μm, and 40 % <2 μm. Co- ultraPEALut (Epitech Group s.r.l.) was dissolved in 10 % eth- anol (Sigma-Aldrich, St Louis, MO, USA) and used at a con- centration of 1 mg/kg in the in vivo study. ED ARTICLE In recent years, the pathophysiology of SCI has been a focus of extensive studies; animal models have been proved to be important tools in this field and are employed to inves- tigate the mechanisms of primary and secondary injury. Pre- vious studies have shown that autophagy plays a key role in secondary injury in both animal models and human tissue by causing progressive degeneration of the spinal cord [15, 16]. However, the molecular pathway of secondary injury and the role of autophagy in the recovery of SCI remain unclear. Fol- lowing acute spinal cord injury, an increase in mTOR expres- sion and p70S6K activity also may be required for functional improvement [17]. The involvement of autophagic mecha- nism underlying spinal neuroprotective effects of palmitoylethanolamide (PEA) and luteolin association was never been investigated. Thus, we studied the autophagic pathway involved in spinal cord compression and to explore the effects of the treatment with a new co-ultramicronized composite of N-PEA and luteolin (Lut) on autophagy expres- sion. This formulation (co-ultraPEALut) is based on associa- tion of anti-inflammatory PEAwith anti-oxidant Lut on a 10:1 mass basis. We have previously demonstrated that treatment with co-ultraPEALut significantly reduced the inflammatory process in an ex vivo model of spinal cord organotypic cul- tures and in experimental mouse models of TBI [18, 7]. Introduction AR Spinal cord injury (SCI) is defined as an acute traumatic lesion of neural elements in the spinal canal (spinal cord and cauda equina), resulting in a change, either temporary or permanent, in normal motor, sensory, or autonomic function. Spinal cord injury usually begins with a sudden, traumatic blow to the spine that causes local segmental damage to the spinal cord, which is called primary injury [1, 2]. The primary damage to the tissue is followed by a second phase of tissue degenera- tion, the secondary injury that can occur over weeks or even months. In secondary injury, acute inflammation can develop into a chronic process if feedback mechanisms fail to inhibit amplification of the inflammatory response. Chronic inflam- mation leads to a continuous influx of neutrophils, macro- phages, lymphocytes, and eosinophils from the circulation, causing more destruction and scarring of tissue [3]. Cell death resulting from all of these mechanisms occurs through necrot- ic and apoptotic phenomena or autophagy. ED A a poss b e t e apeut c ike SCI. RETR Concern about autophagy has recently increased because of its potential role in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, where autophagy may be a protective mechanism [4]. Earlier studies also demon- strated increased autophagic activity at lesion sites after cere- bral hypoxia-ischemia (HI) injury [5], intracerebral hemor- rhage (ICH) [6], and traumatic brain injury (TBI) [7, 8]. * Emanuela Esposito eesposito@unime.it RE 1 Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres, 31, 98166 Messina, Italy Autophagy, literally Bself-eating, is now recognized as an intracellular catabolic mechanism for the degradation, elimi- nation, and recycling of long-lived proteins and unwanted organelles in a cell during development and under stress con- ditions [9, 10]. Therefore, autophagy plays an important role Mol Neurobiol (2016) 53:3783–3792 3784 in homeostasis and also to a kind of cell death, which is known as autophagic cell death [11, 12]. It begins with the formation of double-membraned vesicle that subsequently engulfs cyto- plasmic components, including cytosolic proteins and organ- elles, to become autophagosomes (APs). APs fuse with lyso- somes to form autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. Several key molecular components participate in the initiation, pro- gression, and completion of autophagy, like the mammalian target of rapamycin (mTOR) that inhibits autophagy, whereas Beclin 1 and LC3 promote it [8, 13]. Introduction AR However, autophagy seems to be a double-edged sword that protects cells when it is moderately activated, yet its excessive activation can induce autophagic cell death [14]. dark cycle. The animals were acclimatized to their environ- ment for 1 week and had ad libitum access to tap water and rodent standard diet. The study was approved by the Univer- sity of Messina Review Board for the care of animals. All animal experiments complied with regulations in Italy (D.M. 116192) as well as the EU regulations (O.J. of E.C. L 358/1 12/18/1986). SCI Mice were anesthetized with intraperitoneal administration of ketamine and xylazine (2.6 and 0.16 mg/kg body weight, re- spectively). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles, as previously described [18]. These muscles were dissected away, the spinal cord was exposed via a four-level T5 to T8 laminectomy, and SCI was produced by extradural compression at T6 to T7 level, using an aneurysm clip with a closing force of 24 g. Following surgery, 1.0 cm3 of saline was administered subcu- taneously in order to replace the blood volume lost during the surgery. During recovery from anesthesia, mice were placed on a warm heating pad and covered with a warm towel. The mice were individually housed in a temperature-controlled room at 27 °C. Food and water were provided to the mice ad libitum. During this time period, the animals’ bladders were manually voided twice a day until the mice were able to regain normal bladder function. In all injured groups, the spinal cord was compressed for 1 min. Sham animals were only subjected to laminectomy. Spinal cord tissues were taken at 24 h after trauma. Tissue segments contained the lesion Therefore, the aim of this study was to demonstrate that treatment co-ultraPEALut inhibited autophagy even after SCI, having neuroprotective effect. RET having neuroprotective Methods Animals RE g p Methods Animals CD1 mice (male 25–30 g; Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with stan- dard rodent chow and water. Mice were housed in stainless steel cages in a room kept at 22±1 °C with a 12-h light, 12-h RE Co-ultramicronization Process of PEA and Lut E Fur- thermore in this last model, we showed that co-ultraPEALut reduced autophagy after TBI [7]. TRACTED The dose of co-ultraPEALut (1 mg/kg) used was based on previous in vivo studies [18]. ED Animals CD1 mice (male 25–30 g; Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with stan- dard rodent chow and water. Mice were housed in stainless steel cages in a room kept at 22±1 °C with a 12-h light, 12-h 3785 Mol Neurobiol (2016) 53:3783–3792 ImmunoResearch) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody against β-actin (1:5000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent according to manufacturer’s instructions (SuperSignal West Pico Chemiluminescent Substrate, Pierce). The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc™XRS+software and standardized to β-actin levels. Images of blot signals (8-bit/ 600-dpi resolution) were imported to analysis software (Image Quant TL, v2003). A preparation of commercially available molecular weight markers made of proteins of molecular weight 10 to 250 kDa was used to define molecular weight positions and as reference concentrations for each molecular weight. TICLE (1 cm on each side of the lesion), according to counts of retrogradely labeled neurons at the origin of distinct descend- ing motor pathways and to morphometric assessments of nor- mal residual tissue at the injury epicenter, as previously de- scribed [19]. Immunofluorescence Stainin RT 4. SCI+co-ultraPEALut (10:1): mice were subjected to SCI and administered co-ultraPEALut at the final dose of 1 mg/kg i.p. at 1 and 6 h after SCI (n=30). After deparaffinization and rehydration, detection of GFAP, NeuN, Beclin-1, and MAPLC3-α was carried out after boiling in 0.1 M citrate buffer for 1 min. Nonspecific adsorption was minimized by incubating the section in 2 % (v/v) normal goat serum in PBS for 20 min. Sections were incubated with mouse monoclonal anti-NeuN (1:100, v/v Millipore), or with mouse monoclonal anti-GFAP (1:100, v/v Santa Cruz Biotechnolo- gy), or with polyclonal rabbit anti-Beclin-1 (1:100, v/v Santa Cruz, Biotechnology), or with rabbit anti-MAPLC3α (1:100, v/v Santa Cruz Biotechnology) antibody in a humidified chamber for O/N at 37 °C. Sections were washed with PBS and were incubated with secondary antibody FITC- conjugated anti-mouse Alexa Fluor-488 antibody (1:2000 v/v Molecular Probes, UK) and with TEXAS RED- conjugated anti-rabbit Alexa Fluor-594 antibody (1:1000 in PBS, v/v Molecular Probes, UK) for 1 h at 37 °C. Sections were washed and for nuclear staining 4′,6′-diamidino-2- phenylindole (DAPI; Hoechst, Frankfurt; Germany) 2 μg/ml in PBS was added. Sections were observed and photographed at ×20 magnification using a Leica DM2000 microscope (Leica). All images were digitalized at a resolution of 8 bits into an array of 2560×1920 pixels. Optical sections of fluores- cence specimens were obtained using a HeNe laser (543 nm), a laser UV (361–365 nm), and an argon laser (458 nm) at a 1- min, 2-s scanning speed with up to 8 averages; 1.5 μm sections were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly labeled pixels and applying settings that allowed clear visualization of struc- tural details while keeping the highest pixel intensities close to 200. The same settings were used for all images obtained from the other samples that had been processed in parallel. Digital images were cropped and figure montages prepared using Adobe Photoshop CS5 (Adobe Systems; Palo Alto, CA). ED ART Experimental Groups and Treatments Mice Were Randomly Allocated into the Following Groups Mice Were Randomly Allocated into the Following Groups 1. Sham+vehicle: mice were subjected to laminectomy, but the aneurysm clip was not applied and treated intraperito- neally (i.p.) with vehicle (n=30). 1. Sham+vehicle: mice were subjected to laminectomy, but the aneurysm clip was not applied and treated intraperito- neally (i.p.) with vehicle (n=30). 1. Sham+vehicle: mice were subjected to laminectomy, but the aneurysm clip was not applied and treated intraperito- neally (i.p.) with vehicle (n=30). 2. Sham+co-ultraPEALut (10:1)=same as the Sham+vehi- cle group, but co-ultraPEALut was administered at the final dose of 1 mg/kg i.p. at 1 and 6 h after laminectomy (n=30). 2. Sham+co-ultraPEALut (10:1)=same as the Sham+vehi- cle group, but co-ultraPEALut was administered at the final dose of 1 mg/kg i.p. at 1 and 6 h after laminectomy (n=30). rence concentration e Staining RTIC 3. SCI+vehicle: mice were subjected to laminectomy and the aneurysm clip was applied (n=30). 3. SCI+vehicle: mice were subjected to laminectomy and the aneurysm clip was applied (n=30). Western Blot Analysis Cytosolic and nuclear extracts were prepared with slight mod- ifications of a published procedure [20]. Spinal cord tissue from each mouse was suspended in extraction Buffer A con- taining 0.2 mM PMSF, 0.15 mM pepstatin A, 20 mM leupeptin, 1 mM sodium orthovanadate, homogenized at the highest setting for 2 min, and centrifuged at 12,000 rpm for 4 min at 4 °C. Supernatants represented the cytosolic fraction. The pellets, containing enriched nuclei, were resuspended in Buffer B containing 1 % Triton X-100, 150 mM NaCl, 10 mM Tris–HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM PMSF, 20 mm leupeptin, and 0.2 mM sodium orthovanadate. After centrifugation 10 min at 12,000 rpm at 4 °C, the supernatants that we obtained were the nuclear proteins. Protein concentrations were estimated by the Bio-Rad protein assay using bovine serum albumin as standard. Briefly, samples were heated to 100 °C for 5 min, and equal amounts of protein were separated on 18 % SDS-PAGE gel and transferred to nitrocellulose mem- brane. Specific primary antibody, anti- anti-MAPLC3α (1:500, Santa Cruz Biotechnology), Beclin-1 (1:500; Santa Cruz Biotechnology), anti-p62 (1:1000; Cell Signaling), anti-mTOR (1:1000; Cell Signaling), anti-p70S6K (1:1000; Cell Signaling), and p-Akt (1:1000; Cell Signaling) were mixed in 1× PBS, 5 % w/v nonfat dried milk, 0.1 % Tween- 20 (PMT), and incubated at 4 °C, overnight. After, mem- branes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase- conjugated goat anti-rabbit IgG (1:2000, Jackson RETRACTED 3786 Mol Neurobiol (2016) 53:3783–3792 Materials For histopathological examination by standard hematoxylin and eosin (H&E) staining, the mice were anesthetized with ketamine and xylazine (2.6 and 0.16 mg/kg body weight respectively) 24 h after injury and then perfused transcardially with cold PBS (0.1 M). Tissues were removed under magnified vision, and segments containing the lesion (1 cm on each side of the lesion) were collected in 4 % paraformaldehyde for proper fix- ation and then dehydrated with graded ethanol and embedded in paraffin wax. Sections of 5-μm thickness were cut into longitu- dinal sections for the posterior area of the spinal cord, stained with H&E, and studied using light microscopy (Dialux 22 Leitz). Representative images were shown. Blinded histologic scoring (Fig. 1d) was performed on a 6-point scale: 0, normal; 1, 1–5 eosinophilic neurons within the gray matter area; 2, 5–10 eosinophilic neurons; 3, >10 eosinophilic neurons; 4, a small infarction (less than one-third of the gray matter area); 5, a mod- erate infarction (one-third to one-half of the gray matter area); and 6, a large infarction (more than half of the gray matter area). Unless otherwise stated, all compounds were obtained from Sigma-Aldrich, while co-ultraPEALut was a kind gift from Epitech Group s.r.l. (Saccolongo, Italy). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0.9 % NaCl, Baxter, Milan, Italy) or 10 % dimethyl sulfoxide. E Effect of Co-ultraPEALut on Histological Parameters To evaluate the severity of the trauma at the level of the perilesional area at 24 h after injury, the sections obtained from each group were stained with hematoxylin and eosin (H&E). Compared with the sham group (Fig. 1a, b, see mag- nification higher a1 and b1, respectively), the SCI+vehicle group showed the presence of edema and alteration of the white matter (Fig. 1b, d, see magnification higher b1). Treat- ment with co-ultraPEALut attenuated acute inflammation and injury at 24 h after SCI (Fig. 1c, d, see magnification higher c1). Effect of Co-ultraPEALut on Autophagy After SCI: Expression of Beclin-1, p62, and MAP-LC3 Statistical Evaluation All values in the figures and the text are expressed as mean± SEM. Results shown in the figures are representative of at least three experiments performed on different in vivo exper- imental days. In each experiment, we used five animals per group, unless otherwise indicated. The results were analyzed by one-way analysis of variance followed by a Bonferroni post hoc test for multiple comparisons. A p value of less than 0.05 was considered significant. RTICL Fig. 1 Effects of co-ultraPEALut treatment on histological alterations of the spinal cord tissue 24 h after injury. Compared with the Sham-surgery mice (a, a1), significant histologic damage to the spinal cord was evident in mice subjected to SCI and treated with vehicle (b, b1). In the perilesional area of the SCI mice, edema was evident, as and alteration of the white matter. A significant reduction of SCI-associated damage was observed in the tissue samples collected from mice treated with co- ultraPEALut (c, c1). These data are also visible in the histological score (d). ***p<0.001 vs Sham; ###p<0.001 vs SCI RETRACTED AR of the white matter. A significant reduction of SCI-associated damage was observed in the tissue samples collected from mice treated with co- ultraPEALut (c, c1). These data are also visible in the histological score (d). ***p<0.001 vs Sham; ###p<0.001 vs SCI ED AR ED AR RETRAC of the white matter. A significant reduction of SCI-associated damage was observed in the tissue samples collected from mice treated with co- ultraPEALut (c, c1). These data are also visible in the histological score (d). ***p<0.001 vs Sham; ###p<0.001 vs SCI of the white matter. A significant reduction of SCI-associated damage was observed in the tissue samples collected from mice treated with co- ultraPEALut (c, c1). These data are also visible in the histological score (d). ***p<0.001 vs Sham; ###p<0.001 vs SCI Fig. 1 Effects of co-ultraPEALut treatment on histological alterations of the spinal cord tissue 24 h after injury. Compared with the Sham-surgery mice (a, a1), significant histologic damage to the spinal cord was evident in mice subjected to SCI and treated with vehicle (b, b1). In the perilesional area of the SCI mice, edema was evident, as and alteration Mol Neurobiol (2016) 53:3783–3792 3787 Results autophagy. To compare the level of Beclin-1 protein between the injured and the treated spinal cord section, western blot analysis was performed. We showed that Beclin-1 expression dramatically increased in the spinal cord tissue (T6 to T8 tract) collected from mice subjected to SCI compared to the sham group, while the treatment with co-ultraPEALut significantly reduced SCI-induced Beclin-1 expression (Fig. 2a, see densi- tometry analysis in a1). For more accurate quantification of co-ultraPEALut effects on autophagy process, we examined levels of p62 by western blot analysis. We detected that p62 expression increased after SCI, whereas the treatment with co- ultraPEALut significantly reduced levels of this protein (Fig. 2b, see densitometry analysis in b1). p62 also interacts with a central component of the machine autophagy, autoph- agic marker MAP-LC3, and carries the altered proteins to degradation by autophagy. Therefore, by western blot analy- sis, we showed that the ratio of LC3 I to LC3 II increased in untreated animals 24 h after injury, though this ratio signifi- cantly diminished to near sham levels of expression following co-ultraPEALut treatment (Fig. 2c, see densitometry analysis in c1), suggesting a reversal of autophagic activity upregula- tion seen following SCI. ARTICLE Effect of Co-ultraPEALut on Autophagy After SCI: Expression of Beclin-1, p62, and MAP-LC3 To investigate the mechanisms of autophagy after SCI, we examined levels of proteins involved in the regulation and formation of autophagosomes. Beclin-1 has a central role in autophagy because it interacts with various cofactors inducing Fig. 2 Effect of co-ultraPEALut about proteins promoting autophagy after SCI. Western blot analysis for Beclin-1, p62, and LC3 showed an increase of levels of these proteins in SCI group compared to the Sham group, whereas the treatment with co-ultraPEALut significantly reduced SCI-induced Beclin-1, p62, and LC3 expression (a, a1; b, b1; c, c1 respectively). The data are representative of at least three independent experiments. a **p<0.01 vs Sham; ##p<0.01 vs SCI. b **p<0.01 vs Sham; #p<0.05 vs SCI. c *p<0.05 vs Sham; #p<0.05 vs SCI RETRACTED A ED D RACTED D Fig. 2 Effect of co-ultraPEALut about proteins promoting autophagy after SCI. Western blot analysis for Beclin-1, p62, and LC3 showed an increase of levels of these proteins in SCI group compared to the Sham group, whereas the treatment with co-ultraPEALut significantly reduced SCI-induced Beclin-1, p62, and LC3 expression (a, a1; b, b1; c, c1 respectively). The data are representative of at least three independent experiments. a **p<0.01 vs Sham; ##p<0.01 vs SCI. b **p<0.01 vs Sham; #p<0.05 vs SCI. c *p<0.05 vs Sham; #p<0.05 vs SCI Mol Neurobiol (2016) 53:3783–3792 3788 Effect of Co-ultraPEALut on Beclin-1 and MAP-LC3 Expression by Immunofluorescence Staining upstream signal in the regulation of autophagy which is pos- itively regulated by PI3K/Akt and results in the inhibition of autophagy. Therefore, by western blot analysis, we detected the activity of Akt/mTOR signaling pathways in our model in vivo. Our results showed that p-Akt and mTOR levels de- creased significantly after SCI, compared with the Sham group, while co-ultraPEALut treatment increased p-Akt and mTOR expression, inhibiting autophagy (Fig. 5a, c, see den- sitometry analysis in a1 and b1, respectively). E To better understand the Beclin-1 and MAP-LC3 expression in a specific population of cells, including neurons and astrocytes, the spinal cord sections (T6 to T8 tract) at 24 h after compres- sion were double-stained for Beclin-1 (red) or MAP-LC3 (red) and various cell type markers, such as NeuN (green) for neurons and GFAP (green) for astrocytes (Figs. 3 and 4). In the double staining, the expression of Beclin-1 was observed in NeuN and GFAP-labeled cells of the injured SCI group (Fig. 3g–i and j–l, respectively) compared to Sham (Fig. 3a–c and d–f, respective- ly), whereas a treatment with co-ultraPEALut not only de- creased significantly these proteins expression but also the dou- ble staining is less observed in both NeuN and GFAP (Fig. 3m– o and p–r, respectively). Similarly, at 24 h after SCI, double staining of LC3 revealed a strong co-localization with NeuN and GFAP in SCI group (Fig. 4g–i and j–l, respectively) com- pared to the Sham group (Fig. 4a–c and d–f, respectively), while treatment of co-ultraPEALut decreased significantly these pro- tein expressions (Fig. 4m-o and p-r respectively). Moreover, a downstream target of mTOR signal a p70S6K activity was assessed by western blot analysis. p70S6k activ- ity was suppressed in mice after SCI, whereas the co- ultraPEALut treatment enhanced activation of p70S6K signal- ing (Fig. 5c, see densitometry analysis in c1). CLE y y ) i h i l i j i RTICL Discussion The CNS is sensitive to mechanical injuries causing perma- nent functional deficits such as the case of patients who have SCI. The mechanical forces imparted to the spinal cord cause immediate tissue disruption, with direct axonal and neuronal injury, inducing the death of neurons. Moreover, neurons con- tinue to die for hours or days after SCI, as a consequence of excitotoxicity, vascular dysfunction, and, in particular, be- cause of the ensuing inflammatory response [21]. Inflamma- tory responses are a major component of secondary injury and ED ART Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway After SCI D Treatment with the Co-ultraPEALut reduced Beclin-1 expression (m–r). All images were digitalized at a resolution of 8 bits into an array of 2048×2048 pixels D AR Fig. 4 Co-localization of NeuN/LC3 and GFAP/LC3 after SCI. Results are shown for (a–f) Sham group, (g–l) mice with SCI, and (m–r) mice with SCI treated with co-ultraPEALut. Double-stained sections at 24 h post-injury indicated that LC3 expression increased in both neurons and astrocytes respectively. Orange arrows indicate co-localizations (g–l) and D still unknown. The most plausible explanation is that catabo- lism through autophagy is predominantly survival-supporting, but that an imbalance in cell metabolism, where autophagic cellular consumption exceeds the cellular capacity for synthe- sis, promotes cell death [28]. ED play a central role in regulating the pathogenesis of acute and chronic SCI [22]. Functional recovery after SCI is often poor and, as yet, there are no nonsurgical therapies that have been successfully translated for use in the clinic. Therefore, there is a great need for drugs that will reduce post-traumatic, inflammation-related spinal cord damage because it has been reported that reducing inflammation decreases secondary de- generation and the functional deficit after SCI. In a previous study, we showed that co-ultraPEALut treatment (1 mg/kg) exerted beneficial effects in ex vivo model using spinal cord organotypic slice cultures and in in vivo model of spinal cord compression injury in mice. Briefly, we found that co- ultraPEALut had anti-inflammatory, antioxidant, and neuro- protective properties [18]. ETRACTED Therefore, the aim of the present study was to explore new therapeutic approaches promoting survival of cells. In partic- ular, we wanted to investigate whether the new compound co- ultraPEALut (at the dose of 1 mg/kg) was able to modulate autophagy promoting functional recovery after SCI. In the first step, using H&E staining, we evaluated the effect of co-ultraPEALut on spinal cord at the level of the perilesional area at 24 h after injury. Histological analysis showed that mice, after SCI, presented edema and alteration of the white matter, while mice treated with co-ultraPEALut showed an attenuation of acute inflammation and injury after SCI. Furthermore, recent studies demonstrated that another pos- sible consequence of SCI is energy depletion, and this elicits autophagy [15, 16, 23]. Autophagy is a cellular catabolic deg- radation response to starvation or stress, whereby cellular pro- teins, organelles, and cytoplasm are engulfed, digested, and recycled to sustain cellular metabolism [9, 24]. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway After SCI D The PI3K/Akt/mTOR pathway is an intracellular signaling pathway important in regulating the cell cycle. mTOR is ED Fig. 3 Co-localization of Neun/Beclin-1 and GFAP/Beclin-1 after SCI. Results are shown for a–f Sham group, g–l mice with SCI, and m–r mice with SCI treated with co-ultraPEALut. Double-stained sections at 24 h post injury indicated that Beclin-1 expression increased in both neurons and astrocytes respectively. Orange arrows indicate co-localizations (g–l) and revealed a high co-localization between NeuN/Beclin1 and GFAP/ Beclin-1 double staining. Treatment with the co-ultraPEALut reduced Beclin-1 expression (m–r). All images were digitalized at a resolution of 8 bits into an array of 2048×2048 pixels RETRACTED D TRA and revealed a high co-localization between NeuN/Beclin1 and GFAP/ Beclin-1 double staining. Treatment with the co-ultraPEALut reduced Beclin-1 expression (m–r). All images were digitalized at a resolution of 8 bits into an array of 2048×2048 pixels Fig. 3 Co-localization of Neun/Beclin-1 and GFAP/Beclin-1 after SCI. Results are shown for a–f Sham group, g–l mice with SCI, and m–r mice with SCI treated with co-ultraPEALut. Double-stained sections at 24 h post injury indicated that Beclin-1 expression increased in both neurons and astrocytes respectively. Orange arrows indicate co-localizations (g–l) Mol Neurobiol (2016) 53:3783–3792 3789 Fig. 4 Co-localization of NeuN/LC3 and GFAP/LC3 after SCI. Results are shown for (a–f) Sham group, (g–l) mice with SCI, and (m–r) mice with SCI treated with co-ultraPEALut. Double-stained sections at 24 h post-injury indicated that LC3 expression increased in both neurons and astrocytes respectively. Orange arrows indicate co-localizations (g–l) and revealed a high co-localization between NeuN/LC3 and GFAP/LC3 double staining. Treatment with the Co-ultraPEALut reduced Beclin-1 expression (m–r). All images were digitalized at a resolution of 8 bits into an array of 2048×2048 pixels D ARTICLE FAP/LC3 after SCI. Results e with SCI, and (m–r) mice uble-stained sections at 24 h creased in both neurons and ate co-localizations (g–l) and revealed a high co-localization between NeuN/LC3 and GFAP/LC3 double staining. Treatment with the Co-ultraPEALut reduced Beclin-1 expression (m–r). All images were digitalized at a resolution of 8 bits into an array of 2048×2048 pixels D ARTICLE revealed a high co-localization between NeuN/LC3 and GFAP/LC3 double staining. Treatment with the Co-ultraPEALut reduced Beclin-1 expression (m–r). All images were digitalized at a resolution of 8 bits into an array of 2048×2048 pixels D ARTICLE CLE revealed a high co-localization between NeuN/LC3 and GFAP/LC3 double staining. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway After SCI D Finally, we wanted to evaluate on Akt/mTOR/p70S6K pathway considered a central regulatory p lation involved in regulating cell entiation, and survival [33, 34] regulator of autophagy directly s protein is a central signal integrat point with upstream Akt and dow two most important mediators [8 blot analysis, we detected mTO phorylation of Akt after SCI. O Akt and mTOR levels decreased co-ultraPEALut treatment increa teins. Moreover, p70S6k activity SCI, whereas the co-ultraPEALu tion of p70S6K signaling. These tective role of co-ultraPEALut is excessive autophagy via Akt/mT ulation of protein degradation. o-ultra PEALut about proteins inhibiting autophagy blot analysis for mTOR, p-Akt, and p70S6K showed of these proteins in SCI group compared to the Sham reatment with co-ultraPEALut significantly increased SCI-induced mTOR, p-Akt, and p70S6 respectively). The data are representat experiments. a **p<0.01 vs Sham; # Sham; ##p<0.01 vs SCI. c ***p<0.00 ETRACTED ARTIC TED ARTIC Fig. 5 Effect of co-ultra PEALut about proteins inhibiting autophagy after SCI. Western blot analysis for mTOR, p-Akt, and p70S6K showed a decrease of levels of these proteins in SCI group compared to the Sham group, whereas the treatment with co-ultraPEALut significantly increased ACTE SCI-induced mTOR, p-Akt, and p70S6K expression (a, a1; b, b1; c, c1 respectively). The data are representative of at least three independent experiments. a **p<0.01 vs Sham; #p<0.05 vs SCI. b **p<0.01 vs Sham; ##p<0.01 vs SCI. c ***p<0.001 vs Sham; ##p<0.01 vs SCI E Finally, we wanted to evaluate the effect of co-ultraPEALut on Akt/mTOR/p70S6K pathway that in autophagy has been considered a central regulatory pathway of the protein trans- lation involved in regulating cell proliferation, growth, differ- entiation, and survival [33, 34]. mTOR kinase is a master regulator of autophagy directly suppressing autophagy. This protein is a central signal integrator that functions as a check- point with upstream Akt and downstream p70S6K being the two most important mediators [8]. Therefore, using western blot analysis, we detected mTOR expression and the phos- phorylation of Akt after SCI. Our results indicated that p- Akt and mTOR levels decreased significantly after SCI, while co-ultraPEALut treatment increased expression of these pro- teins. Moreover, p70S6k activity was suppressed in mice after SCI, whereas the co-ultraPEALut treatment enhanced activa- tion of p70S6K signaling. These data confirmed that the pro- tective role of co-ultraPEALut is associated with inhibition of excessive autophagy via Akt/mTOR/p70S6K signals and reg- ulation of protein degradation. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway After SCI D Constitutive, basal autophagy also has an important homeostatic function, by maintaining protein and organelle quality control. Al- though most evidence supports a role for autophagy in sus- taining cell survival, paradoxically, cell death resulting from progressive cellular consumption has been attributed to unre- strained autophagy [25–27]. The mechanisms that regulate the mutually opposed survival and death roles for autophagy are RET In the second step, we studied the effects of this new com- pound on autophagy markers after SCI. At first, we examined the levels of proteins that promote autophagy such as Beclin-1 and MAP-LC3. Beclin-1 is a protein that participates in the regulation of autophagy and has an important role in develop- ment, tumorigenesis, and neurodegeneration [29]. Beclin-1 functions as a scaffold for the formation of the PI3K complex, one of the first components recruited during the development of autophagosomes. During autophagy initiation and autophagosome formation, Beclin-1 binds microtubule- Mol Neurobiol (2016) 53:3783–3792 3790 Fig. 5 Effect of co-ultra PEALut about proteins inhibiting autophagy after SCI. Western blot analysis for mTOR, p-Akt, and p70S6K showed a decrease of levels of these proteins in SCI group compared to the Sham group, whereas the treatment with co-ultraPEALut significantly increased SCI-induced mTOR, p-Akt, and p70S6K expression (a, a1; b, b1; c, c1 respectively). The data are representative of at least three independent experiments. a **p<0.01 vs Sham; #p<0.05 vs SCI. b **p<0.01 vs Sham; ##p<0.01 vs SCI. c ***p<0.001 vs Sham; ##p<0.01 vs SCI ACTED ARTICLE ICLE n 1A/1B-light chain 3 I (LC3I) that is con- mbrane-bound form (LC3II), which then inter- iquitin-binding protein p62/sequestosome 1 62 accumulates when autophagy is inhibited vels can be observed when autophagy is in- be used as a marker to study autophagic flux y, we observed an increase of the expression 3, and p62 after SCI that dramatically de- atment with co-ultraPEALut. Furthermore, sess whether Beclin-1 and MAP-LC3 were cific cells. Therefore, the spinal cord sections pression were double-stained for Beclin-1 or various cell type markers, such as NeuN for AP for astrocytes. By immunofluorescence served a strong co-localization of Beclin-1 un and GFAP. These results showed that after on of these two proteins increases in neurons while treatment with co-ultraPEALut reduces lin-1 and LC3 in both cell types. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway After SCI D associated protein 1A/1B-light chain 3 I (LC3I) that is con- verted to its membrane-bound form (LC3II), which then inter- acts with the ubiquitin-binding protein p62/sequestosome 1 [30–32]. Since p62 accumulates when autophagy is inhibited and decreased levels can be observed when autophagy is in- duced, p62 may be used as a marker to study autophagic flux [32]. In this study, we observed an increase of the expression of Beclin-1, LC3, and p62 after SCI that dramatically de- creased after treatment with co-ultraPEALut. Furthermore, we wanted to assess whether Beclin-1 and MAP-LC3 were expressed in specific cells. Therefore, the spinal cord sections at 24 h after compression were double-stained for Beclin-1 or MAP-LC3 and various cell type markers, such as NeuN for neurons and GFAP for astrocytes. By immunofluorescence staining, we observed a strong co-localization of Beclin-1 and LC3 with Neun and GFAP. These results showed that after SCI, the expression of these two proteins increases in neurons and astrocytes, while treatment with co-ultraPEALut reduces the levels of Beclin-1 and LC3 in both cell types. RETRAC associated protein 1A/1B-light chain 3 I (LC3I) that is con- verted to its membrane-bound form (LC3II), which then inter- acts with the ubiquitin-binding protein p62/sequestosome 1 [30–32]. Since p62 accumulates when autophagy is inhibited and decreased levels can be observed when autophagy is in- duced, p62 may be used as a marker to study autophagic flux [32]. In this study, we observed an increase of the expression of Beclin-1, LC3, and p62 after SCI that dramatically de- creased after treatment with co-ultraPEALut. Furthermore, we wanted to assess whether Beclin-1 and MAP-LC3 were expressed in specific cells. Therefore, the spinal cord sections at 24 h after compression were double-stained for Beclin-1 or MAP-LC3 and various cell type markers, such as NeuN for neurons and GFAP for astrocytes. By immunofluorescence staining, we observed a strong co-localization of Beclin-1 and LC3 with Neun and GFAP. These results showed that after SCI, the expression of these two proteins increases in neurons and astrocytes, while treatment with co-ultraPEALut reduces the levels of Beclin-1 and LC3 in both cell types. RETRAC Mol Neurobiol (2016) 53:3783–3792 3791 antioxidant gamma-glutamylcysteinyl ethyl ester. 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J Neuroinflammation 10:91. doi:10.1186/ 1742-2094-10-91 E Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway After SCI D In addition, our results suggest that SCI induces autophagic cell death at the lesion site; therefore, co-ultraPEALut treatment inhibiting autophagy in in vivo model of SCI has protective effect. In conclusion, co-ultraPEALut could be considered as a possible therapeutic target in an acute traumatic lesion like SCI. 9. Levine B, Klionsky DJ (2004) Development by self-digestion: mo- lecular mechanisms and biological functions of autophagy. Dev Cell 6(4):463–477 10. Melendez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B (2003) Autophagy genes are essential for dauer develop- ment and life-span extension in C. elegans. Science 301(5638): 1387–1391. doi:10.1126/science.1087782 11. Gozuacik D, Kimchi A (2004) Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23(16):2891–2906. doi:10.1038/ sj.onc.1207521 E 12. Larsen KE, Sulzer D (2002) Autophagy in neurons: a review. H Histopathol 17(3):897–908 LE Acknowledgments The authors would like to thank Maria Antonietta Medici for her excellent technical assistance during this study and Mr Francesco Soraci for his secretarial and administrative assistance and Miss Valentina Malvagni for her editorial assistance with the manuscript. This study was supported by PON 01_02512. 13. Park JM, Tougeron D, Huang S, Okamoto K, Sinicrope FA (2014) Beclin 1 and UVRAG confer protection from radiation-induced DNA damage and maintain centrosome stability in colorectal can- cer cells. PLoS One 9(6):e100819. doi:10.1371/journal.pone. 0100819 ICLE Compliance with Ethical Standards 14. Ferraro E, Cecconi F (2007) Autophagic and apoptotic response to stress signals in mammalian cells. Arch Biochem Biophys 462(2): 210–219 RTIC Conflict of Interest The authors declare that they have no conflict of interest. RETRACT References Lai Y, Hickey RW, Chen Y, Bayir H, Sullivan ML, Chu CT, Kochanek PM, Dixon CE et al (2008) Autophagy is increased after traumatic brain injury in mice and is partially inhibited by the 26. Debnath J, Baehrecke EH, Kroemer G (2005) Does autophagy contribute to cell death? Autophagy 1(2):66–74 3792 Mol Neurobiol (2016) 53:3783–3792 protein expression in colon carcinomas from patients receiving 5- fluorouracil as adjuvant chemotherapy. Cancer Biol Ther 14(2): 100–107. doi:10.4161/cbt.22954 protein expression in colon carcinomas from patients receiving 5- fluorouracil as adjuvant chemotherapy. Cancer Biol Ther 14(2): 100–107. doi:10.4161/cbt.22954 27. Reef S, Zalckvar E, Shifman O, Bialik S, Sabanay H, Oren M, Kimchi A (2006) A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death. Mol Cell 22(4): 463–475. doi:10.1016/j.molcel.2006.04.014 protein expression in colon carcinomas from patients receiving 5- fluorouracil as adjuvant chemotherapy. Cancer Biol Ther 14(2): 100–107. doi:10.4161/cbt.22954 31. Tanida I, Ueno T, Kominami E (2008) LC3 and Autophagy. Methods Mol Biol 445:77–88. doi:10.1007/978-1-59745-157-4_4 31. Tanida I, Ueno T, Kominami E (2008) LC3 and Autophagy. Methods Mol Biol 445:77–88. doi:10.1007/978-1-59745-157-4_4 28. Mathew R, Karantza-Wadsworth V, White E (2007) Role of au- tophagy in cancer. Nat Rev Cancer 7(12):961–967. doi:10.1038/ nrc2254 32. Bjorkoy G, Lamark T, Pankiv S, Overvatn A, Brech A, Johansen T (2009) Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol 452:181-197. doi:10.1016/S0076-6879(08) 03612-4S0076-6879(08)03612-4 29. Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT, Heintz N, Yue Z (2009) Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat Cell Biol 11(4):468–476. doi:10.1038/ncb1854 33. Pyronnet S, Sonenberg N (2001) Cell-cycle-dependent translational control. Curr Opin Genet Dev 11(1):13–18 E 34. Schmelzle T, Hall MN (2000) TOR, a central con growth. Cell 103(2):253–262 LE 30. Park JM, Huang S, Wu TT, Foster NR, Sinicrope FA (2013) Prognostic impact of Beclin 1, p62/sequestosome 1 and LC3
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Hand Washing Induces a Clean Slate Effect in Moral Judgments: A Pupillometry and Eye-Tracking Study
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Hand Washing Induces a Clean Slate Effect in Moral Judgments: A Pupillometry and Eye-Tracking Study received: 31 December 2014 accepted: 16 April 2015 Published: 21 May 2015 Kai Kaspar1,2, Vanessa Krapp2 & Peter König2,3 Physical cleansing is commonly understood to protect us against physical contamination. However, recent studies showed additional effects on moral judgments. Under the heading of the “Macbeth effect” direct links between bodily cleansing and one’s own moral purity have been demonstrated. Here we investigate (1) how moral judgments develop over time and how they are altered by hand washing, (2) whether changes in moral judgments can be explained by altered information sampling from the environment, and (3) whether hand washing affects emotional arousal. Using a pre-post control group design, we found that morality ratings of morally good and bad scenes acquired more extreme values in the control group over time, an effect that was fully counteracted by intermediate hand washing. This result supports the notion of a clean slate effect by hand washing. Thereby, eye- tracking data did not uncover differences in eye movement behavior that may explain differences in moral judgments. Thus, the clean slate effect is not due to altered information sampling from the environment. Finally, compared to the control group, pupil diameter decreased after hand washing, thus demonstrating a direct physiological effect. The results shed light on the physiological mechanisms behind this type of embodiment phenomenon. The fundamental goal of physical cleansing is to protect us against physical contamination and, hence, to foster physiological health. However, empirical studies showed that physical cleansing also touches psychological aspects. Already, Shakespeare described in his drama that Lady Macbeth repeatedly experi- enced the desire to wash her hands after she incited her husband to kill the King of Scotland. Accordingly, Zhong and Liljenquist1 found that moral impurity actually elicits the desire for cleansing-related prod- ucts. This so-called “Macbeth effect” stimulated further research on the relationship between physical and moral purity. In a follow-up study, they demonstrated that washing one’s hands after recalling an unethical deed of the past reduced the motivation to volunteer, thus indicating that hand washing already restored a suitable moral self-image and, hence, reduced the desire to compensate the unethical deed by voluntary help. More recently, Reuven et al.2 replicated this finding, while the effect was stronger in participants with obsessive-compulsive disorder. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ www.nature.com/scientificreports/ this Macbeth effect was accompanied by an active cortical network that comprises sensorimotor brain areas, thus indicating an embodiment of the moral-purity metaphor. Also, Lobel et al.5 found that more money was donated to charity before rather than after bathing for religious purification. Finally, Lee and Schwarz6 found that the Macbeth effect seems to be specific with respect to the motor modality that is primarily involved in the moral transgression. The desire for mouthwash was high after communicat- ing a lie via voice mail, while the desire for hand sanitizer was higher after lying in an email. All these observations demonstrate a direct link between one’s own bodily cleanliness and moral purity. Physical cleansing seems to have the power to restore the perception of one’s own moral self that, in turn, may reduce or even eliminate the Macbeth-like cognitions and behavior.f g Research on embodied cognition provides a simple explanation for this effect: Williams et al.7 stated that “early sensorimotor experiences serve as the foundation for the later development of more abstract concepts and goals” (p. 1257). In early childhood, we learn the concept of physical purity and how to achieve this goal. This knowledge serves as the scaffold for the later development of conceptually related abstract ideas such as moral purity. Accordingly, Lakoff and Johnson8 proposed in their work on concep- tual metaphors that abstract concepts are built upon earlier experiences that are often body-related. As a consequence, established associations between the physical and abstract cognitive concept emerge that are assumed to be stable across the whole lifespan9 and are presumably grounded on the same neuro- anatomical foundation10. Similarly, according to Rozin’s body-to-soul-preadaptation theory of disgust11, a neuronal circuitry, developed for the processing of the originally food-related emotion of disgust, was preadapted for an extension to other threats beyond the rejection of food, such as moral threats. Indeed, Schnall et al.12 found that the feelings of disgust increased the severity of moral judgments relative to controls. Correspondingly, Moll et al.13 found that strongly overlapping brain regions were activated by pure disgust (without moral connotations) and the moral emotion of indignation. The strong conceptual and neuroanatomical relationship between moral and physical purity might explain why physical cleans- ing can affect moral reasoning and vice versa.f gf g Importantly, the psychological effect of physical cleansing is not limited to the moral domain. www.nature.com/scientificreports/ Recent studies showed that washing one’s hands can wash off traces of the past in general. Lee and Schwarz14 found that washing can reduce post-decisional dissonance effects and Xu et al.15 showed that the expe- rience of bad luck and good luck can be washed away. Kaspar16 found that hand washing after failure in an anagram task did not only increase optimism but also hampered future performance. The author assumes that hand washing may have “lowered participants’ need to work harder in order to restore their perception of competence” (p. 71). Finally, Florack et al.17 found that hand washing can reduce decision preferences that are biased by ownership. Given these findings, the Macbeth effect may only be one signature of a more general “clean slate effect,” as termed by Lee and Schwarz14. Correspondingly, Xu et al.15 concluded that hand washing removes both desirable as well as undesirable traces of the past. Similarly, Florack et al.17 stated that “the physical action of hand washing resets the cognitive system to a more neutral state” (p. 288). Hence, hand washing apparently does not only help to wash away our sins1 but also has the power to generally attenuate the effects of past events.f p g yf p However, in addition to the idea of a general clean slate effect, it has been shown that hand washing, as well as the mere activation of the cleanliness concept, can alter moral judgments. This aspect is crucial, as it includes the question of how cleanliness treatments work in a specific context. Zhong et al.18 reported that study participants who had either actually cleansed their hands or merely visualized themselves in a clean versus dirty state rated critical social issues, such as pornography and smoking, to be more morally wrong. Also, participants who were primed for cleanliness reported a higher moral self-image that medi- ated the effect of the cleanliness priming on moral judgments. This result indicates that the attribution of physical purity to oneself generalizes to the impression of moral purity as well. Consequently, when we feel to be morally clean, this may license harsher moral judgments. y y j g In contrast, Schnall et al.19 found that certain moral actions were judged less severe, in contrast to a control condition, when the concept of physical cleanliness was activated either by a sentence unscram- bling task or by washing one’s hands after experiencing disgust. Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 Hand Washing Induces a Clean Slate Effect in Moral Judgments: A Pupillometry and Eye-Tracking Study In a study by Gollwitzer and Melzer3, inexperienced video game players felt higher moral distress after playing a video game that included violence against humans (versus objects), and they selected more hygiene products in a subsequent product choice task than frequent video game players. Moreover, in a study by Denke et al.4, participants enacted a sequence of scenarios that included either a moral deed (telling the truth) or an immoral one (lying). Immediately after a scenario ended, they rated the desirability of different products, while only cleansing-related products were rated as more desirable after an immoral act than after a moral one. At the same time, 1Social and Media Psychology, Department of Psychology, University of Cologne, Cologne, Germany. 2Neurobiopsychology, Institute of Cognitive Science, University of Osnabrück, Osnabrück, Germany. 3Department of Neurophysiology and Pathophysiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Correspondence and requests for materials should be addressed to K.K. (email: kkaspar@uni-koeln.de) Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 1 1 The Present Study y In contrast to previous studies on cleanliness effects, we applied a pre-post control group design. Hitherto, it is unclear how moral judgments evolve across several issues of similar morality. Smith et al.22 found that increasing the familiarity of information about a person leads to less systematic or analytic process- ing of this information, thus resulting in more stereotyped judgments and less individualized judgments. Correspondingly, we assume that stereotyping in the moral domain would be reflected by more extreme judgments when judging numerous morally good and bad actions in a sequence. In such a situation, the notion of a clean slate effect suggests that hand washing in the meantime would counter increasing stereotyping, thus leading to less extreme judgments after hand washing due to a reset of the cognitive moral system. In contrast, the notion of an enhanced moral self by cleansing one’s hands would predict even more severe moral judgments. However, previous studies have mainly focused on immoral actions and, hence, the question arises as to whether the effect of hand washing would also generalize to mor- ally good issues. Indeed, according to the idea of moral consistency, moral self-regard may motivate to behave in a way that helps to maintain a high moral self5. In this sense, hand washing may also lead to more extreme judgments in the case of morally good actions in order to perpetuate one’s high moral self by emphasizing the praiseworthiness of moral actions.f y p g p Moreover, we asked whether hand washing modulates information sampling or whether it affects moral judgements at a later stage of the judgment process. Two extreme scenarios are conceivable. The act of hand washing could alter sampling information from the environment. The unchanged process of moral judgement would act on a different set of sampled information and potentially reach a different verdict. Alternatively, the act of hand washing could be neutral with respect to the sampling of informa- tion, but the process of moral judgement would evaluate identical information differently. Only the latter case would exemplify a direct link between hand washing and moral judgments. In fact, we propose to interpret the effects of washing in terms of information sampling and weighting. The hitherto reported effects showed that the act of cleansing did not literally wash away what had already happened in the past. Instead, washing led to a specific (re-)weighting of the available information. The Present Study For example, hand washing did not undo an unethical deed that participants had previously committed1, did not change previous decisions14, and did not substitute a failure experience by success16, but it changed the weighting of unethical deeds, reduced the need to devaluate non-chosen options, and increased one’s optimism to be more successful in the future, respectively. In each of these cases, we can assume that the effect was based on a selective sampling of information. In fact, as highlighted by Lindskog et al.23, “several accounts of human judgment and decision making assume that people sample information from memory prior to making a judgment or decision” (p. 783). However, we do not only selectively sample informa- tion from memory but also from the environment, while the current information sampling depends on the individual’s attention, capacity, and processing goals24. Thus, the effects of hand washing may base on sampling biases on both stages. Following Smith et al.22, we expected that increasing the familiarity of information (induced by a long sequence of moral vs. immoral scenes) would lead to a less systematic processing of information and result in more stereotyped (i.e., extreme) moral judgments (see above). If hand washing affects information sampling from memory (e.g., when classifying a specific scene as very immoral compared to social norms stored in memory), it should not show an effect on the visual exploration of moral and immoral scenes. If, however, hand washing affects information sampling from the environment, changes on the level of eye movements should be traceable. According to the notion of a clean slate effect, washing should reset one’s cognitive state to a more neutral one17 and, hence, increase peoples’ openness for each individual scene so that they are more motivated to visually explore it before making a moral judgment. Correspondingly, Ybarra25 stated that “being cognitively open refers to the tendency to seek out and integrate additional information into a current judgment” (p. 431). In fact, when people repeatedly observe visual scenes, their viewing activity decreases, while higher interest in visual scenes elicits a more active exploration of the scenes26. In contrast, if washing elicits the impression of one’s own moral cleanliness, even more severe moral judgments are conceivable, which could result from an additionally lowered engagement in stimulus exploration.f y g g p Finally, does hand washing affect the physiological level or is its impact limited to purely disembodied cognitive processes? www.nature.com/scientificreports/ Zhong et al.18 proposed a simple expla- nation for the seemingly contradicting results; in their study, the activation of the cleanliness concept apparently restored one’s own moral self-image, and in the work of Schnall et al.19, the state of cleanliness was perhaps attributed to the judged target but not to the self of the judging person. Hence, it seems to be crucial who is viewed as clean. Johnson et al.20 failed to replicate the results of Schnall et al.19, but in a more recent replication study, Huang21 showed that less severe moral judgments only occurred when study participants invested low effort in the sentence unscrambling task. We suppose that low task involvement facilitated the attribution of cleanliness to the judged targets.f j g g To conclude, activating cleanliness cognitions with a priming procedure showed mixed effects that are presumably signatures of different attribution processes. In contrast, washing one’s own hands makes the attribution of a clean state to oneself indubitable. Thereby, two mechanisms have been posited so far: on the one hand, washing may lead to an increase in one’s perceived moral purity and, hence, makes judgments about others’ moral transgressions more severe. On the other hand, washing may lead to a general clean slate effect, thus attenuating the impression of past events and setting the cognitive system to a more neutral state. The present study contrasts these two potential mechanisms in the moral domain by changing the experimental paradigm and methodology. 2 www.nature.com/scientificreports/ Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 Methods All h The display resolution was 1280 ×  960 pixels, the refresh rate was 85 Hz, and the distance to the screen was set at 80 cm without a headrest to facilitate normal viewing conditions. The computer running the experiment was connected to the host computer (Pentium 4, Dell Inc., Round Rock, TX, USA) with EyeLink software via a local network. yt An EyeLink II system (SR Research, Ontario, Canada) recorded subjects’ pupil size and eye move- ments at a sampling rate of 500 Hz and compensated for head movements. In order to calibrate the sys- tem, the subjects made saccades to a grid of 13 fixation spots on the screen, which appeared one by one in a random order. The tracking of the eye, which provided the lower validation error, started as soon as this value was below the 0.5° visual angle. A fixation spot appeared after each trial in the middle of the screen in order to control for slow drifts in measured eye movements. In the cases of an error larger than 1°, calibration and validation were repeated. Procedure. Upon arrival, the subjects were introduced into the study procedure and had to sign a consent form. As a recent study revealed the strong impact of mood on how people observe complex images31, the subjects initially rated their current mood on four 5-point Likert-items to assess their cur- rent happiness, alertness, calmness, and listlessness32. This measurement enabled us to check whether potential differences in the dependent variables between the washing and non-washing group were con- taminated by pre-experimental differences in subjects’ mood. No difference was revealed, all t(38) ≤  1.29, p ≥  0.206. Afterward, the subjects sat down in front of a screen. The experimenter instructed them to freely observe each of the following images and to subsequently rate the morality of the depicted scene by a key press. Then, the eye-tracker was calibrated and validated. Each of the 72 scenes was presented for six seconds in accordance to previous studies (e.g.,26,31,33) and were followed by a screen that asked to rate the morality of the previous scene on a 9-point scale from 1 (very immoral) to 9 (very moral). The next scene was presented after a corresponding key press. After the subjects had observed half of the scenes (block 1), a break was introduced. Methods All h All the participants gave written informed consent to participate in this study. We performed the study in accordance with the Declaration of Helsinki and national guidelines of the German Psychological Society. The experimental methods were approved by the Ethical Committee of the University of Osnabrück (Germany). Participants. Forty subjects (16 males), who were naive to the purpose of the study, participated for course credits. Their average age was 22.25 years (SD =  3.85). We selected the number of subjects accord- ing to previous studies in the field that reported significant differences between a real cleansing condition and a control condition in which no hand cleansing was applied. Across four studies,14 (Study 1;15 Study 1 and 2;19 Study 2), a sample size of n = 20 per condition was found to be sufficient. All subjects had normal or corrected-to-normal visual acuity and passed the Ishihara Test for Color Blindness29. They were randomly assigned to one of the two experimental conditions (washing group vs. no washing group). Material and Apparatuses. The subjects observed 72 complex images, 36 immoral scenes, and 36 moral scenes. The immoral scenes were selected in accordance to the morality ratings of different nega- tive social issues18, and the moral scenes were selected according to the notion that morally good actions are those that bring about a great amount of happiness and the least amount of physical or psychological pain for other persons or the environment. The scenes depicted human behavior that is either morally bad (e.g., physically beating someone up, polluting the environment, stealing something, taking illegal drugs) or good (e.g., helping old people, handing a gift, caring for children, laughing together). The images were partially borrowed from the International Affective Picture Set (IAPS,30) and partially borrowed from the internet. All images had a resolution of 1280 ×  960 and were full-colored. Two independent raters categorized all images and completely agreed. Inter-rater agreement was additionally validated by a sam- ple of 20 subjects (not included in the eye-tracking session) who observed all images in a randomized order and rated each image on a 9-point scale from 1 (very immoral) to 9 (very moral). On average, all 36 immoral scenes were rated ≤ 3.95 (SD =  1.79), and all 36 moral scenes were rated ≥ 5.60 (SD =  1.57). Images were presented on a 21-in. Samsung SyncMaster 1100 DF 2004 CRT Monitor (Samsung Electronics, Seoul, South Korea). The Present Study This is an important question, as the majority of results rests on self-reports by the experimental subjects being susceptible to many external factors. Previous studies exclusively focused on the self-reported emotional effects of cleanliness and provided mixed results. While Liljenquist et al.27 found no effect of a clean-scented room on positive and negative affect, Zhong and Liljenquist1, as well as Reuven et al.2, found reduced negative emotions by hand washing after recalling an unethical deed of the past. According to the clean slate effect of washing, the level of participants’ arousal should also be reset to a more neutral state. To summarize, the present study addresses the three questions described above: (1) we used a pre-post control group design to investigate whether hand washing would cushion increasing stereotyp- ing in moral judgments, as suggested by Smith et al.22. Accordingly, we hypothesized that the difference in morality ratings for moral versus immoral scenes increases over time. Thereby, hand washing may induce a generalized “clean slate effect”14,15 for immoral as well as moral judgments that counteract this tendency or, alternatively, washing may elicit even more extreme judgments due to the impression of an enhanced moral self-image18 (Hypothesis 1, Change in Morality Ratings). (2) We applied eye-tracking methodology to clarify whether the sampling and processing of information is actually influenced by Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 3 www.nature.com/scientificreports/ hand washing, thus providing an objective measurement. This allows testing of the hypothesis that the impact of hand washing is limited to the formation of moral judgments and does not touch early pro- cesses of information gathering (Hypothesis 2, Information Sampling). (3) We tracked pupil size before and after hand washing as an objective measurement for subjects’ emotional arousal28. If the clean slate effect of hand washing also affects physiological arousal, pupil diameter should decrease after hand wash- ing (Hypothesis 3, Physiological Effects). Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 Results H h Hypothesis 1: Change in Morality Ratings. We initially calculated an IMAGE SET (moral vs. immoral scenes) x TIME POINT (before vs. after the break) x WASHING CONDITION (washing vs. no washing during the break) mixed-measures ANOVA (Greenhouse-Geisser applied). As expected, we found a significant effect of the image set on the morality ratings, F(1, 38) =  703.67, p <  0.001, ηp 2 =  0.95, with much higher ratings for the moral images (M =  7.38, SD =  0.84) than for the immoral images (M =  2.24, SD =  0.66) (see Fig. 1). Moreover, we found an interaction between the image set and time point, F(1, 38) =  4.93, p =  0.032, ηp 2 =  0.12, a marginally significant interaction between image set and washing condition, F(1, 38) =  2.78, p =  0.104, ηp 2 =  0.07, as well as a three-way interaction, F(1, 38) =  4.21, p =  0.047, ηp 2 =  0.10. No further effects occurred, all F(1, 38) ≤  0.60, p ≥  0.444, ηp 2 ≤  0.02. Because we were primarily interested in the potential within-subject changes between the first and sec- ond block of the experiment, we subsequently investigated whether the morality ratings changed within the washing group and the no washing group. In fact, the no washing group showed more extreme morality ratings in block 2 (t2) than in block 1 (t1) in both directions. The ratings for the moral scenes increased from the first to the second block, t(19) =  − 2.11, p =  0.049, d =  0.47, while the ratings for the immoral scenes decreased, t(19) =  2.21, p =  0.040, d =  0.49. In contrast, in the washing group, neither the morality ratings of the moral scenes changed nor the morality ratings for the immoral images, both t(19) ≤  0.57, p ≥  0.578, d ≤  0.13.f p≥ ≤ To make this interaction more visible, we computed the difference scores between t2 (block 2) and t1 (block 1), which served as a dependent variable in a 2 ×  2 (IMAGE SET x WASHING CONDITION) ANOVA. As shown in Fig. Methods All h The subjects were told that they had to perform a sensorimotor coor- dination task in order to maintain concentration and attention for the second part of the eye-tracking session. The subjects played Jenga for two minutes, but half of them previously washed their hands, alleg- edly due to hygienic reasons (washing group), whereas the control group (no washing group) waited for an additional two minutes. After the break, the subjects continued with scene observation and evaluation (block 2). The sequence of scenes was constant across the subjects in favor of the between-subject com- parison (washing vs. no washing group). Randomized scene sequences usually produce additional noise Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 4 www.nature.com/scientificreports/ in the eye-tracking data, and they do not counterbalance the potential priming effects in the viewing behavior from one scene to the next (cf.31). However, the order of scenes was pseudo-randomized within the subjects; that is, three of six successively presented scenes depicted immoral content, while the next immoral scene was not predictable. p Moreover, as we were interested in a within-subject effect of hand washing between the first and sec- ond half of the eye-tracking session, we ensured that both subsets of scenes (18 immoral and 18 moral scenes before as well as after the break) did not differ in visual saliency. Therefore, each of the 72 scenes was analyzed by means of a saliency map computation. The standard algorithm34 predicts the fixation patterns by a decomposition of pictures into several physical features (e.g., contrast and orientation). Additionally, we applied a more recent graph-based visual saliency algorithm (GBVS) to predict the fixations with an even higher reliability35. Both algorithms revealed no difference between the scenes of the first and second eye-tracking part, both t(35) ≤  0.10, p ≥  0.924.i i y g p p Finally, in order to check whether the image ratings depended on the specific image sequence in the eye-tracking session, a third group (comparison group) of 20 subjects with a mean age of 20.75 (SD =  2.65) observed and rated all images in a randomized order (without eye-tracking and an intermediate break). Eye Movement Parameters. In order to appropriately describe the viewing behavior of study par- ticipants, we selected parameters that are both very sensitive to the emotional and motivational states of the observer as well as a suitable indicator for global viewing activity26,31. Methods All h In accordance with previous studies on human vision, we therefore focused on the mean length of saccades as an indicator for the visual step size that participants use to scan the scenes. The detection of a saccade was based on the following three default measures: amplitude of at least 0.1° visual angle, with a velocity of at least 30°/s, and with an acceleration of at least 8000°/s2. The minimal saccade velocity was 25°/s after saccade onset. All these values had to be sustained for at least 4 ms for a valid saccade. In contrast, fixations were defined as periods without saccades36. We also investigated the mean duration of fixations because it is a sensitive indicator for information processing depth. It is believed that longer fixation durations indicate a deeper and more extensive processing of visual stimuli37,38. The eye-tracker detected and automatically parameterized fixations and saccades during each fixation. The first fixation of each trial was excluded from the analysis because its localization was determined by the preceding fixation spot used for drift correction. Finally, we applied an entropy measurement to quantify the extent of the spatial exploration of the scenes. Entropy has the advantage of measuring the spread of fixation distributions without the necessity to make assumptions about scene-specific geometrical arrangements. Higher entropy values indicate a more extensive fixation distribution; that is, a more spatially extended exploration of a scene. Thereby, the absolute values in entropy are negligible, as they depend on image resolution and the size of the Gaussian kernel that we used to convolve the fixation distribution map (leading to a fixation density map that is used for entropy calculation). This procedure is described in detail elsewhere39. Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The effect of hand washing on morality ratings. The left panel depicts absolute values, the right panel depicts the change in morality ratings from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines indicate the standard error of the mean. Figure 1. The effect of hand washing on morality ratings. The left panel depicts absolute values, the right panel depicts the change in morality ratings from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines indicate the standard error of the mean. the image sequence was randomized across subjects. In this group, the difference scores for the moral and immoral scenes (mean of last 36 images vs. first 36 images) also differed significantly, t(19) =  2.38, p =  0.028, d =  0.53, with increased morality ratings for moral scenes (+ 0.158), but slightly decreased ratings for immoral scenes (− 0.053). g ( ) To conclude, on a scale ranging from very immoral to very moral, we found much higher morality ratings for the moral scenes than for the immoral scenes, thus validating our stimulus set. Even more importantly, we found more stereotyped morality judgments over time (block 2 vs. block 1), but this effect disappeared when participants washed their hands during the break. Thus, the results support the notion of a clean slate effect. Hand washing apparently reset the dynamic in participants’ morality judgments. Hypothesis 2: Information Sampling. With respect to the mean duration of fixations, we initially calculated a 2 ×  2 ×  2 (IMAGE SET x TIME POINT x WASHING CONDITION) ANOVA. As shown in Fig. 2, we found a significant main effect of the image set, F(1, 38) =  103.60, p <  0.001, ηp 2 =  0.73, with longer fixations on the immoral versus moral scenes. We also found a main effect of the time point, F(1, 38) =  4.56, p =  0.039, ηp 2 =  0.11, with longer fixations in block 2 versus block 1. The image set and time point also significantly interacted, F(1, 38) =  15.08, p <  0.001, ηp 2 =  0.28, whereby the effect of the time point (i.e., longer fixation durations in block 2 vs. www.nature.com/scientificreports/ block 1) was only present on moral scenes, t(39) =  4.36, p <  0.001, d =  0.69, but not on immoral scenes, t(39) =  − 0.38, p =  0.70, d =  0.06. The ANOVA did not show further significant effects, all F(1, 38) ≤  0.47, p ≥  0.495, ηp 2 ≤  0.01, except a just significant interaction between image set and washing condition, F(1, 38) =  4.00, p =  0.053, ηp 2 =  0.10, with a higher difference between immoral and moral scenes in the no washing group versus washing group. However, and importantly, this slight interaction has to be interpreted with caution. Although the three-way interaction did not reach statistical significance, it is not apparent how much of the group difference is due to between-subject differences that already existed from the outset; that is, before the cleanliness treatment was applied to the washing group. Thus, to clarify whether hand washing actu- ally affected participants’ fixation durations, we again calculated the difference scores between the two blocks (t2 minus t1) that represent the temporal change and thus correct for the group differences that might have already existed before the cleanliness treatment. We used the difference scores as a depend- ent variable in a 2 ×  2 (IMAGE SET ×  WASHING CONDITION) ANOVA. We found no effect of the washing condition and no interaction, both F(1, 38) ≤  0.47, p ≥  0.495, ηp 2 ≤  0.01, but the difference in the fixation duration between block 1 and block 2 differed between the moral and immoral scenes, F(1, 38) =  15.08, p <  0.001, ηp 2 =  0.28, thus reflecting the image set by time point interaction reported above. As shown by Fig. 2, in both experimental groups, the fixation durations on moral scenes were longer after the break, both t(19) =  2.76, p ≤  0.012, d ≥  0.62, while there was no difference between the two groups, t(38) =  0.99, p =  0.330, d =  0.31. Regarding the immoral scenes, the difference scores neither differed from zero within the two groups, both |t|(19) ≤  0.39, p ≥  0.700, d ≤  0.09, nor between them, t(19) =  0.11, p =  0.912, d =  0.04. Consequently, hand washing did not significantly affect the duration of fixations. Results H h 1, we again found no main effect of the washing condition, F(1, 38) =  0.03, p =  0.866, ηp 2 <  0.01, but the difference scores revealed a significant main effect of the image set, F(1, 38) =  4.93, p =  0.032, ηp 2 =  0.12, with a negative difference score for the moral scenes, but a positive dif- ference score for the immoral scenes. This main effect reflects the image set by the time point interaction found in the initial three-way ANOVA. Moreover, the image set interacted with the washing condition, F(1, 38) =  4.21, p =  0.047, ηp 2 =  0.10, thus reflecting the three-way interaction previously found. p ηpl g y p y Finally, in order to verify that the tendency toward more extreme morality ratings in the no washing group was not an artefact of the constant sequence of scenes, we tested the comparison group, in which Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 5 www.nature.com/scientificreports/ I h d h l h f d h d h ifi In the next step, we investigated the length of saccades that participants made to scan the scenes. The 2 ×  2 ×  2 (IMAGE SET x TIME POINT x WASHING CONDITION) ANOVA showed a main 6 Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ Figure 2. The mean duration of fixations. The left panel depicts absolute values, the right panel depicts the change in fixation duration from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines indicate the standard error of the mean. Figure 2. The mean duration of fixations. The left panel depicts absolute values, the right panel depicts the change in fixation duration from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines indicate the standard error of the mean. Figure 3. The mean length of saccades (left panel) and the mean entropy quantifying the extent of spatial exploration of scenes (right panel). Vertical lines indicate the standard error of the mean. Figure 3. The mean length of saccades (left panel) and the mean entropy quantifying the extent of spatial exploration of scenes (right panel). Vertical lines indicate the standard error of the mean. effect of the time point, F(1, 38) =  23.71, p <  0.001, ηp 2 =  0.38, with shorter saccades in block 2 versus 1 (see Fig. 3). Moreover, a main effect of the image set occurred, F(1, 38) =  31.42, p <  0.001, ηp 2 =  0.45, with longer saccades on immoral versus moral scenes. No further effects were found, F(1, 38) ≤  2.05, p ≥  0.160, ηp 2 ≤  0.05. Accordingly, when using difference scores (t2 minus t1) in a 2 ×  2 (IMAGE SET x WASHING CONDITION) ANOVA, no effect remained, all F(1, 38) ≤  2.05, all p ≥  0.160, all ηp 2 ≤  0.05, because calculating the difference scores naturally eliminated the main effects of the time point and image set. ll d h f h l l f fi d b Th Finally, we investigated the extent of the spatial exploration of images quantified by entropy. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. The effect of hand washing on pupil size. The left panel depicts absolute values, the right panel depicts the change in pupil size from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines indicate the standard error of the mean. Figure 4. The effect of hand washing on pupil size. The left panel depicts absolute values, the right panel depicts the change in pupil size from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines indicate the standard error of the mean. To sum up, the viewing behavior changed between block 1 and block 2 in terms of longer fixations on the moral (but not immoral) scenes, which were paralleled by a less spatially extensive explora- tion (entropy). Moreover, saccades were longer before versus after the break, and they were longer on immoral versus moral scenes. However, and in contrast to the morality judgments, hand washing did not influence eye movement parameters. Consequently, the impact of hand washing did not touch early processes of information sampling from the environment. Hypothesis 3: Physiological Effects. We initially calculated a 2 ×  2 ×  2 (IMAGE SET ×  TIME POINT ×  WASHING CONDITION) ANOVA with pupil size as the dependent variable. As shown by Fig. 4, we found a significant main effect of the image set, F(1, 38) =  196.51, p <  0.001, ηp 2 =  0.84, with larger pupil sizes when observing immoral versus moral scenes. We also found a main effect of the time point, F(1, 38) =  53.91, p <  0.001, ηp 2 =  0.59, with larger pupil sizes in block 1 versus block 2. Moreover, we found a marginally significant main effect of the washing condition, F(1, 38) =  2.89, p =  0.097, ηp 2 =  0.07, with larger pupil sizes in the washing versus no washing group. Also, we found an interaction between the time point and washing condition, F(1, 38) =  4.61, p =  0.038, ηp 2 =  0.11. The three-way interaction was marginally significant, F(1, 38) =  2.78, p =  0.104, ηp 2 =  0.07. No interaction between image set and washing condition, as well as between image set and time point, occurred, both F(1, 38) ≤  0.22, p ≥  0.643, ηp 2 ≤  0.01. www.nature.com/scientificreports/ p ηp In order to further scrutinize the interactions with regard to the impact of the washing treatment, we again computed the difference scores (t2 minus t1) serving as the dependent variable in a 2 ×  2 (IMAGE SET ×  WASHING CONDITION) ANOVA. This analysis revealed no main effect of the image set, F(1, 38) =  0.22, p =  0.643, ηp 2 =  0.01, but a marginally significant interaction, F(1, 38) =  2.78, p =  0.104, ηp 2 =  0.07, that reflected the three-way interaction reported above. Finally, the washing condition showed a significant main effect, F(1, 38) =  4.61, p =  0.038, ηp 2 =  0.11, that reflected the washing condition by time point interaction reported above. As Fig. 4 shows, in both conditions (washing and no washing), the difference in pupil size was significantly negative (one-sample t-tests against zero: all p <  0.001), thus indicating a smaller pupil size after the break. However, the decrease in pupil size was significantly larger when subjects had washed their hands between block 1 and block 2. Consequently, hand washing also affected the physiological level. Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ The 2 ×  2 ×  2 (IMAGE SET ×  TIME POINT ×  WASHING CONDITION) ANOVA again showed a main effect of the time point, F(1, 38) =  9.13, p =  0.004, ηp 2 =  0.19, a main effect of the image set, F(1, 38) =  79.62, p <  0.001, ηp 2 =  0.68, and an interaction between these two factors, F(1, 38) =  20.65, p <  0.001, ηp 2 =  0.35. As depicted in Fig. 3, the subjects explored moral scenes more spatially extensively in block 1 versus block 2, t(39) =  5.06, p <  0.001, d =  0.80, while the immoral scenes were equally extensively explored before versus after the break, t(39) =  − 0.302, p =  0.765, d =  0.05. The ANOVA did not show further significant effects, all F(1, 38) ≤  1.29, all p ≥  0.264, all ηp 2 ≤  0.03. 7 Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 7 Discussion Hand washing is a daily ritual whose fundamental goal is to protect us against physical and biological contamination and, hence, foster physiological health. However, empirical evidence showed that hand washing also affects moral judgments. Unmoral deeds enhance the desire for cleansing products in terms of a Macbeth effect1,3,4, and the impact of washing goes beyond judgment formation, as it induces a kind of clean slate effect in several domains14–17. The present study addresses three critical questions focused on the original domain of moral judgments.f First, we found that hand washing between the two blocks induced a clean slate effect in the form of eliminating the tendency for more extreme moral judgments over time. This finding supports previous studies that reported clean slate effects regarding cognitive dissonance14, good and bad luck15, failure experience16, and product evaluation17. In each case, physical cleansing washed away traces of the past by resetting previous experiences. The present within-subject design revealed such a general clean slate 8 Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ effect in the domain of moral judgments, thus generalizing to the judgment of other people’s bad and good deeds (Hypothesis 1). Second, we found more extreme moral judgments over time for moral and immoral scenes as long as no hand washing took place in the meantime. This finding corresponds to the results of Smith et al.22, who found that increased familiarity of information fosters stereotyped judgments. Smith et al. explained this by changes in information processing. According to their findings, increasing familiar- ity of information leads to less systematic or analytic processing, thus resulting in more stereotyped judgments. Indeed, we found changes in viewing activity as a signature of image processing from the first to the second block. This effect was, however, mainly limited to the moral scenes: fixation duration increased and the amount of spatial exploration decreased on the moral images over time while no change occurred on the immoral scenes. Moreover, saccades were shorter in block 2 versus 1, and they were longer on immoral versus moral scenes. This result pattern matches studies showing that images of negative valence are observed more actively31. Beyond this, we found no hint that hand washing affects eye-tracking parameters. This result is evidence against an effect of physical cleansing on information sampling from the environment. Discussion Instead, the effect of hand washing seems to affect the selection and/or weighting of information stored in memory (e.g., social norms).h Third, the present study provides objective evidence for an impact of physical cleansing on the phys- iological level. While previous studies limited the focus to self-reports, we analyzed pupil size as an indicator for emotional arousal28. We found that pupil size decreased from the first to the second meas- urement block, while hand washing in between the measurement blocks additionally decreased pupil size (Hypothesis 3). Presumably, the subjects successively habituated to the (im)moral scenes indicated by a downregulation of arousal. The immoral scenes are especially arousing, as shown by the norm values of the IAPS manual30. Hand washing seems to facilitate emotional downregulation, as also suggested by Zhong and Liljenquist1, as well as Reuven et al.2, who found attenuated reports of negative emotions when subjects washed their hands after they had recalled an unethical deed of the past. Consequently, the clean slate effect is more than a cognitive reset, and hand washing actually affects the physiological level.ii f g g yf p y g Given the present findings, future studies in the field of embodied cognition research should fathom the psychophysiological effects of phenomena that have been primarily investigated on the behavioral level so far – such as physical cleansing. A bulk of studies has already shown that the effects of bodily sensations are not limited to cognitive processes but they also affect emotional states (cf.10,40). The effect of hand washing on pupil size and the unaffected eye movement behavior indicate a fruitful avenue for follow-up studies. Uncovering the psychophysiological mechanisms behind embodiment phenomena not only increases our understanding of the cognitive consequences of bodily sensations such as morality judgments. It also helps to exploit the potential for therapeutic and other applied purposes. References 2. Reuven, O., Liberman, N. & Dar, R. The effect of physical cleaning on threatened morality in individuals with obsessive compulsive disorder. Clinical Psychological Science 2, 224–229 (2013).t y g 3. Gollwitzer, M. & Melzer, A. Macbeth and the joystick: evidence for moral cleansing after playing a violent video game. Journa of Experimental Social Psychology 48, 1356–1360 (2012). f p y gy 4. Denke, C., Rotte, M., Heinze, H. J., & Schaefer, M. Lying and the subsequent desire for toothpaste: activity cortex predicts embodiment of the moral-purity metaphor. Cerebral Cortex (2014). doi: 10.1093/cercor/b f y gy 4. Denke, C., Rotte, M., Heinze, H. J., & Schaefer, M. 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Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 9 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Kaspar, K. et al. Hand Washing Induces a Clean Slate Effect in Moral udgments: A Pupillometry and Eye-Tracking Study. Sci. Rep. 5, 10471; doi: 10.1038/srep10471 (2015). How to cite this article: Kaspar, K. et al. Hand Washing Induces a Clean Slate Effect in Moral Judgments: A Pupillometry and Eye-Tracking Study. Sci. Rep. 5, 10471; doi: 10.1038/srep10471 (201 This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://lirias.kuleuven.be/bitstream/20.500.12942/694156/2/Cerebrospinal%20fluid%20tau%20levels%20are%20associated%20with%20abnormal%20neuronal%20plasticity%20markers%20in%20Alzheimers%20disease.pdf
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Cerebrospinal fluid tau levels are associated with abnormal neuronal plasticity markers in Alzheimer’s disease
Molecular neurodegeneration
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Abstract Background:  Increased total tau (t-tau) in cerebrospinal fluid (CSF) is a key characteristic of Alzheimer’s disease (AD) and is considered to result from neurodegeneration. T-tau levels, however, can be increased in very early disease stages, when neurodegeneration is limited, and can be normal in advanced disease stages. This suggests that t-tau levels may be driven by other mechanisms as well. Because tau pathophysiology is emerging as treatment target for AD, we aimed to clarify molecular processes associated with CSF t-tau levels. Methods:  We performed a proteomic, genomic, and imaging study in 1380 individuals with AD, in the preclinical, prodromal, and mild dementia stage, and 380 controls from the Alzheimer’s Disease Neuroimaging Initiative and EMIF-AD Multimodality Biomarker Discovery study. Results:  We found that, relative to controls, AD individuals with increased t-tau had increased CSF concentrations of over 400 proteins enriched for neuronal plasticity processes. In contrast, AD individuals with normal t-tau had decreased levels of these plasticity proteins and showed increased concentrations of proteins indicative of blood– brain barrier and blood-CSF barrier dysfunction, relative to controls. The distinct proteomic profiles were already present in the preclinical AD stage and persisted in prodromal and dementia stages implying that they reflect disease traits rather than disease states. Dysregulated plasticity proteins were associated with SUZ12 and REST signaling, suggesting aberrant gene repression. GWAS analyses contrasting AD individuals with and without increased t-tau highlighted several genes involved in the regulation of gene expression. Targeted analyses of SNP rs9877502 in GMNC, associated with t-tau levels previously, correlated in individuals with AD with CSF concentrations of 591 plasticity associated proteins. The number of APOE-e4 alleles, however, was not associated with the concentration of plasticity related proteins. *Correspondence: pj.visser@amsterdamumc.nl; pj.visser@maastrichtuniversity.nl 1 Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, PO Box 7057 1007, MB, Amsterdam, The Netherlands Full list of author information is available at the end of the article *Correspondence: pj.visser@amsterdamumc.nl; pj.visser@maastrichtuniversity.nl 1 Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, PO Box 7057 1007, MB, Amsterdam, The Netherlands Full list of author information is available at the end of the article © The Author(s) 2022. Visser et al. Molecular Neurodegeneration (2022) 17:27 https://doi.org/10.1186/s13024-022-00521-3 Open Access Cerebrospinal fluid tau levels are associated with abnormal neuronal plasticity markers in Alzheimer’s disease Pieter Jelle Visser1,2,3*  , Lianne M. Reus1, Johan Gobom4,5, Iris Jansen6, Ellen Dicks1, Sven J. van der Lee1,7, Magda Tsolaki8, Frans R. J. Verhey2, Julius Popp9,10, Pablo Martinez‑Lage11, Rik Vandenberghe12,13, Alberto Lleó14, José Luís Molinuevo15,16, Sebastiaan Engelborghs17,18, Yvonne Freund‑Levi3,19, Lutz Froelich20, Kristel Sleegers21,22, Valerija Dobricic23, Simon Lovestone24, Johannes Streffer17,25, Stephanie J. B. Vos2, Isabelle Bos2, ADNI, August B. Smit26, Kaj Blennow4,5, Philip Scheltens1, Charlotte E. Teunissen27, Lars Bertram22,28, Henrik Zetterberg4,5,29,30 and Betty M. Tijms1 CSF analysis CSF samples in EMIF-AD MBD were collected according to the BIOMARKAPD protocol, [12, 18] and in ADNI as described elsewhere [19]. Abstract 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 Conclusions:  CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and blood–brain and blood-CSF barrier dysfunction. Future trials may need to stratify on CSF t-tau status, as AD individuals with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite CSF prot‑ eomic profiles. Keywords:  Alzheimer’s disease, Molecular mechanisms, Biomarker discovery, Heterogeneity, Neuronal plasticity, Cerebrospinal fluid proteomics Conclusions:  CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and blood–brain and blood-CSF barrier dysfunction. Future trials may need to stratify on CSF t-tau status, as AD individuals with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite CSF prot‑ eomic profiles. Keywords:  Alzheimer’s disease, Molecular mechanisms, Biomarker discovery, Heterogeneity, Neuronal plasticity, Cerebrospinal fluid proteomics Clinical assessment Global cognition was assessed by the Mini-Mental State Examination (MMSE) [14] and ADAS-Cog 11-item ver- sion (ADNI) [15]. As a measure for memory function we used the delayed recall of the logical memory subscale II of the Wechsler Memory Scale (ADNI), [16] or center specific verbal word learning tasks (EMIF-AD MBD) [12]. We selected the Clinical Dementia Rating (CDR) scale sum of boxes score as a measure of disease severity [17]. Group definition and staging We selected individuals with AD pathology defined as abnormally decreased CSF amyloid beta 1–42 (Aβ1-42), and we subdivided this group into those with abnor- mally increased total tau (t-tau) and those with normal t-tau. Based on cognitive performance, AD individuals were classified in 3 clinical stages as preclinical AD (nor- mal cognition), prodromal AD (MCI) and mild AD-type dementia according to study specific criteria [12, 13]. The control group consisted of individuals with normal cog- nition, and normal CSF Aβ1-42 and t-tau levels. Backgroundh collaboration under the supervision of Principle Inves- tigator Michael W. Weiner, MD. The primary goal of ADNI is to study whether serial biological markers, and clinical and neuropsychological measures can be com- bined to measure progression of MCI and early AD and has enrolled 2850 individuals, see www.​adni-​info.​org. The EMIF-AD MBD study aimed to identify markers for diagnosis and prognosis of predementia AD. It combined existing clinical data, samples and scans of 1218 individu- als with normal cognition, MCI or mild dementia from prospective cohort studies [12]. g The amyloid cascade hypothesis poses that Alzheimer’s disease (AD) starts with amyloid beta (Aβ) aggregation followed by tau pathology [1]. Increased total tau (t-tau) levels in cerebrospinal fluid (CSF) are supposed to be caused by axonal loss [2]. Still, 25% of the AD individuals with mild cognitive impairment (MCI) or dementia have normal t-tau levels despite axonal loss [3, 4]. Moreover, CSF t-tau is abnormally increased in 50% of preclinical AD individuals, when neurodegeneration is limited [5]. Furthermore, our study in cognitively normal monozy- gotic twins discordant for amyloid aggregation, suggested that CSF t-tau levels may rise even before amyloid aggre- gation reaches abnormal levels [6]. An alternative expla- nation for the increase in t-tau in AD may be increased gene expression. A SILK study found that elevated CSF t-tau levels in AD were associated with increased pro- duction rather than from spilling of intracellular tau by dying neurons [7]. A study with induced pluripotent stem cells (iPSC) showed that AD individuals had increased tau expression in neuronal progenitor cells compared to controls [8]. Increased t-tau levels may also result of increased neuronal activity, [9] or increased neuronal plasticity as t-tau has an high expression in brain regions with plastic potential [10]. As the drivers of increased tau levels in AD remain largely unknown, and because tau-related mechanisms are emerging as new treatment targets for AD, we aimed to clarify molecular processes associated with CSF tau dysregulation. To this end, we performed a proteomic, genomic and imaging study including 1380 individuals in the AD continuum, [11] defined by abnormal CSF Aβ1-42, spanning the clinical spectrum from preclinical AD to mild dementia, and 380 controls with normal cognition and normal CSF Aβ1-42 and t-tau from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and the European Medical Information Framework for Alzheimer’s disease (EMIF-AD) Multi- modality Biomarker Discovery (MBD) study [2]. Study participants CSF Aβ1-42 and t-tau were measured locally with INNOTEST ELISA or INNOBIA AlzBio3 (Fujirebio, Ghent, Belgium). Cut-offs for Aβ1-42 and t-tau were We selected 961 individuals from ADNI (adni.loni. usc.edu) and 799 individuals from the EMIF-AD MBD study [12]. ADNI started in 2003 as a public–private Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 3 of 16 cohort-specific in EMIF-AD MBD [12]. Aβ1-42 cut-offs were determined for each cohort using Gaussian mix- ture modelling [20]. We measured neurogranin, neuro- filament-light, YKL-40, Aβ38 and Aβ40 by ELISA [2]. We performed untargeted mass spectrometry using tan- dem mass tag (TMT) with 10 + 1 plexing as previously described, using high-pH reverse phase HPLC for peptide prefractionation [20, 21]. Peptides were mapped to 2535 proteins using the software ProteomeDiscoverer v.2.2. (Thermo Scientific), using Mascot (MatrixScience) for protein identification (precursor Dm tolerance 15 ppm, fragment tolerance 0.05  Da, max missed cleavage sites 2), searching the human subset of the UniProtKB Swis- sProt database (www.​unipr​ot.​org). Percolator (MatrixSci- ence) was used for scoring peptide specific matches, and a strict 1% false discovery rate (FDR) was set as threshold for identification. For reporter ion quantification the fol- lowing settings were used: Integration tolerance 20 ppm; Integration Method Most Confident Centroid; normalize on the reference protein average. The median (IQR) CV for these proteins was 5.6 (3.8, 8.0). cohort-specific in EMIF-AD MBD [12]. Aβ1-42 cut-offs were determined for each cohort using Gaussian mix- ture modelling [20]. We measured neurogranin, neuro- filament-light, YKL-40, Aβ38 and Aβ40 by ELISA [2]. We performed untargeted mass spectrometry using tan- dem mass tag (TMT) with 10 + 1 plexing as previously described, using high-pH reverse phase HPLC for peptide prefractionation [20, 21]. Peptides were mapped to 2535 proteins using the software ProteomeDiscoverer v.2.2. (Thermo Scientific), using Mascot (MatrixScience) for protein identification (precursor Dm tolerance 15 ppm, fragment tolerance 0.05  Da, max missed cleavage sites 2), searching the human subset of the UniProtKB Swis- sProt database (www.​unipr​ot.​org). Percolator (MatrixSci- ence) was used for scoring peptide specific matches, and a strict 1% false discovery rate (FDR) was set as threshold for identification. For reporter ion quantification the fol- lowing settings were used: Integration tolerance 20 ppm; Integration Method Most Confident Centroid; normalize on the reference protein average. The median (IQR) CV for these proteins was 5.6 (3.8, 8.0). MBD sample). For related proteins that had identical values due to fragment a-specificity (e.g. Proteomic process scores (PPS) Proteins and protein fragments (ADNI) values were standardized according the mean and standard deviation values of the control group and expressed as z-scores, with a score of 0 indicating the average concentration of the control group, z-scores > 0 higher concentrations, and z-scores < 0 lower concentrations than controls. For ADNI, we averaged peptides that mapped to the same protein into a composite protein score when they cor- related with r > 0.5, and included them as single peptides otherwise [20]. When the same protein was measured by different platforms in ADNI, values were averaged if they correlated with r > 0.5 and otherwise we included them as separate proteins. From the EMIF-AD MBD TMT mass spectrometry analysis proteins were included when observed in at least 92 individuals (30% of EMIF-AD For illustration purposes we combined proteins from selected GO-BPs, and proteins associated with BBB dys- function and BACE1 substrates into a PPS by averaging z-scores of individual proteins belonging to the process that differed between AD individuals with normal or increased t-tau in cross-sectional analysis in the total group. ADNI Aβ1-42 and t-tau were measured on the xMAP platform (Luminex Corp, Austin, TX). Cut-offs for Aβ1-42 and t-tau were used as published [19]. Changes in t-tau levels over time were assessed in ADNI using longitudinal samples measured within the same batch. YKL-40, neurogranin, APP beta, neurogranin, neurofilament light, alpha synu- clein, HBB, CFH, sTREM2, VILIP-1, and BACE1 were measured by ELISA or related assays, 190 analytes were analyzed by the Human DiscoveryMAP panel (MAP- RBM) and 225 proteins were analyzed by targeted mass spectroscopy [22, 23]. We used the quality checked and finalised ‘Normalized Intensity’ data as described in https://​adni.​loni.​usc.​edu/​wp-​conte​nt/​uploa​ds/​2012/​ 01/​2011D​ec28-​Bioma​rkers-​Conso​rtium-​Data-​Primer-​ FINAL1.​pdf Study participants ACTA1 and ACTA2) we randomly selected one protein for analysis. Other annotations Proteins associated with high choroid plexus expression were defined based on the Allen Brain Atlas, [32] through Harmonizome [29] and ABAEnrichment analysis [33]. Blood–Brain barrier (BBB) associated proteins were selected from reference [34–36]; BACE1 substrates were selected from reference [37, 38]; Alpha secretase sub- strates were selected from reference [39, 40]; and gamma secretase substrates were selected from reference [41]. Predominant cell‑type of protein production Based on the BRAIN RNASeq database (http://​www.​ brain​rnaseq.​org), [31] we labelled proteins as being pre- dominantly produced by a cell type when levels were higher than 40% of older individuals produced across cell types. Proteomic annotation Enrichment analysis We included proteins for enrichment analyses that were increased or decreased at p < 0.05 relative to the contrast tested. Biological process enrichment analyses  We performed pathway enrichment analyses for Gene Ontology biologi- cal processes (GO-BP) using the online Panther applica- tion and SynGo [24–27]. Transcription factor enrichment analyses  We per- formed transcription factor enrichment analysis using ChEA through Harmonizome through Enrichr [28–30]. Genotype imputation and quality control Data processing and quality control was performed using GenomeStudio Software (v2.0.04, Illumina, Inc.), as described previously [44]. To identify ethnic outliers, a principal component analysis (PCA) of ancestry was per- formed based on 1000 Genomes clustering, phase 3 using PLINK [44]. Individuals of non-European descent and family relations up to second degree were excluded. After filtering, the ADNI genotype data included 747 individu- als and EMIF-AD MBD 931 individuals. SNPs were locally imputed using Minimac 3 to the Haplotype Reference Consortium reference panel [46]. To account for population structure, principal com- ponents (PC1-PC20) were computed on a subset of relatively uncorrelated (r2 < 0.2) single nucleotide poly- morphisms (SNPs). For ADNI subjects, Apolipopro- tein E (APOE) ε4 genotype was assessed with two SNPs; rs429358 the ‘ε4-allele’ and rs7412 the ‘ε2 allele’. APOE ε4 genotypes in EMIF-AD MBD were generated as described elsewhere [44]. Genomic assessment EMIF-AD MBD samples were genotyped using the Illu- mina Global Screening array (Illumina, Incl) and 936 samples passed post-experiment QC [44]. ADNI samples were genotyped using the Illumina 2.5-M or Illumina OmniQuad array [45]. Statistics Our main outcomes are the biological process and tran- scription enrichment analyses for which we report FDR corrected p-values. For other analysis we report uncor- rected p-values. Test statistics can be found in the sup- plemental tables. Group comparisons between AD subgroups and controls were performed using ANOVA correcting for age and gender. Change in cognition and imaging markers were assessed with linear mixed models including as main terms group, time, and the interaction group by time, and correcting for age and gender, and additionally for level of education for cognitive markers. We investigated within the AD individuals with normal t-tau and AD individuals with increased t-tau whether protein concentrations increased or decreased with increasing disease severity ranging from preclinical AD, prodromal AD, to mild AD-dementia with linear models. A protein was considered to decrease or increase with disease severity if the linear trend was statistically signifi- cant or if there was a difference in concentration between preclinical AD and prodromal AD, preclinical AD and mild AD-dementia, or between prodromal AD and mild AD-dementia and the trend analysis supported the change with severity. Associations between GRS scores Polygenic and other genetic risk score analysis Polygenic and other genetic risk score analysis Polygenic risk scores (PGRS) for AD were computed for each subject using PRSice (V2.3) [48]. PGRS were cal- culated by adding the sum of each allele weighted by the strength of its association with AD risk as calculated previously [49]. AD cases were defined as patients clini- cally diagnosed with AD-type dementia or individuals with a parental history of AD (i.e., AD-by-proxy) [49]. Clumping was performed prior to calculating PGRS, to remove SNPs that are in linkage disequilibrium (r2 > 0.1) within a slicing 1 M bp window. After clumping, PGRS were computed using various SNP inclusion thresholds. In order to explore how specific genetic alterations were associated with CSF proteomic profiles, we calculated for a selection of genesets based on MAGMA analysis (see results), gene-specific risk scores (GRS) including only SNPs located on the respective genes with clumping as described above and computation of GRS using various SNP inclusion thresholds. We used weights from one cohort to generate GRS in the other cohort. In order to reduce the number of tests, GRS scores were selected for SNP inclusion threshold that best differentiated within AD between abnormally increased and normal t-tau. Neuroimaging analysis We studied neuroimaging data only from ADNI, because this study collected longitudinal scans. As a measure of brain atrophy, we took cortical thickness data from 34 cortical areas from the longitudinal processing pipeline in Freesurfer version 4.3 for 1.5 T T1-weighted MRI scans, and v5.1 for 3  T T1-weighted MRI scans (http://​adni.​ Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 4 of 16 of the meta-analysis summary statistics. We considered for further analysis gene scores based on at least 6 SNPs and geneset scores with information on at least 6 genes. loni.​usc.​edu). As a measure of amyloid accumulation, we used region-specific SUVR values for florbetapir bind- ing assessed by PET imaging in 34 cortical areas as par- cellated by Freesurfer v4.5.0 [42]. As a measure of brain metabolism we analysed fluorodeoxyglucose (FDG)-PET scans, [43] and determined average glucose metabolism for brain areas standardized to the average uptake in the vermis and brain stem according to the Desikan-Killiany atlas (http://​adni.​loni.​usc.​edu/​metho​ds/​pet-​analy​sis-​ method/​pet-​analy​sis/). loni.​usc.​edu). As a measure of amyloid accumulation, we used region-specific SUVR values for florbetapir bind- ing assessed by PET imaging in 34 cortical areas as par- cellated by Freesurfer v4.5.0 [42]. As a measure of brain metabolism we analysed fluorodeoxyglucose (FDG)-PET scans, [43] and determined average glucose metabolism for brain areas standardized to the average uptake in the vermis and brain stem according to the Desikan-Killiany atlas (http://​adni.​loni.​usc.​edu/​metho​ds/​pet-​analy​sis-​ method/​pet-​analy​sis/). GWAS and post‑GWAS analyses Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD individuals with normal tau from controls. Venn diagram shows number of proteins that differed Fig. 1  Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD individuals with normal tau from controls. Venn diagram shows number of proteins that differed and CSF protein levels were tested with linear models adjusted for age and sex. decreased levels of 2 proteins in ADNI (1% of proteins measured) and of 43 proteins in EMIF-AD MBD (3% of proteins measured) (Fig. 1, Data S2). AD individuals with normal CSF t-tau, showed an opposite pattern with increased levels of only 7 proteins in ADNI (3% of pro- teins measured) and of 60 proteins in EMIF-AD MBD (4% of proteins measured) and decreases in 107 proteins in ADNI (43% of proteins measured) and of 411 proteins in EMIF-AD MBD (28% of proteins measured). Around 50% of the proteins with increased levels in individuals with increased t-tau had decreased levels in individu- als with normal t-tau. This indicates that t-tau groups show disruptions in the same molecular processes, but in opposite directions. GWAS and post‑GWAS analyses Genome-wide association study (GWAS) analyses on increased CSF t-tau in subjects with abnormal Aβ1-42 were performed separately in ADNI (abnormal Aβ1-42 and increased t-tau n = 246, abnormal Aβ1-42 and nor- mal t-tau n = 238) and EMIF-AD MBD (abnormal Aβ1-42 and increased t-tau n = 294, abnormal Aβ1-42 and nor- mal t-tau n = 155) using PLINK software (V1.90). We used a logistic regression model including PC1-PC3 and sex. Genome-wide significance was defined as p ≤ 5e-08. Meta-analysis on ADNI and EMIF-AD MBD GWAS summary statistics was performed using METAL [47]. MAGMA software was used to compute gene scores and geneset scores for biological pathways based on p-values Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 5 of 16 Fig. 1  Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD individuals with normal tau from controls. Venn diagram shows number of proteins that differed Fig. 1  Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD individuals with normal tau from controls. Venn diagram shows number of proteins that differed Fig. 1  Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Results Of the individuals with AD, 788 (57%) had increased CSF t-tau levels and 592 (43%) normal t-tau levels. Abnormal and normal t-tau groups showed generally similar demo- graphics and baseline cognitive performance (Data S1a). We first determined stability of t-tau levels over time in 499 ADNI participants who had repeated CSF sam- pling up to 7 years. CSF t-tau increased in the prodromal AD stage at similar rates for both t-tau groups, and this increase did not differ from the increase in t-tau in the control group (Figure S1a). At last follow-up, 20% of the AD individuals with normal t-tau at baseline developed abnormally increased t-tau at follow-up and these indi- viduals had t-tau levels just below the cut-point at base- line (Figure S1b). Taken together, these findings suggest that CSF t-tau levels reflect a trait rather than a stage. Enrichment analyses with GO-BP for CSF proteins increased in the AD group with abnormal t-tau rela- tive to controls, showed in both ADNI and EMIF-AD MBD involvement of plasticity-related processes such as nervous system development, axonogenesis, syn- apse assembly, myelination, gliogenesis, angiogenesis, mitogen-activated protein kinase (MAPK) signaling, cell-cycle, gene expression and glycolysis (Fig. 2 selec- tion of representative processes; Figure S2 shows synaptic processes; Data S3a, S3b show all enriched processes). Abnormality in each of these processes has been reported before in separate studies, [8, 50–55] and our findings now suggest that they are part of com- mon plasticity response. We also observed involvement l We then studied CSF proteome profiles for t-tau sub- groups in 559 individuals with proteomic data (Data S1b). This included targeted proteomics of 248 proteins in ADNI and targeted and untargeted proteomics of 1458 proteins in EMIF-AD MBD (Data S2). Compared to controls, AD individuals with increased CSF t-tau had increased levels of 130 proteins in ADNI (52% of ADNI proteins measured) and of 477 proteins in EMIF-AD MBD (33% of EMIF-AD MBD proteins measured) and Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 6 of 16 Fig. 2  Association of selected GO biological processes and transcription factors with CSF proteins, MAGMA geneset score, and cell type. Results A,B GO-BP and SUZ12 and REST transcription factors enriched for proteins in AD individuals with increased t-tau (a + t +) or normal t-tau (a + t-) with increased (red) or decreased (blue) concentrations relative to controls in the total sample (A) or in the preclinical AD stage (B). P-values of all GO-BPs are listed in Data S3a. C P-value of association of GO-BP with abnormal t-tau in GWAS-based MAGMA genset analysis. P-values of all GO-BPs are listed in Data S4d. D Proportion of proteins with cell-type specific expression. A: ADNI cohort, E: EMIF-AD MBD cohort; A + t + : abnormal Aβ1-42 and increased t-tau; A + t-: abnormal Aβ1-42 and normal t-tau; CN A + t + : abnormal Aβ1-42 and increased t-tau in preclinical AD stage; CN A + t-: abnormal Aβ1-42 and normal t-tau in preclinical AD stage. Oligo = oligodendrocyte; endothelial = endothelial cell. (protein enrichment) and Data S4d (MAGMA analysis) Fig. 2  Association of selected GO biological processes and transcription factors with CSF proteins, MAGMA geneset score, and cell type. A,B GO-BP and SUZ12 and REST transcription factors enriched for proteins in AD individuals with increased t-tau (a + t +) or normal t-tau (a + t-) with increased (red) or decreased (blue) concentrations relative to controls in the total sample (A) or in the preclinical AD stage (B). P-values of all GO-BPs are listed in Data S3a. C P-value of association of GO-BP with abnormal t-tau in GWAS-based MAGMA genset analysis. P-values of all GO-BPs are listed in Data S4d. D Proportion of proteins with cell-type specific expression. A: ADNI cohort, E: EMIF-AD MBD cohort; A + t + : abnormal Aβ1-42 and increased t-tau; A + t-: abnormal Aβ1-42 and normal t-tau; CN A + t + : abnormal Aβ1-42 and increased t-tau in preclinical AD stage; CN A + t-: abnormal Aβ1-42 and normal t-tau in preclinical AD stage. Oligo = oligodendrocyte; endothelial = endothelial cell. (protein enrichment) and Data S4d (MAGMA analysis) through histone acetylation [58, 59]. Previous studies showed evidence of REST/SUZ12 de-repression in AD brain tissue, iPSC neurons from individuals with sporadic AD, and tangle bearing AD neurons [8, 60, 61]. Results Analysis of cell-type specific proteins revealed that proteins of all major brain cell types differed in con- centration between AD individuals with increased and normal t-tau and that plasticity proteins were typically neuron-specific (Fig. 2, Data S2). i We then investigated whether the opposite protein pat- terns could already be detected in the preclinical stage of AD. We found that relative to controls, preclinical AD individuals with increased t-tau had higher levels of plasticity proteins while preclinical AD individuals with normal t-tau showed lower plasticity protein levels. Pre- clinical AD individuals with normal t-tau also showed higher concentrations of proteins associated with bar- rier dysfunction (Table 1, Fig. 2b, Fig. 3, Data S2 column BK-BN, Data S3a for all enriched GO-BP). These findings indicate that both aberrant neuronal plasticity processes and barrier dysfunction are very early events in AD. i We continued analysis by comparing CSF protein con- centrations between AD individuals with increased t-tau and normal t-tau directly. AD individuals with increased t-tau showed, relative to those with normal t-tau, an increase in the concentration of 167 proteins in ADNI (67% of ADNI proteins measured) and of 768 proteins in EMIF-AD MBD (53% of EMIF-AD MBD proteins meas- ured) and a decrease in the concentration of 2 proteins in ADNI (1% of proteins measured) and of 136 proteins in EMIF-AD MBD (9% of proteins measured). These We next investigated how protein concentrations changed with increasing disease severity stages. In AD individuals with increased t-tau levels, the concentra- tion of 59 (ADNI) to 129 proteins (EMIF-AD MBD) decreased with increasing disease severity and the con- centration of 2 (ADNI) to 34 proteins (EMIF-AD MBD) Table 1  Top 20 proteins that differed in preclinical AD from the control group according to CSF t-tau status T-tau Total tau, CN Control group with normal cognition, normal Aβ1-42 and normal t-tau a Protein associated with Blood Brain Barrier dysfunction b Proteins with high expression in choroid plexus. Results Interest- ingly, of the proteins with increased gene expression in these previous studies that were measured in our CSF study, the majority showed increased CSF concentrations in AD individuals with increased t-tau: 4 of 4 (CALB1, NRXN3, SCN3B, SNAP25) proteins from reference [60]; 56 of 67 (84%) proteins from reference [8]; and 200 of 238 (84%) proteins from reference [61] (annotated in data S2, column DF-DG). of cytokine signaling, leukocyte activation, oxidative stress, mitochondrial dysfunction, and apoptosis. Amy- loid production was increased, reflected by increased levels of amyloid precursor protein (APP), Aβ1-40, and Aβ1-38, and increased levels and activity of BACE1, and increased concentrations in substrates of the main secretases involved in Aβ metabolism (i.e., BACE1, alpha and gamma secretase; annotated in Data S2, column DA-DC). Increased Aβ production is known to set off the amyloid cascade in autosomal dominant AD, [56] and our data suggest that increased amyloid production may also play a role in sporadic AD [57]. To identify potential driv- ers of increased protein levels we performed CHeA tran- scription factor-binding site enrichment analysis, which indicated SUZ12 (p-FDR corrected = 1.62E-11) and REST (p-FDR corrected = 1.04E-9) as most significantly enriched. SUZ12 and REST repress gene transcription AD individuals with normal t-tau showed, relative to the control group, decreased levels of proteins associ- ated with neuronal plasticity and regulation of MAPK signaling, with concomitant lower levels of APP, Aβ1-40 and Aβ1-38, and secretase substrates (Fig. 2, Figure S2; Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 7 of 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 Data S3a, S3b for all enriched processes). Proteins with decreased concentrations in the AD group with normal t-tau, also converged on SUZ12 and REST transcription factors, suggesting that increased gene repression activ- ity is driving this CSF proteomic profile. Furthermore, the AD group with normal t-tau showed 67 proteins with increased levels relative to controls. Twenty of these pro- teins were associated with blood–brain barrier (BBB) dysfunction (annotated in Data S2, column DD) and 25 proteins had a high expression in choroid plexus (ABAE- nrichment minimum pFWER = 0.004), suggesting blood- CSF barrier (BCSFB) dysfunction (annotated in Data S2, column DE). Increased proteins were mainly seen in the EMIF-AD MBD cohort, as ADNI proteomics was tar- geted for brain specific proteins. proteins were enriched for the same processes that dif- fered for each group relative to controls (Figure S3, Data S3a). Results Full list of proteins is provided in Data S2, column BK-BN Contrast Cohort Number of proteins that differ Top 20 proteins with largest effect size Preclinical AD with increased t-tau  Proteins with increased concentration relative to CN EMIF-AD MBD 262 YWHAH, LAMP5, PCSK5, CHI3L1, SMOC1, ADCYAP1, SPP1, GDA, CRYM, TAGLN3, PLXNA2, PCDH8, HPRT1, CPD, GAP43, ENPP5, CAMK2A, PKM, MELTF, NAXE ADNI 93 NRGN, PKM, VSNL1, GOT1, ALDOA, SPP1, GOT2, HGF, NEO1, NCSTN, MOG, APLP2, SOD1, BACE1, APP, CHI3L1, FABP3, ENO2, NCAM2, SPON1  Proteins with decreased concentration relative to CN EMIF-AD MBD 19 S100A6, ADAMDEC1, GPLD1, ANXA5, KNG1, SERPINA4, HSPA1A, IGHV4-30–2, CPB2, IGHV4-30–4, FLNA, IGHV3-30, PRAP1, IGLV3-10, HSPA6, ABI3BP, IGHV2-70D, HSPA7, PON1 ADNI 2 GOLM1, LEP Preclinical AD with normal t-tau  Proteins with increased concentration relative to CN EMIF-AD MBD 36 SLC39A12b, ­ADIPOQa, ANGPTL7, ­LGI1b, ­CD9b, KRT24, ­IFI30a, TTR​b, ­FOLR1b, NCMAP, ­SLC5A5b, SELPLG, ­ENPP2b, ­F5b, CTSA, KRT12, KRT9, ­SIAEb, COL15A1, KRT10 ADNI 3 EDN1, APOE-e4, ­FGF4b  Proteins with decreased concentration relative to CN EMIF-AD MBD 102 HS6ST1, CDH9, ASTN1, PLXNA1, APP, PMP2, STMN3, CDH8, PRKAR1A, GABARAPL2, GALNT6, PITHD1, GALNT1, CPM, MMP17, FAM19A2, CD99, FURIN, TAGLN, CDH12 ADNI 24 abeta1-40, abeta1-38, ADGRL1, CADM3, NEO1, NPTX1, MCAM, CHGB, PCSK1, NEGR1, L1CAM, UBB, PTPRN, CAC‑ NA2D1, TIMP1, PAM, BTD, VEGFA, DAG1, NBL1 Table 1  Top 20 proteins that differed in preclinical AD from the control group according to CSF t-tau status Number of proteins that differ Preclinical AD with increased t-tau Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 8 of 16 CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Nervous system development CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Gene expression CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Cytokine-mediated signalling CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Leukocyte activation CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Angiogenesis CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Myelination CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Blood-brain barrier CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS BACE1 substrates Fig. 3  Association of protein process score (PPS) with disease severity. Data are based on cross-sectional analysis and combines data from ADNI and EMIF-AD MBD. Results In grey PPS of control group with normal cognition and normal CSF Aβ1-42 and t-tau; In red PPS of AD individuals with increased t-tau; In blue PPS of AD individuals with normal t-tau. CN = cognitively normal; MCI = mild cognitive impairment; Dem = dementia CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Nervous system development nt CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Gene expression CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Cytokine-mediated signallin ng CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Leukocyte activation Cytokine-mediated signalling PPS PPS PPS CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Angiogenesis CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS BACE1 substrates CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Myelination CN MCI Dem -4 -3 -2 -1 0 1 2 3 4 PPS Blood-brain barrier Myelination PPS PPS PPS PPS Fig. 3  Association of protein process score (PPS) with disease severity. Data are based on cross-sectional analysis and combines data from ADNI and EMIF-AD MBD. In grey PPS of control group with normal cognition and normal CSF Aβ1-42 and t-tau; In red PPS of AD individuals with increased t-tau; In blue PPS of AD individuals with normal t-tau. CN = cognitively normal; MCI = mild cognitive impairment; Dem = dementia Presence of the APOE ε4 allele, the major genetic AD risk factor, was more common in AD individuals with increased t-tau compared to AD individuals with nor- mal t-tau (66% vs 53% p < 0.001) and was in both AD groups more common than in controls (17%, p < 0.001). Compared to controls, AD individuals with increased t-tau and with normal t-tau had both higher AD PGRS across SNP inclusion thresholds (Fig. 4a, Data S4a) [49]. PGRS did not differ between AD individuals with increased t-tau and normal t-tau after stratification for clinical stage and correction for APOE ε4 carriership and age (Data S4a), suggesting that these groups have a similar AD genetic risk architecture. Next, we per- formed an exploratory GWAS within AD individuals to identify other potential genetic markers associated with increased t-tau. We found tentative associations with SNPs in the APBB2 gene (nominal p-value 1.63–2.92E- 06, Data S4b). Results The top 3 genes associated with increased t-tau from gene-based analyses included, in addition to APBB2, TBC1D10B (nominal p = 2.9E-04), a Rab GTPA activating protein involved in MAPK signaling, and LRP3 (nominal p = 3.36E-04), a low-density lipoprotein receptor protein (Data S4c). The top 3 GO-BP gene-sets associated with increased t-tau from MAGMA analysis were positive regulation of cellular process (self-con- tained p = 1.07E-04), protein K29-linked ubiquitination (self-contained p = 1.52E-04) and negative regulation of actin nucleation (self-contained p = 1.57E-04, Data S4d). Twenty-eight percent of GO-BPs enriched for increased. Proteins of which the concentration decreased with disease severity were enriched for neuronal plastic- ity and proteins of which the concentration increased were enriched for mitochondrial outer membrane per- meabilization and complement activation (Fig. 3, Data S2, S3a (all enriched GO-BP), Figure S3). In AD individu- als with normal t-tau, the concentration of 48 (ADNI) to 265 (EMIF-AD MBD) proteins decreased with increas- ing disease severity and the concentration of 10 proteins (ADNI) to 54 (EMIF-AD MBD) increased with disease severity (Fig. 3, Data S2, S3a (all enriched GO-BP), Fig- ure S3). Proteins of which the concentration decreased with disease severity were enriched for neuronal plas- ticity and proteins of which the concentration increased proteins included BBB related proteins (n = 32) and pro- teins with increased expression in the choroid plexus (n = 14). Because the concentration of plasticity pro- teins decreased in both AD individuals with increased t-tau and normal t-tau, differences in plasticity proteins between the groups that were present in the preclinical AD stage persisted in the prodromal and dementia stage (Fig. 3). The decrease in concentration of neuronal plas- ticity proteins with disease severity aligns with the obser- vation of decreased gene expression of neuronal plasticity proteins with increasing disease severity in post-mortem AD studies [57, 62]. Next, we investigated whether genetic factors were related to increased t-tau in AD individuals in the com- bined ADNI and EMIF-AD MBD cohorts (n = 1067). Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 9 of 16 Fig. 4  Genetic analysis. A PGRS across SNP thresholds SNP weights were based on [49]. Full data (including data after correction for APOE ε4 genotype and demographics, and separate for cohort and clinical stage) are shown in Supplemental Data S4a. Results a + t + : abnormal Aβ1-42 and increased t-tau (red); a + t-: abnormal Aβ1-42 and normal t-tau (blue). B Number of proteins associated with genetic risk score (GRS) and processes associated with these proteins. Left site shows number of proteins with a positive correlation with CSF t-tau GWAS-based GRS (coloured outer ring) and right site indicates to which GO-biological processes (GO-BP) these proteins below (GO-BP CSF, grey outer ring). Shown are the 10 GRS with the highest number of positive correlations with CSF proteins. The size of the arrows visualizes the number of correlations between GRS and protein concentration. For example, the GO-BP gene expression GRS showed a positive correlation with 256 proteins and these proteins were predominantly associated with nervous system development, gene expression and cell adhesion. Arrows to CSF GO-BP are shown in case the GRS correlated with at least 8 proteins from the CSF GO-BP. Supporting data in Data S5a. ECM = Extracellular matrix Fig. 4  Genetic analysis. A PGRS across SNP thresholds SNP weights were based on [49]. Full data (including data after correction for APOE ε4 genotype and demographics, and separate for cohort and clinical stage) are shown in Supplemental Data S4a. a + t + : abnormal Aβ1-42 and increased t-tau (red); a + t-: abnormal Aβ1-42 and normal t-tau (blue). B Number of proteins associated with genetic risk score (GRS) and processes associated with these proteins. Left site shows number of proteins with a positive correlation with CSF t-tau GWAS-based GRS (coloured outer ring) and right site indicates to which GO-biological processes (GO-BP) these proteins below (GO-BP CSF, grey outer ring). Shown are the 10 GRS with the highest number of positive correlations with CSF proteins. The size of the arrows visualizes the number of correlations between GRS and protein concentration. For example, the GO-BP gene expression GRS showed a positive correlation with 256 proteins and these proteins were predominantly associated with nervous system development, gene expression and cell adhesion. Arrows to CSF GO-BP are shown in case the GRS correlated with at least 8 proteins from the CSF GO-BP. Supporting data in Data S5a. ECM = Extracellular matrix lowest self-contained p-value. For each of the GRS, we used weights from one cohort to generate a GRS in the other cohort. We then correlated the individual GRS of each gene or GO-BP with individual protein concen- trations (Data S5a). Results A positive correlation means that a higher GRS is associated with a higher protein con- centration. The largest number of positive correlations of GRS with CSF protein concentrations were observed for the GO-BP gene expression GRS (288 proteins), the GO-BP regulation of MAPK cascade GRS (146 proteins), and the GO-BP histone H3 acetylation GRS (127 proteins, Fig. 4b, left coloured part, Data S5a). The proteins that showed a positive correlation with these GRS were typically associated with nervous sys- tem development, gene expression, and cell adhesion CSF proteins that differed in concentration between AD t-tau groups, differed also in GO-BP gene-set score between the t-tau groups (Fig. 2; Data S3a). This sug- gests that interindividual differences in CSF t-tau concentrations may have partly a genetic basis. To investigate whether genetic markers influenced CSF protein concentrations, we correlated subject level GRS with subject level CSF protein concentrations in 188 individuals with abnormal Aβ1-42. We used the GRS of the top 3 genes, the GRS of the top 3 GO-BP and the GRS of 9 GO-BPs that differed between abnormal and normal t-tau groups in both CSF and MAGMA geneset analysis in Fig. 2. Given the association of CSF profiles with REST and SUZ12 we also selected the GRS of 2 GO-BPs associated with histone acetylation with the CSF proteins that differed in concentration between AD t-tau groups, differed also in GO-BP gene-set score between the t-tau groups (Fig. 2; Data S3a). This sug- gests that interindividual differences in CSF t-tau concentrations may have partly a genetic basis. To investigate whether genetic markers influenced CSF protein concentrations, we correlated subject level GRS with subject level CSF protein concentrations in 188 individuals with abnormal Aβ1-42. We used the GRS of the top 3 genes, the GRS of the top 3 GO-BP and the GRS of 9 GO-BPs that differed between abnormal and normal t-tau groups in both CSF and MAGMA geneset analysis in Fig. 2. Given the association of CSF profiles with REST and SUZ12 we also selected the GRS of 2 GO-BPs associated with histone acetylation with the Visser et al. Molecular Neurodegeneration (2022) 17:27 Visser et al. Molecular Neurodegeneration (2022) 17:27 Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 10 of 16 Page 10 of 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 (Fig. 4b, right grey part). Results These findings support the notion that the increase in plasticity-related processes in CSF has in part a genetic background. Moreover, we tested the association of SNP rs9877502 in GMNC, previously associated with CSF t-tau levels without stratification for amyloid aggregation, with tau status [44, 63, 64]. Consistent with previous data, the A-allele frequency at this site was more common in AD indi- viduals with increased t-tau compared to those with normal t-tau (nominal p-value = 0.02). We then asso- ciated this SNP with CSF protein levels using additive models. 591 of 1705 proteins (35%) showed an increase in CSF level with increasing number of rs9877502 A-alleles (Data S5b). These proteins were enriched for neuronal plasticity related processes (Data S5c), and SUZ12 (p-FDR corrected = 5.27e-24) and REST (p-FDR corrected = 6.34e-18) signaling. 105 proteins (6%) showed a negative correlation with the number of rs9877502 A-alleles, including 36 proteins associ- ated with BBB dysfunction and 36 immunoglobulins. In a smilar way, we associated the number of APOE-e4 alleles with CSF protein levels in individuals with AD. Forty-five proteins showed an increase in CSF levels with increasing number of APOE-e4 alleles (Data S5b) and these proteins were enriched for GO-BP learning or memory (p-FDR corrected = 0.046). The concentra- tion of 36 proteins showed a negative correlation with the number of APOE-e4 alleles (Data S5b) and were not enriched for any GO-BP. Results from our molecular process and transcription factor enrichment analysis suggested that the increased levels of neuroplasticity proteins in the AD group with increased t-tau could result from an increase in mitogenic MAPK signaling, or from a reduction in gene repression by REST and SUZ12. The de-repression of gene expres- sion by REST/SUZ12 in AD has previously been reported in post-mortem AD studies and in iPSC neurons from individuals with sporadic AD [8, 60, 61]. Our finding that a subset of proteins coded by genes with increased expression in these previous studies had increased CSF concentrations in AD individuals with increased t-tau as well, further supports the role of REST/SUZ12 de-repres- sion in AD. Both APP and tau are regulated by REST or SUZ12, and this may explain why the increases in levels of Aβ production markers and tau occur in tandem [6]. Results The increased concentration of neuronal plasticity pro- teins might reflect a compensatory mechanism triggered by tau-induced synaptic dysfunction such as impairment of presynaptic vesicle release, trafficking of glutamatergic receptors, and maturation of dendritic spines [65]. AD neuropathological studies suggested that such a plastic- ity response is likely pathological and results in aberrant synaptic sprouting (both at axons and at dendrites), dis- organised capillairies and an abnormal cortical myelin architecture [51, 52, 66]. Increased expression of APP and tau may initiate a vicious cycle. APP and Aβ peptides can activate MAPK signaling, [53, 67, 68] which may fur- ther increase APP production[69, 70]. The APP intracel- lular domain (AICD) stimulates gene transcription, [71] and may increase expression of APP and BACE1 [72]. Abnormal tau may increase gene expression through depletion of H3K9me2, altered spatial open chromatin organization, or altered H3K9 acetylation [73, 74].h Finally, we studied the effect of t-tau on disease pro- gression using longitudinal ADNI data. Relative to controls, AD groups with increased t-tau and normal t-tau showed both faster memory decline and PET-amyloid accumulation in the preclinical stage and showed faster decline in all cognitive and imaging meas- ures in the prodromal and dementia stage. Compared to AD individuals with normal t-tau, those with increased t-tau declined faster on cognitive tests and in glucose metabolism in the prodromal AD stage and showed a faster decline in cortical thickness in the prodromal and dementia stage (Fig. 5, Data S6). g y [ ] The group of AD individuals with normal t-tau was characterized by decreased concentrations of plasticity related proteins and increased concentrations of BBB and BCSFB permeability related proteins. The increase in barrier permeability may be caused by accumula- tion of Aβ in vessel walls and toxic effects of Aβ on cells that constitute these barriers [75, 76]. This can loosen tight junctions and increase paracellular transport [75, 77–79]. Impaired BBB and BCSFB function may lead to hypoplasticity through impairments in their physi- ological role in glucose transport, capillary perfusion, and neurogenesis, [79] and possibly also by increasing REST signaling, which can be triggered by ischemia [80]. The increase in concentrations of CSF proteins that are produced in the choroid plexus suggests BCSFB dysfunction. The choroid plexus is involved in the clearance of Aβ, [81] and the increase in concentra- tion of proteins expressed in the choroid plexus could be a response to Aβ induced inflammation [82–84]. Discussion Our main finding is that AD individuals with increased CSF t-tau levels showed increased concen- trations of proteins associated with neuronal plas- ticity, while AD individuals with normal t-tau levels showed decreased concentrations of plasticity pro- teins. These opposite CSF proteomic profiles were already observed in the preclinical stage of AD, and persisted in prodromal and mild dementia stages, indicating that they reflect different disease traits rather than different disease states. Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 11 of 16 Fig. 5  Longitudinal cognitive and imaging changes according to diagnostic group and clinical stage Upper images: change in cognitive markers. A MMSE, B CDR sum of boxes, C Logical memory, and D ADAS-Cog. Change of controls are shown in grey, of AD individuals with increased t-tau in red (a + t +), and of AD individuals with normal t-tau (a + t-) in blue. 1. Slope p < 0.05 different from control; 2. Slope p < 0.05 different from 0; 3. Slope p < 0.05 different between AD individuals with increased t-tau (a + t +) and AD individuals with normal t-tau (a + t-). Lower images: Annual rate of change in imaging markers. E amyloid aggregation, F cortical thickness and G FDG-PET glucose metabolism. PET amyloid data are not shown for AD individuals with dementia with normal t-tau because of low number of participants (n = 6). Data are shown in Data S6. Data are from ADNI only. CN = normal cognition; MCI = mild cognitive impairment; a + t +  = abnormal Aβ1-42 and increased t-tau; a + t- = abnormal Aβ1-42 and normal t-tau Fig. 5  Longitudinal cognitive and imaging changes according to diagnostic group and clinical stage Upper images: change in cognitive markers. A MMSE, B CDR sum of boxes, C Logical memory, and D ADAS-Cog. Change of controls are shown in grey, of AD individuals with increased t-tau in red (a + t +), and of AD individuals with normal t-tau (a + t-) in blue. 1. Slope p < 0.05 different from control; 2. Slope p < 0.05 different from 0; 3. Slope p < 0.05 different between AD individuals with increased t-tau (a + t +) and AD individuals with normal t-tau (a + t-). Lower images: Annual rate of change in imaging markers. E amyloid aggregation, F cortical thickness and G FDG-PET glucose metabolism. Discussion The genetic pattern associated with t-tau levels in individuals with AD is thus different from the genetic processes asso- ciated with AD-type dementia, which include immunity, lipid metabolism, and intracellular trafficking [49]. This indicates that within the AD genetic risk architecture other genetic processes could influence AD phenotypes. levels in this and other datasets) [44, 63, 64] with levels of 696 CSF proteins in AD individuals. GMNC is involved in neuronal plasticity and regulation of gene expres- sion [89, 90]. We also found that GRS associated with t-tau were involved in neuronal plasticity and that these GRS showed an association with the concentration of plasticity-related proteins. On the contrary, the number of APOEe4 alleles was not associated with the concen- tration of proteins involved in neuronal plasticity. The genetic pattern associated with t-tau levels in individuals with AD is thus different from the genetic processes asso- ciated with AD-type dementia, which include immunity, lipid metabolism, and intracellular trafficking [49]. This indicates that within the AD genetic risk architecture other genetic processes could influence AD phenotypes. study showed that increased CSF t-tau levels were associ- ated with long-term depression (LTD) after transcranial magnetic stimulation in the motor cortex [96]. As this study was performed in demented individuals, it is possi- ble that LTD reflects synaptic failure. Future studies that combine electrophysiological measures with proteomic data in human beings are required to study this question in more detail. We further found that AD individuals with increased t-tau also showed increased cytokine signaling and leu- kocyte activation. This activation could result from sol- uble tau oligomers, [97] although it is also possible that both increased t-tau levels and microglia activation result from amyloid pathology. A strength of the study is the large sample size available for proteomic analysis and the use of two independent cohorts. Even though these cohorts performed proteom- ics in a different way (targeted proteomics in ADNI, non- targeted proteomics in EMIF-AD MBD) and the overlap in proteins measured in both studies was limited, enrich- ment analysis showed very similar results between the cohorts (Fig. 2), which supports the robustness of our findings. The consistency across cohorts was also pre- sent on the protein level. Discussion Of the 167 proteins measured in both cohorts, only 7 proteins (5%) showed a difference between the cohorts on both major outcomes (AD with normal t-tau vs control and AD with increased t-tau ver- sus controls, Data S2). A limitation is that CSF proteom- ics was performed cross-sectionally and the differences between disease stages need to be confirmed in longitu- dinal studies. In addition, the association between change in CSF protein concentrations and biological processes needs to be further addressed in functional studies. The sample size for genetic analysis was small and larger sam- ples are needed to confirm these results. l As we found a wide range of processes deregulated, it is possible that this may reflect an aspecific response to brain pathology. This seems, however, unlikely as the same set of proteins that was increased in AD individu- als with increased t-tau was decreased in AD individuals with normal t-tau suggesting that these processes have a common denominator. In addition, we found that these proteins were enriched for SUZ12 and REST transcrip- tion factors, which are master regulators of neuronal plasticity. Moreover, abnormalities in nervous system development related processes, myelination, angiogen- esis, MAPK signaling, cell-cycle, gene expression and glycolysis have all been reported in separate AD post- mortem, animal or cell studies [8, 50–55]. Still, some dysregulated processes may only be indirectly involved in plasticity. For example, increased levels of proteins associated with glycolysis could be a response to the high energy demand resulting from increased neuronal activ- ity [91]. It is unlikely that the increased levels of plastic- ity proteins in AD individuals with increased t-tau simply reflect cell death as levels of plasticity-associated proteins were highest in the preclinical AD stage, when neurode- generation is limited, and decreased with increasing dis- ease severity. Discussion PET amyloid data are not shown for AD individuals with dementia with normal t-tau because of low number of participants (n = 6). Data are shown in Data S6. Data are from ADNI only. CN = normal cognition; MCI = mild cognitive impairment; a + t +  = abnormal Aβ1-42 and increased t-tau; a + t- = abnormal Aβ1-42 and normal t-tau Our GWAS and GRS analyses suggested that levels of tau and neuroplasticity proteins partly depend on genes involved in gene expression, although no genome-wide significant signals were identified. APBB2 showed the strongest association with CSF t-tau in these analyses. While markers in APBB2 do not show strong evidence for association with AD risk in GWAS comparing con- trols with individuals with AD-dementia [49], the gene represents an interesting functional candidate as APBB2 binds AICD. Alternative splicing of APBB2 increased the Aβ42/ Aβ40 ratio [87], and overexpression increased Aβ1-40, APP, and AICD levels and changed gene expres- sion [68, 87, 88]. In addition, we found associations of rs9877502 in GMNC (previously associated with t-tau AD individuals with normal t-tau may have cerebral amyloid angiopathy (CAA), a condition in which Aβ is deposited in cerebral and leptomeningeal blood vessels [85]. CAA is common in individuals with AD and cor- relates with Aβ plaque deposition and neurofibrillary tangles [85]. CAA has been associated with normal lev- els of t-tau levels and a decrease in Aβ40 levels, which was also observed in our AD individuals with normal t-tau [86]. Although t-tau levels were within normal limits, this group showed a typical AD genetic pattern and a progressive disease course with increases in cog- nitive and AD biomarkers abnormalities, which sup- ports the view that this is a subtype of AD, and not a different disease entity. Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 12 of 16 Visser et al. Molecular Neurodegeneration Page 12 of 16 levels in this and other datasets) [44, 63, 64] with levels of 696 CSF proteins in AD individuals. GMNC is involved in neuronal plasticity and regulation of gene expres- sion [89, 90]. We also found that GRS associated with t-tau were involved in neuronal plasticity and that these GRS showed an association with the concentration of plasticity-related proteins. On the contrary, the number of APOEe4 alleles was not associated with the concen- tration of proteins involved in neuronal plasticity. Funding g This work has been supported by ZonMW Memorabel grant programme #73305056 (BMT), #733050512 (SL) and #733050824 (BMT and PJV), the Swedish Research Council (#2018–02532, HZ), the European Research Council (#681712, HZ) and Swedish State Support for Clinical Research (#ALFGBG-720931, HZ), the Alzheimerfonden (Grant no. AF-930934) and Dtiftelsen Gamla tjänarinnor (JG), and the Innovative Medicines Initiative Joint Undertaking under EMIF grant agreement #115372 (PJV, HZ, SV, IB). HZ is a Wallenberg Academy Scholar. The Leuven cohort was funded by Stichting Alzheimer Onderzoek (#11020, #15005, #13007) and the Vlaamse Impulsfi‑ nanciering voor Netwerken voor Dementie-onderzoek (IWT #135043). The Lausanne cohort was supported by the Swiss National Research Foundation SNF (#320030_141179). Data was used for this project of which collection and sharing was funded by the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (National Institutes of Health Grant U01 AG024904) and DOD ADNI (Depart‑ ment of Defense award number W81XWH-12–2-0012). ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the following: AbbVie, Alzheimer’s Association; Alzheimer’s Drug Discovery Foundation; Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; Cer‑ eSpir, Inc.; Cogstate; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Roche Ltd and its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research & Development LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer Inc.; Piramal Imaging; Servier; Takeda Phar‑ maceutical Company; and Transition Therapeutics. The Canadian Institutes of Health Research is providing funds to support ADNI clinical sites in Canada. Private sector contributions are facilitated by the Foundation for the National Institutes of Health (www.​fnih.​org). The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimer’s Therapeutic Research Institute at the University of Southern California. ADNI data are disseminated by the Laboratory for Neuro Imaging at the University of Southern California. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s13024-​022-​00521-3. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s13024-​022-​00521-3. Additional file 1: Data S1. Participant characteristics. S1a: Characteristics of individuals with CSF Aβ1-42 and tau measurements available; S1b: Characteristics of individuals with CSF proteomic data. Data S2. Protein annotation and statistics of group comparisons of protein levels. Data S3a. Full list of GO biological processes associated with proteins that differ according to group and clinical stage. Data S3b. SynGO enriched synaptic cellular components and biological processes that differ accord‑ ing to group. Data S4a. Estimated marginal means of AD GWAS-based polygenic risk scores in controls, AD individuals with increased t-tau and AD individuals with normal t-tau. Data S4b. Top 1000 SNPS from GWAS on AD individuals with increased t-tau and normal t-tau in pooled ADNI and EMIF-AD MBD cohorts. Data S4c. Difference in MAGMA gene score between AD individuals with increased t-tau and normal t-tau based on t-tau GWAS in pooled ADNI and EMIF-AD MBD cohorts. Data S4d. Difference in GO biological process MAGMA geneset score between AD individuals with increased t-tau and normal t-tau based on t-tau GWAS in pooled ADNI and EMIF-AD MBD cohorts. Data S5a. Correlation between genetic risk score and CSF protein level in individuals with abnormal Aβ1- 42. Data S5b. Association of the number of GMNC rs9877502-A risk alleles and number of APOE-e4 alleles with CSF protein concentrations in a linear model in individuals with AD. Data S5c. GO-BP processes enriched for proteins that have a positive or negative association with the number of rs9877502-A risk alleles in an additive model. Data S6. Annual change in imaging measures. Acknowledgements We thank Drs. Andre Franke and Michael Wittig as well as Mrs. Tanja Wesse and Sanaz Sedghpour Sabet for their help with the genome-wide genotyping experi‑ ments. Further, we thank Dr. Fabian Kilpert and Shengjun Hong for his help with the processing and management of the genome-wide genotyping data. Data used in preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A complete listing of ADNI investigators can be found at: http://​adni.​loni.​ usc.​edu/​wpcon​tent/​uploa​ds/​how_​to_​apply/​ADNI_​Ackno​wledg​ement_​List.​pdf Conclusions CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and blood– brain and blood-CSF barrier dysfunction. The asso- ciation of increased CSF t-tau with neuronal plasticity protein levels provides support for the role of tau in neuronal plasticity and gene expression and implies that increased CSF t-tau levels may not simply reflect axonal loss [8, 73, 74]. It will be of critical importance to stratify future trials on t-tau status, as individuals with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite proteomic profiles. The dosing of amyloid and tau antibodies may need to be tailored to t-tau levels, because of differences in BBB/ BCSFB integrity between those with increased and nor- mal t-tau. Treatments with drugs that target amyloid production, hyperexcitation, or hyperplasticity, such The observation that increased t-tau levels are associ- ated with an increase in concentration of plasticity pro- teins, aligns with observations that tau is associated with increased neuronal excitability in neurophysiological studies [92]. In an AD mice model it was shown that tau could be an enabler of neural network dysfunction caused by amyloid pathology as tau reduction reduced amyloid- related overexcitation [92, 93]. Tau may also have direct effects on excitability as overexpression of human tau in mice models increased delta/theta power and reduc- tion of tau expression counteracted these changes [94]. Another study showed that nuclear translocation of tau led to increased expression of VgluT1, which is involved in glutamatergic synaptic transmission [95]. A clinical Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 13 of 16 as bace inhibitors, histone modifiers and anti-epileptic drugs, could only be effective in AD individuals with increased t-tau in the predementia stage, when the CSF concentration of amyloid production markers and plas- ticity related proteins are highest. Retrospective analysis of previous trial data according to CSF t-tau status may lead to a better understanding why effects in these trials were absent or minimal and whether AD individuals with increased t-tau and normal t-tau have a different treat- ment response. Future studies on the mechanisms that lead to t-tau associated proteomic profiles, such as REST and SUZ12 signaling, will help to clarify AD pathophysi- ology and may eventually lead to novel drug targets. Conclusions REST transcription factors associated with proteins that differed between AD individuals with increased t-tau and normal t-tau and proteins that changed with disease severity. REST transcription factors associated with proteins that differed between AD individuals with increased t-tau and normal t-tau and proteins that changed with disease severity. Abbreviations AD Al h i ’ d AD: Alzheimer’s disease; ADNI: Alzheimer’s Disease Neuroimaging Initiative; AICD: APP intracellular domain; APOE: Apolipoprotein E; APP: Amyloid precur‑ sor protein; Aβ: Amyloid beta; BBB: Blood–Brain barrier; BCSFB: Blood-CSF barrier; CAA​: Cerebral amyloid angiopathy; CDR: Clinical Dementia Rating; CN: Cognitively normal; CSF: Cerebrospinal fluid; ECM: Extracellular matrix; EMIF- AD: European Medical Information Framework for Alzheimers disease; FDG: Fluorodeoxyglucose; FDR: False discovery rate; GO-BP: Gene Ontology biologi‑ cal processes; GRS: Gene-specific risk scores; GWAS: Genome-wide associa‑ tion study; iPSC: Induced pluripotent stem cells; LD: Linkage disequilibrium; LTD: Long-term depression; MAPK: Mitogen-activated protein kinase; MBD: Multimodality Biomarker Discovery; MCI: Mild cognitive impairment; MMSE: Mini-Mental State Examination; PC1: Principal component; PCA: Principal component analysis; PET: Positron Emission Tomography; PGRS: Polygenic risk scores; PPS: Proteomic process scores; SNP: Single nucleotide polymorphisms; SUVR: Standardized uptake value; T-tau: Total tau; TMT: Tandem mass tag. Authors’ contributions Conceptualization: PJV, BMT. Methodology proteomics EMIF-AD: JG, HZ. Formal analyses: BMT, PJV, LR, JG, VD, SH, ED. Resources: JG, LR, IJ, ED, SH, SL, VD, MT, FRJV, JP, PMLA, RV, AL, JLM, SE, YFL, LF, KS, LB, SL, JS, SV, IB, DP, GS, KB, PS, CET, HZ, PJV. Data curation: SV, IB. Writing—Original Draft: BMT, PJV. Writing— Review & Editing: all authors. Supervision: PJV, BMT. Funding acquisition: PJV, SL, HZ, JB, KB, LB, KS. All authors read and approved the final manuscript. Author details 6. Konijnenberg E, Tomassen J, den Braber A, Ten Kate M, Yaqub M, Mulder SD, Nivard MG, Vanderstichele H, Lammertsma AA, Teunissen CE, et al. Onset of Preclinical Alzheimer Disease in Monozygotic Twins. Ann Neurol. 2021;89:987–1000. 1 Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, PO Box 7057 1007, MB, Amster‑ dam, The Netherlands. 2 Alzheimer Center Limburg, School for Mental Health and Neuroscience, Maastricht University, PO Box 616, 6200, MD, Maastricht, The Netherlands. 3 Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Karolinska Institutet, Stockholm, Sweden. 4 Clini‑ cal Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. 5 Department of Psychiatry and Neurochemistry, Institute of Neu‑ roscience and Physiology, Sahlgrenska Academy at the University of Goth‑ enburg, Mölndal, Sweden. 6 Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, Amsterdam, the Netherlands. 7 Section Genomics of Neurodegen‑ erative Diseases and Aging, Department of Clinical Genetics, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, the Netherlands. 8 1St Department of Neurology, AHEPA University Hospital, Thessaloniki, Makedonia, Greece. 9 Old Age Psychiatry, University Hospital Lausanne, Lausanne, Switzerland. 10 Depart‑ ment of Geriatric Psychiatry, University Hospital of Psychiatry and University of Zürich, Zürich, Switzerland. 11 Fundación CITA-Alzhéimer Fundazioa, San Sebastian, Spain. 12 Neurology Service, University Hospitals Leuven, Leuven, Belgium. 13 Laboratory for Cognitive Neurology, Department of Neurosciences, KU Leuven, Leuven, Belgium. 14 IIB-Sant Pau, Hospital de La Santa Creu I Sant Pau, Universitat Autonoma de Barcelona, Barcelona, Spain. 15 Barcelonaβeta Brain Research Center (BBRC), Barcelona, Spain. 16 Alzheimer’s Disease Unit and Other Cognitive Disorders Unit, Hospital Clinic de Barcelona, Barcelona, Spain. 17 Reference Center for Biological Markers of Dementia (BIODEM), Institute Born‑Bunge, University of Antwerp, Antwerp, Belgium. 18 Depart‑ ment of Neurology, UZ Brussel and Center for Neurosciences, Vrije Universiteit Brussel, Brussels, Belgium. 19 Department of Psychiatry at School of Medical Sci‑ ences, Örebro University, Örebro, Sweden. 20 Department of Geriatric Psychiatry, Zentralinstitut Für Seelische Gesundheit, University of Heidelberg, Mannheim, Germany. 21 Complex Genetics Group, VIB Center for Molecular Neurology, VIB, Antwerp, Belgium. 22 Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. 23 Lübeck Interdisciplinary Platform for Genome Analytics, Institutes of Neurogenetics and Cardiogenetics, University of Lübeck, Lübeck, Germany. 24 Jansen UK, High Wycombe, UK. 25 AC Immune SA, Lausanne, Switzerland. 26 Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. 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Raw proteomic data from EMIF-AD MBD has been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier https://​doi.​org/​ 10.​6019/​PXD01​9910. Access to other EMIF-AD MBD data can be requested from the authors. Datasharing may be restricted by consent given by research participants within each contributing cohort and by European GDPR regula‑ tions, which currently exclude data sharing with a number of non-European countries. Statistical data can be found in the supplementary information files. via the PRIDE partner repository with the dataset identifier https://​doi.​org/​ 10.​6019/​PXD01​9910. Access to other EMIF-AD MBD data can be requested from the authors. Datasharing may be restricted by consent given by research participants within each contributing cohort and by European GDPR regula‑ tions, which currently exclude data sharing with a number of non-European countries. Statistical data can be found in the supplementary information files. Additional file 2: Figure S1. Longitudinal change in CSF total tau. Figure S2. Enrichment of synaptic processes in individuals with AD according to t-tau status. Figure S3. Enriched GO biological processes and SUZ12 and Additional file 2: Figure S1. Longitudinal change in CSF total tau. Figure S2. Enrichment of synaptic processes in individuals with AD according to t-tau status. Figure S3. Enriched GO biological processes and SUZ12 and Page 14 of 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 Visser et al. Molecular Neurodegeneration Competing interests h h d l h 5. Vos SJ, Xiong C, Visser PJ, Jasielec MS, Hassenstab J, Grant EA, Cairns NJ, Morris JC, Holtzman DM, Fagan AM. Preclinical Alzheimer’s disease and its outcome: a longitudinal cohort study. Lancet Neurol. 2013;12(10):957–65. The authors declare that they have no competing interests. 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Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
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Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana Yan Chen1,*, Ming He2,*, Zhao-Qun Li3, Ya-Nan Zhang4 & Peng He2 received: 01 February 2016 accepted: 19 May 2016 Published: 09 June 2016 Periplaneta americana is a notorious urban pest prevalent in human habitats; very little is known about its chemosensory mechanism. Employing the advanced next-generation sequencing technique, in the present study, we conducted transcriptome sequencing and analysis of the antennae of the adult males and females as well as their mouthparts using an Illumina platform. This resulted in the discovery of a huge number of the members of all major known chemosensory receptor families in P. americana, including 96 odorant receptors (ORs), 53 ionotropic receptors (IRs), and 33 gustatory receptors (GRs). Tissue expression profiles showed most of them mainly expressed in antennae and phylogenetic analysis demonstrated the expansion in the clade distinguishing them from other functionally well-known Lepidoptera species. A high percentage of chemosensory receptor genes (ORs in particular) showing female antenna bias in mRNA expression was observed. Our results provide a basis for further investigations on how P. americana coordinates its chemosensory receptor genes in chemical communication with environments, and for development of novel pest management approaches. Insects communicate with their environment through chemosensation to help them accomplish a large number of essential physiological processes, such as mate-finding, host location, and alarming their conspecifics. Insects employ three major groups of chemosensory receptors, namely odorant receptors (ORs), variant ionotropic receptors (IRs), and gustatory receptors (GRs)1–3. Insect ORs and GRs possess seven transmembrane domains; compared to mammalian G-protein coupled receptors, orientation of these domains is reverse, with an intracel- lular N-terminus and an extracellular C-terminus4. Analysis of available insect genome sequences indicates that the OR family has undergone rapid evolution in a species-specific manner. A highly variable number of insect ORs have been identified in different insect species; for example, 62 ORs occur in Drosophila melanogaster5, 79 in Anopheles gambiae6, 170 in Apis mellifera7, 259 in Tribolium castaneum8, and 66 in Danaus plexippus9. In D. melanogaster antenna, only one OR gene is expressed in most dendritic membranes of the olfactory sensory neurons (OSNs)10. OR co-receptor (ORco), being one of the most conserved OR genes among various species, pre-dates other ORs that appear only in winged insects11. ORco interacts with other ligand-specific ORs to form an ORx-ORco complex, which functions as a ligand-gated cation channel12. Pheromone receptors (PRs), one of the sub-classes of ORs, are specifically activated by sex pheromone components and have been widely studied in Lepidoptera13,14. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 01 February 2016 accepted: 19 May 2016 Published: 09 June 2016 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Tissues ♂Antennae ♀Antennae Mouthparts Total size 6.67 Gb 6.29 Gb 6.35 Gb GC content 36.80% 36.82% 41.15% Number of transcripts 304,023 Total unigene count 248,192 Genes with homologues in NR 40,294 Total transcript nucleotides 210,868,904 Total unigene nucleotides 143,462,349 N50 transcript length 1155 nt N50 unigene length 795 nt Longest unigene length 32,380 nt Median unigene length 327 nt Table 1. Summary of P. americana transcriptome assembly. Table 1. Summary of P. americana transcriptome assembly. GRs, the other members of the chemosensory superfamily, are likely to have the same ancestor as ORs and also have a seven transmembrane structure15. Insect GRs mainly respond to non-volatile ligands with the exception of CO2. Based on the known substances to which they respond, GRs were grouped with sugar receptors16, CO2 receptors17, and bitter or other receptor18. Although GRs do not have a common coreceptor such as ORco, they are also supposed to form heteromeric complexes, without fixed combinations19. IR, a new family of olfactory receptors, has the same ancestor as the mammalian ionotropic glutamate receptors (iGluRs)20. IRs are involved in insect odorant reception, as was first shown by the combined misexpression experiments and their subcel- lular localization in olfactory organs in D. melanogaster20. Further research showed that IRs differed from ORs in responding to carboxylic acids and amines21. IRs can be classified into two distinct subfamilies with different ancestors, namely, the conserved “antennal IRs” and the species-specific “divergent IRs.” The antennal IRs may represent the original olfactory receptor family of insects. IR8a and IR25a, belonging to antennal IRs, are two IR co-receptors22; they usually exhibit higher expression than other IRs23,24. Besides the olfactory function, IRs also play a key role in gustation. In D. melanogaster, IR52c and IR52d are co-expressed in the taste sensilla of the foreleg of males and regulate sexual behaviour, whereas IR76b is involved in salt tasting25. Cockroaches are a big group of ancient insects of the order Blattodea. Of the approximately 4,600 cockroach spe- cies, about 30 are associated with human habitats. American cockroach, Periplaneta americana, one of the notori- ous urban pests is common and difficult to control. It can tolerate a wide range of environments, from Arctic cold to tropical heat. Periplanone-A and periplanone-B are two major sex-pheromone components emitted from the female P. americana, which trigger strong electroantennogram responses and behavioural response of conspecific male individuals26. www.nature.com/scientificreports/ To avoid toxic baits, cockroaches rapidly evolved their gustatory system responsible for glucose aversion27. However, little is known about the chemosensory mechanism of these essential chemicals (such as sex pheromone components, periplanone-A and periplanone-B, and glucose) in this species. Only few che- mosensory receptor gene fragments have been identified in another cockroach species, Blattella germanica28. Therefore, in the present study, we sequenced and analyzed three transcriptomes of the adults of P. americana using next-generation sequencing, and identified 190 putative chemosensory receptor genes. These included 96 ORs, 61 iGluRs/IRs, and 33 GRs. To understand their potential functions, we conducted gene ontology (GO) annotation and scanned the tissue-specific expression of these sequences, as well. We also examined their phy- logenetic relationships with some other insect species. Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana Yan Chen1,*, Ming He2,*, Zhao-Qun Li3, Ya-Nan Zhang4 & Peng He2 Not much is known about the recognition mechanism in other insect species with sex phero- mones of different chemical structure. 1Key Lab of Optimal Utilization of Natural Medicine Resources, Department of Pharmacology of Chinese Material Medica, Guizhou Medical University, Huaxi District, Guiyang, Guizhou 550025, People’s Republic of China. 2State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, People’s Republic of China. 3Key Laboratory of Tea Biology and Resource Utilization, Ministry of Agriculture, Tea Research Institute, Chinese Academy of Agricultural Science, Hangzhou, 310008, People’s Republic of China. 4College of Life Sciences, Huaibei Normal University, Huaibei, 235000, People’s Republic of China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.-N.Z. (email: ynzhang_ insect@163.com) or P.H. (email: phe1@gzu.edu.cn) 1 Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Results S In the molecular function category, the genes expressed in the three organs were mostly enriched for binding (e.g., nucleotide, ion, and odorant binding) and catalytic activity (e.g., hydrolase and oxidoreductase). In the biological process category, most common were the cellular and metabolic processes. In the cellular compo- nent terms, the most abundant were cell and cell part (Fig. 1B). Identification of P. americana OR/IR/GR genes. The unigenes related to candidate olfactory receptors (ORs/IRs/GRs) were identified based on the keyword searches of the BLASTx and Pfam family annotations. To avoid missing any P. americana OR/IR/GR genes, the predicted unigene protein sequences were also analyzed using PSI-BLASTp with OR/IR/GR sequences from B. germanica30 and Z. nevadensis31. In all, we identified 190 unigenes belonging to the chemosensory receptor family in the transcriptome of male antennae, female antennae, and mouthparts of P. americana. These included 96 ORs (including one ORco gene), 61 IRs/iGluRs (53 IRs and 8 iGluRs), and 33 GRs, all of which shared high identities with other insect OR (~34–88%), IR (~33–95%), and GR (~29–84%) genes after re-BLASTx identification. Information on the OR, IR, and GR genes including the unigene references, lengths, and the best BLASTx hits are listed in Supplementary Table S2. Tissue expression profile of P. americana OR/IR/GR genes. Specific tissue expression pattern usually indicates specific gene functions. Thus, quantitative polymerase chain reaction (qPCR) was conducted to investi- gate the tissue expression profile of the candidate P. americana OR/IR/GR genes (Figs 2–4). For OR and IR genes, we selected four tissues, namely the male antennae, female antennae, maxillary palps (Mp), and legs (Le). The results indicated that these OR or IR-encoding genes were expressed exclusively in the antennae (especially in female antennae) of the olfactory organ. Based on statistical analysis, 79 of the 85 P. americana (Pame) ORs and 43 of the 46 PameIRs showed antenna-biased expression pattern (11 PameORs and 4 PameIRs were not amplified in all the tested tissues despite attempts using multiple primer pairs). Of these, PameORco had the highest expres- sion among all the ORs. Only 4 PameORs and one PameIRs were dominantly expressed in the male antennae, 53 PameORs and 30 PameIRs were female antennae-biased, and 22 PameORs and 11 PameIRs were equal expressed in the male and female antennae (Figs 2 and 3). Results S Sequencing and assembly of transcriptome. Three transcriptomes from male antennae (mA), female antennae (fA), and mouthparts (Mo) were sequenced using the Illumina HiSeq™​ 4000 platform (Illumina, Tianjin, China) and assembled with Trinity (version: r20140413p1)29. About 45.91 (mA), 43.45 (fA), and 43.17 (Mo) million reads were obtained for each transcriptome. After filtering, 44.45 (mA), 41.92 (fA), and 42.34 (Mo) million clean reads were generated, which comprised of 6.67 (mA), 6.29 (fA), and 6.35 (Mo) gigabases (Gb), with a longest unigene length of 32,380 nt and a median length of 327 nt after combined assembly of these three tran- scriptome. Finally, these reads were assembled into 304,023 transcripts and 248,192 unigenes, with N50 lengths of 1,155 and 795 nt, respectively (Table 1). In addition, the unigenes with a sequence length >​500 nt accounted for 29.14% of the transcriptome assembly (Supplementary Fig. S1). The transcriptome raw reads have been deposited with the NCBI SRA database (accession number: SRR3089536, SRR3089537, and SRR3089538). Homology analysis and gene ontology annotation. BLASTx homology searches of all the 248,192 unigenes showed that 40, 294 (16.23%) had homologous genes in the non-redundant (NR) protein database with a cut-off E-value of 10−5. The best match percentage (35.32%) was with Zootermopsis nevadensis sequences, fol- lowed by sequences from Acyrthosiphon pisum (5.30%), Stegodyphus mimosaum (4.10%), T. castaneum (3.10%), and Diaphorina citri (2.60%) (Fig. 1A). p g Gene ontology (GO) annotations for all the unigenes were obtained using the Blast2GO pipeline, according to the BLASTx search against NR. The GO annotations were used to classify the transcripts into functional groups according to specific GO categories. Among the 248,192 unigenes, 38,307 (15.43%) could be assigned to various Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 2 cientificreports/ Figure 1. Annotation summaries for P. americana unigenes. (A) Species distribution of unigenes with the best hit annotation terms in the non-redundant (NR) database. (B) Gene ontology (GO) classifications of P. americana unigenes. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Annotation summaries for P. americana unigenes. (A) Species distribution of unigenes with the best hit annotation terms in the non-redundant (NR) database. (B) Gene ontology (GO) classifications of P. americana unigenes. Figure 1. Annotation summaries for P. americana unigenes. (A) Species distribution of unigenes with the best hit annotation terms in the non-redundant (NR) database. (B) Gene ontology (GO) classifications of P. americana unigenes. GO terms. Results S Due to their predominant expression in male antennae (the Male antennae/Female antennae mRNA expression level >​10), two other highly expressing PameORs, PameOR1, and PameOR2, with expression levels in the male antennae being 16.8- and 36.9-fold higher than in the female antennae, respectively (Fig. 2A–C) were observed, suggesting the two ORs may participate in female sex phero- mones recognition. Seven PameORs, PameOR17, 23, 33, 40, 43, 90, and 92, three PameIRs, PameIR23, 24, and 25 (Fig. 3A–C) demonstrated female-specific expression (Female antennae/Male antennae >​10). We also found 2 PameORs (PameOR63 and 67) and 4 PameIRs (PameIR1, 10, 16 and 17) to be dominantly expressed in the major Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 3 cientificreports/ Figure 2. Tissue expression profile of PameOR genes based on relative mRNA quantity. (A) Heat map illustrating the Log10 transformation of mRNA expression levels of the PameORs in different tissues. (B) Relative mRNA expression level of all the PameOR genes; the level of PameOR1 expression in the male antennae was set as 1. (C) Relative mRNA expression level of each PameOR gene in a represented stack; the level of PameOR in the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. www.nature.com/scientificreports/ Figure 2. Tissue expression profile of PameOR genes based on relative mRNA quantity. (A) Heat map illustrating the Log10 transformation of mRNA expression levels of the PameORs in different tissues. (B) Relative mRNA expression level of all the PameOR genes; the level of PameOR1 expression in the male antennae was set as 1. (C) Relative mRNA expression level of each PameOR gene in a represented stack; the level of PameOR in the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. chemosensory organs not only in the antennae but also in the maxillary palps. However, 4 PameORs (PameOR17, 74, 75, and 81), 4 PameIRs (PameIR21, 31, 35 and 42), and all 6 iGluRs (PameiGluR1~PameiGluR6) were not observed to have an obvious chemosensory organ-biased expression. Five tissues were selected for investigation of PameGR genes: antennae mixed from both the sexes, maxillary palps, labial palps (Lp), mouthparts (Mor, without maxillary palps and labial palps), and legs. The results showed that 21 of the 32 PameGRs expressed majorly in the maxillary palps/labial palps (PameGR27 was not detected in all the tested tissues; Fig. Results S 4A–C); this included dominant expression of PameGR20, 26, and 28 in the maxillary palps and labial palp-biased expression of PameGR19. y g p Five tissues were selected for investigation of PameGR genes: antennae mixed from both the sexes, maxillary palps, labial palps (Lp), mouthparts (Mor, without maxillary palps and labial palps), and legs. The results showed that 21 of the 32 PameGRs expressed majorly in the maxillary palps/labial palps (PameGR27 was not detected in all the tested tissues; Fig. 4A–C); this included dominant expression of PameGR20, 26, and 28 in the maxillary palps and labial palp-biased expression of PameGR19. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 4 www.nature.com/scientificreports/ Phylogenetic analysis To further unco er the functional role of P americana OR/IR/GR genes ph loge Figure 3. Tissue expression profile of PameIR genes based on relative mRNA quantity. (A) Heat map illustrating the Log10 transformation of mRNA expression levels of the PameIRs in different tissues. (B) Relative mRNA expression levels among all the PameIR genes; the expression level of PameIR1 in the male antennae was set as 1. (C) Relative mRNA expression level of each PameIR gene in a represented stack; the relative expression of PameIR in the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. Figure 3. Tissue expression profile of PameIR genes based on relative mRNA quantity. (A) Heat map illustrating the Log10 transformation of mRNA expression levels of the PameIRs in different tissues. (B) Relative mRNA expression levels among all the PameIR genes; the expression level of PameIR1 in the male antennae was set as 1. (C) Relative mRNA expression level of each PameIR gene in a represented stack; the relative expression of PameIR in the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. Phylogenetic analysis. To further uncover the functional role of P. americana OR/IR/GR genes, phyloge- netic trees were constructed using sequences of typical OR/IR/GR genes from other insect species for which the whole genome or transcriptome data were available. Eighty-five PameORs were observed to be distributed in five major groups (Group A, B, C, D, and E) with other insect species ORs (Fig. 5). Bombyx mori PR and A. pisum OR groups were found to be independent groups without any orthologues of PameORs. Results S Group C was the ORco group, which is the most conventional OR among various species; only one ORco gene, PameORco, was grouped in the same sub-group with another cockroach, B. germanica, BgerORco gene. PameOR55 was the only PameOR in Group A, which was grouped with 6 BmorORs and 23 DmelORs. PameOR75 and PameOR90 belonged to Group D with 29 BmorORs, 3 DmelORs, and 4 ZnevORs. Group B was a Blattaria-specific group with 62 PameORs, 26 ZnevORs, and 2 BgerORs. Group E was also a Blattaria-specific group that included 19 PameORs, 30 ZnevORs, and 3 BgerORs. Phylogenetic analysis. To further uncover the functional role of P. americana OR/IR/GR genes, phyloge- netic trees were constructed using sequences of typical OR/IR/GR genes from other insect species for which the whole genome or transcriptome data were available. Eighty-five PameORs were observed to be distributed in five major groups (Group A, B, C, D, and E) with other insect species ORs (Fig. 5). Bombyx mori PR and A. pisum OR groups were found to be independent groups without any orthologues of PameORs. Group C was the ORco group, which is the most conventional OR among various species; only one ORco gene, PameORco, was grouped in the same sub-group with another cockroach, B. germanica, BgerORco gene. PameOR55 was the only PameOR in Group A, which was grouped with 6 BmorORs and 23 DmelORs. PameOR75 and PameOR90 belonged to Group D with 29 BmorORs, 3 DmelORs, and 4 ZnevORs. Group B was a Blattaria-specific group with 62 PameORs, 26 ZnevORs, and 2 BgerORs. Group E was also a Blattaria-specific group that included 19 PameORs, 30 ZnevORs, and 3 BgerORs. Four major iGluR groups were the antennal IR, divergent IR, NMDA iGluR, and non-NMDA iGluR groups. IR8a and IR25a were co-receptors present in the IR group. Two PameIRs, PameIR8 and PameIR25, were observed to be distributed in these two co-receptors groups, respectively. Six PameIRs, PameIR26, 27, 31, 38, 51, and 53, were distributed in the IR75 and IR64a groups. PameIR1 and PameIR10 are the orthologous genes of antennal Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 5 www.nature.com/scientificreports/ ntificreports/ Figure 4. Tissue expression profile of PameGR genes based on relative mRNA quantity. (A) Heat map illustrating the Log10 transformation of mRNA expression levels of the PameGRs in different tissues. Results S (B) Relative mRNA expression level of all the PameGR genes; the level of expression of PameGR1 in the antennae was set as 1. (C) Relative mRNA expression level of each PameGR gene in a represented stack; the level of expression of PameGR in the antennae was set as 1. An, antennae, Mp, maxillary palps, Lp, labial palps Mor, mouthparts without Mp and Lp, Le, Legs. Figure 4. Tissue expression profile of PameGR genes based on relative mRNA quantity. (A) Heat map illustrating the Log10 transformation of mRNA expression levels of the PameGRs in different tissues. (B) Relative mRNA expression level of all the PameGR genes; the level of expression of PameGR1 in the antennae was set as 1. (C) Relative mRNA expression level of each PameGR gene in a represented stack; the level of expression of PameGR in the antennae was set as 1. An, antennae, Mp, maxillary palps, Lp, labial palps Mor, mouthparts without Mp and Lp, Le, Legs. IR21a and IR68a groups, respectively. PameIR6, 15, 44, and 48 were four genes that were grouped with the anten- nal IR41a. However, no orthologous genes of IR87a, IR7, IR41a, IR93, and IR76b were found in P. americana. Two major divergent IR groups of Blattaria were found in the IR tree, namely the A and B group. Group A contained 26 PameIRs and 49 ZnevIRs. Group B contained 11 PameIRs and 48 ZnevIRs (Fig. 6). p ( g ) In the GR phylogenetic tree, 16 PameGRs and 3 PameGRs were distributed in the sugar and CO2 receptors, respectively (Fig. 7). No orthologues of fructose receptors were noticed. The other 12 PameGRs were grouped in another clade “bitter/other receptors” without functionally identified orthologues. Discussion In the present study, we determined the repertoire of chemosensory receptor families (ORs, IRs, and GRs) in a notorious urban pest, P. americana. These receptor genes have potential significance as targets for developing new pest control strategies, as well as for elucidating the molecular mechanisms that underlie insect-host inter- actions. Transcriptomes (19.31 Gb in total) of three chemosensory organs, male antennae, female antennae, and mouthparts, were sequenced; this was higher than the transcriptomes processed in most of the other studies32–34. After extensive sequencing and assembly using the Trinity software, we identified 96 ORs, 61 IRs/iGluRs (53 IRs Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 6 www.nature.com/scientificreports/ Figure 5. Phylogenetic tree of P. americana PameORs and other typical insect ORs. PameORs are highlighted in red. Species abbreviations: Apis, A. pisum; Pame, P. americana; Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Tdom, Lygus lineolaris; Bmor, B. mori. Figure 5. Phylogenetic tree of P. americana PameORs and other typical insect ORs. PameORs are highlighted in red. Species abbreviations: Apis, A. pisum; Pame, P. americana; Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Tdom, Lygus lineolaris; Bmor, B. mori. and 8 iGluRs), and 33 GRs in this species. The number of ORs was much higher than in the two other Blattaria species, Z. nevadensis (60 ORs, genome data)31 and B. germanica (5 ORs, antennal transcriptome data)30. It was also higher than the 62 ORs found in D. melanogaster15 and the 79 ORs in A. gambiae6,35, but was much lower than the number of ORs reported in T. castaneum (259 ORs)8, A. mellifera (170 ORs)7, and L. moratoria (142 ORs)36. The number of IRs was lower than the numbers found in Z. nevadensis (136 IRs)31 and higher than those found in B. germanica (5 IRs, antennal transcriptome data)30, but it was much higher than the 14 IRs found in A. gossypii24 and Sogatella furcifera23, 57 in D. melanogaster20,22,37, and 22 in A. gambiae38. The number of GRs was much lower than that in Z. nevadensis (75 GRs)31, D. melanogaster (68 GRs)15, Anopheles gambiae (72 GRs)6, and B. mori (65 GRs)39, but was higher than in A. mellifera (13 GRs)7. Unlike other ancient insects, which either lack ORco (Lepismachilis y-signata) or possess three ORco genes (Thermobia domestica)11, only one ORco gene was found in P. americana, which has implies a fully developed ORx/ORco-based olfactory system. Discussion This result is same as that observed in another cockroach, B. germanica, where also only one ORco gene, BgerOR1, an orthologue of PameORco, was present in the antennal transcriptome30. IR8a and IR25a are also coreceptors in the IR system20. PameIR8 and PameIR25 were found clustered in the IR8a and IR25a group with members from other insect species, which are candidate IR coreceptors in P. americana and the orthologous genes, ZnevIR8, ZnevIR25, and BgerIR4 for the IR coreceptors from two other Blattaria species. These findings suggest that the adaptation of distinct species to their hosts has led to the diversification of ORs, IRs, and GRs during their evolution. and 8 iGluRs), and 33 GRs in this species. The number of ORs was much higher than in the two other Blattaria species, Z. nevadensis (60 ORs, genome data)31 and B. germanica (5 ORs, antennal transcriptome data)30. It was also higher than the 62 ORs found in D. melanogaster15 and the 79 ORs in A. gambiae6,35, but was much lower than the number of ORs reported in T. castaneum (259 ORs)8, A. mellifera (170 ORs)7, and L. moratoria (142 ORs)36. The number of IRs was lower than the numbers found in Z. nevadensis (136 IRs)31 and higher than those found in B. germanica (5 IRs, antennal transcriptome data)30, but it was much higher than the 14 IRs found in A. gossypii24 and Sogatella furcifera23, 57 in D. melanogaster20,22,37, and 22 in A. gambiae38. The number of GRs was much lower than that in Z. nevadensis (75 GRs)31, D. melanogaster (68 GRs)15, Anopheles gambiae (72 GRs)6, and B. mori (65 GRs)39, but was higher than in A. mellifera (13 GRs)7. Unlike other ancient insects, which either lack ORco (Lepismachilis y-signata) or possess three ORco genes (Thermobia domestica)11, only one ORco gene was found in P. americana, which has implies a fully developed ORx/ORco-based olfactory system. This result is same as that observed in another cockroach, B. germanica, where also only one ORco gene, BgerOR1, an orthologue of PameORco, was present in the antennal transcriptome30. IR8a and IR25a are also coreceptors in the IR system20. PameIR8 and PameIR25 were found clustered in the IR8a and IR25a group with members from other insect species, which are candidate IR coreceptors in P. americana and the orthologous genes, ZnevIR8, ZnevIR25, and BgerIR4 for the IR coreceptors from two other Blattaria species. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Discussion Two PameORs (PameOR63 and PameOR67) and four PameIRs (PameIR1, 10, 16, and 17) were expressed both in the antennae and maxillary palps, indicating that they might play key roles not only in olfaction but also in gustation, as previously reported in mosquito46 and fly47. Moreover, 4 PameORs (17, 74, 75, and 81) and 4 PameIRs (21, 31, 35 and 42, and six iGluRs (iGluR1– 6) exhibited non-chemosensory organ-biased expression. All of them expressed in the legs to a certain extent, which was consistent with some previous studies36,47,48. However, we could not ignore their olfactory function; as reported earlier, IR expression in the legs affected the mating behaviour of fly47.i roles of OR/IR in olfaction, most Locusta migratoria OR/IR genes displayed olfactory tissue specific expression. However, our results showed that Pame OR/IR genes not only exhibited antenna-biased expression patterns, but also antennae/maxillary palps-biased and other expression patterns. Two PameORs (PameOR63 and PameOR67) and four PameIRs (PameIR1, 10, 16, and 17) were expressed both in the antennae and maxillary palps, indicating that they might play key roles not only in olfaction but also in gustation, as previously reported in mosquito46 and fly47. Moreover, 4 PameORs (17, 74, 75, and 81) and 4 PameIRs (21, 31, 35 and 42, and six iGluRs (iGluR1– 6) exhibited non-chemosensory organ-biased expression. All of them expressed in the legs to a certain extent, which was consistent with some previous studies36,47,48. However, we could not ignore their olfactory function; as reported earlier, IR expression in the legs affected the mating behaviour of fly47.i fl PameGRs expressed mainly in the gustatory organs Mp and Lp, which was consistent with the findings in D. melanogaster49. Insect GRs have been classed in four groups: CO2-, fructose-, non-fructose sugar- receptors, and bitter/other receptors50. Based on the analysis of phylogenetic tree, we could assign a functional group to most of the PameGRs. The CO2 receptors are relatively conserved among the insect species51. Three putative CO2 receptors, PameGR4, 13, and 28 were identified in P. americana. However, only PameGR28 displayed maxillary palps-biased expression, indicating that it had a greater possibility to function as a CO2 receptor than the other two putative receptors. They exhibited apparent orthologous relationships with the Z. nevadensis GR genes, PameGR4/ZnevGR16 and PameGR13/PameGR28/ZnevGR7. The presence of three CO2 receptors was consistent with the number of these receptors in most other insect species, except D. Discussion These findings suggest that the adaptation of distinct species to their hosts has led to the diversification of ORs, IRs, and GRs during their evolution. i In some lepidopteran species, the expressions of ORs in male antennae are mostly higher than or equal to that in female antennae; for example, in Spodoptera litura, 17 of the 25 ORs are male antenna-biased and only 2 of 25 ORs show female-biased expression40. In contrast, in B. mori, only 10/47 of the ORs (female/male >​2) show a female-biased expression41. In P. americana, a lot of PameORs (53 out of 85) and PameIRs (30 out of 57) have a female antennae-biased expression; this was in contrast to the findings in M. sexta (7/70, RNAseq data, male/ female >​10)42 and B. mori (2/68, RT-PCR data, female specific)43. In another study on B. germanica, the authors selected two ORs, BgerOR1 and BgerOR2, and one IR, BgerIR5, to investigate their tissue expression pattern by qPCR. All these three genes displayed female antenna-biased expression30. However, this female antenna-biased expression pattern is expected, as in some hymenopteran species, such as the two parasitoid wasps, Chouioia cunea44 and Microplitis mediator45, ORs and IRs have been reported to play a crucial role in female individuals for finding suitable host to lay eggs. However, cockroaches, which are the early ancestors, lack the internal oviposi- tors. Thus, we hypothesize that the female adults would find more food sources, using food volatiles as cues for the olfactory receptors, to meet the energy requirements of pregnant or egg-carrying females. Consistent with the Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 7 www.nature.com/scientificreports/ Figure 6. Phylogenetic tree of P. americana PameIRs and other typical insect iGluRs and IRs. PameIRs are highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori. Figure 6. Phylogenetic tree of P. americana PameIRs and other typical insect iGluRs and IRs. PameIRs are highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori. roles of OR/IR in olfaction, most Locusta migratoria OR/IR genes displayed olfactory tissue specific expression. However, our results showed that Pame OR/IR genes not only exhibited antenna-biased expression patterns, but also antennae/maxillary palps-biased and other expression patterns. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Discussion We hypothesize that some other GRs, not present in this group, might play a role in detecting fructose. are several lineage-specific expansions of Blattaria species; the P. americana and Z. nevadensis bitter/other receptors are common with bitter receptors of other insect species39,42,54. Eleven orthologous gene pairs (ZnevGR16/PameGR4, ZnevGR7/PameGR13/PameGR28, ZnevGR14/PameGR19, ZnevGR15/PameGR10, ZnevGR18/PameGR11, ZnevGR10/PameGR21, ZnevGR8/PameGR30, ZnevGR12/PameGR3, ZnevGR34/PameGR20, ZnevGR47/PameGR22, and ZnevGR69/PameGR2/PameGR15) were observed in these two species indicating that the bitter/other receptors are conserved. BmorGR9 in B. mori55 and DmelGR43a in D. melanogaster were defined as fructose receptors16. It has recently been reported that DmelGR43a can respond to a range of sugars19. However, we did not find any fructose receptor orthologue in P. americana, or in the genome of other known species, Z. nevadensis and A. pisum. We hypothesize that some other GRs, not present in this group, might play a role in detecting fructose. yp p g p g p y g Adults of female P. americana release two major active sex pheromone components, periplanone-A and periplanone-B, to solicit the male adults26. Thus, like in the lepidopterans, male antenna predominate expression is the standard to find PR13,56. PameOR1 and PameOR2, the higher expressing PameORs in the same clade, are proposed to be the candidate PRs, with their expression levels in the male antennae being much higher than in the female antennae and feebly detected in other tissues. Certainly, the putative PR function needs further elucidation by in vivo and in vitro studies. Besides ORs, IRs, such as IR52c and IR52d of D. melanogaster, are also involved in the mating behaviour47. In P. americana, we observed only one IR, PameIR2, to possess male-antennae dominant expression. This IR belonged to the divergent IR of the Blattaria-specific group A with no orthologous genes, and far separated from DmelIR52c and Dmel52d. However, whether the two major sex pheromone components activate the two respective PRs or PameIR2 needs to be investigated further.i p g In conclusion, based on the analyses of transcriptomic data, we identified a large number totally 190 new chemosensory receptors in the urban pest, P. americana; these included 96 ORs, 61 iGluRs/IRs, and 33 GRs, which is much number than many other insect species. Our results provide a valuable resource for investigating and elucidating the mechanisms of olfaction and gustation in P. americana. Discussion melanogaster, which only has two receptors, DmelGR21a and DmelGR63a51. The sugar receptors (SRs) were first reported in D. melanogaster. Eight genes, DmelGR5a, 64a-f, and 61, which are active for a number of non-fructose sugars in vivo by coding for a single GR or for heterodimers19. Fourteen putative SRs were identified in P. americana, which were present in same group in the phylogenetic tree as the DmelSRs. The number of SRs is variable in different insect species due to the distinct host and food they have; two have been reported in A. mellifera7, five in M. sexta42, B. mori39, and Z. nevadensis31, eight in D. melanogaster19 and A. gambiae6, 10 in D. plexippus42, and 16 in T. castaneum52. Eleven of the 14 SRs in P. americana showed Mp/Lp-biased expression pattern, including equal expression of 10 SRs in Mp and Lp. One SR was predominantly expressed in Mp, indicating the Mp/Lp are both important organs to sense sugars. Consistent with this observation, the sugar-binding sites are present in contact with chemosensory hairs on the maxillary palps of P. americana53. Four pairs of ort- hologues were observed, namely PameGR7/PameGR9/PameGR29/BmorGR4, PameGR25/ZnevGR6/ZnevGR5, PameGR12/PameGR23/ZnevGR1, and PameGR6/ZnevGR2. However, the functional information about these orthol- ogous genes is not available. The other 15 PameGRs remain clustered with the predicted bitter/other receptors. There Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 8 www.nature.com/scientificreports/ Figure 7. Phylogenetic tree of P. americana PameGRs and other typical insect GRs. PameGRs are highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori, Agam, A. gambiae. Figure 7. Phylogenetic tree of P. americana PameGRs and other typical insect GRs. PameGRs are highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori, Agam, A. gambiae. are several lineage-specific expansions of Blattaria species; the P. americana and Z. nevadensis bitter/other receptors are common with bitter receptors of other insect species39,42,54. Eleven orthologous gene pairs (ZnevGR16/PameGR4, ZnevGR7/PameGR13/PameGR28, ZnevGR14/PameGR19, ZnevGR15/PameGR10, ZnevGR18/PameGR11, ZnevGR10/PameGR21, ZnevGR8/PameGR30, ZnevGR12/PameGR3, ZnevGR34/PameGR20, ZnevGR47/PameGR22, and ZnevGR69/PameGR2/PameGR15) were observed in these two species indicating that the bitter/other receptors are conserved. BmorGR9 in B. mori55 and DmelGR43a in D. melanogaster were defined as fructose receptors16. It has recently been reported that DmelGR43a can respond to a range of sugars19. However, we did not find any fructose receptor orthologue in P. americana, or in the genome of other known species, Z. nevadensis and A. pisum. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Methods Insect samples. P. americana were purchased from an insect rearing factory in Anhui province, China. We collected 200 antennae and mouthparts each, from the adults of both the sexes (male/female =​ 1:1) for tran- scriptome sequencing. We dissected various tissues (male/female =​ 1:1, approximately 100 antennae of both the genders, 80 maxillary palps, 80 labial palps, and 80 mouthparts without maxillary palps and labial palps, and 100 legs for each replicate experiment) collected from the adults under a microscope in three replicates for each tissue type. The tissue samples were stored in liquid nitrogen at −​80 °C until further use. Insect samples. P. americana were purchased from an insect rearing factory in Anhui province, China. We collected 200 antennae and mouthparts each, from the adults of both the sexes (male/female =​ 1:1) for tran- scriptome sequencing. We dissected various tissues (male/female =​ 1:1, approximately 100 antennae of both the genders, 80 maxillary palps, 80 labial palps, and 80 mouthparts without maxillary palps and labial palps, and 100 legs for each replicate experiment) collected from the adults under a microscope in three replicates for each tissue type. The tissue samples were stored in liquid nitrogen at −​80 °C until further use. cDNA library construction and Illumina sequencing. Total RNA was extracted using TRIzol reagent (Invitrogen Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA degradation and contamina- tion was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The cDNA library construction and Illumina sequencing of the samples were performed by Novogene Bioinformatics Technology Co. Ltd., Beijing, China. A total amount of 3 μ​g RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was puri- fied from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using diva- lent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×​). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Methods Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining over- hangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′​ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150–200 bp length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Thereafter, 3 μ​L USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR. PCR was per- formed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, the PCR products were purified (AMPure XP system) and the library quality was assessed on an Agilent Bioanalyzer 2100 system. De novo assembly of short reads and gene annotation. Raw data (raw reads) in the fastq format were first processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing the reads containing adapter, reads containing ploy-N, and low quality reads from the raw data. Simultaneously, Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality. De novo transcriptome assembly was performed using the short reads assembly program Trinity (version: r20140413p1) with min_kmer_cov set to 2 by default and all other parameters were set to default values. The overlap settings used for the assembly were 30 bp and 80% similarity, and all the other parameters were set to their default values. Unigenes >​150 bp were aligned by BLASTx with protein databases, including Nr, Swiss-Prot, KEGG, and COG (e-value <​10−5), to identify proteins with high sequence identity and to assign putative functional anno- tations. Next, we used the Blast2GO program (version: b2g4pipe_v2.5, e-value =​ 1.0E-6) (https://www.blast2go. com/) to obtain GO annotations of the unigenes and we obtained the GO functional classifications using WEGO software (http://wego.genomics.org.cn/cgi-bin/wego/index.pl). Phylogenetic analysis. Amino acid sequences of the selected ORs, iGluRs/IRs, and GRs were aligned with the MAFFT (E-INS-I parameter)57. Thereafter, PhyML 3.1 with LG substitution model was used to construct a maximum likelihood phylogenetic tree using Bayesian analysis. The OR dataset comprised ORs in the available databases from: A. pisum58,59, B. germanica30, D. melanogaster15, B. mori41, Z. nevadensis31, and T. domestica11. The IR dataset comprised IRs from B. germanica30, B. Discussion As a crucial first step towards under- standing of their functions, we also conducted a comprehensive examination of the tissue expression patterns and phylogenetic tree of these olfactory receptor genes, which demonstrated that most of these OR, IR, and GR genes were expressed in the chemosensory organs and most OR and IR genes are female antenna-biased expres- sion indicating their key role in female physiological behaviours. Our findings provide the foundation for future research into the olfactory and gustatory system of P. americana, such as the calling behaviour. It could also help in identifying a large number of potential target genes for controlling this notorious pest. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ were calculated according to the 2−ΔΔCq method. The relative fold-changes in the different tissues were calculated and normalized based on the transcript levels in the male antennae. were calculated according to the 2−ΔΔCq method. The relative fold-changes in the different tissues were calculated and normalized based on the transcript levels in the male antennae. Statistical analysis. Data (mean ±​ SE) from various samples were subjected to one-way nested analysis of variance (ANOVA) followed by a least significant difference test (LSD) for mean comparison using SPSS Statistics 17.0 (IBM, Chicago, IL, USA). Statistical analysis. Data (mean ±​ SE) from various samples were subjected to one-way nested analysis of variance (ANOVA) followed by a least significant difference test (LSD) for mean comparison using SPSS Statistics 17.0 (IBM, Chicago, IL, USA). References 1. Benton, R. Multigene Family Evolution: Perspectives from Insect Chemoreceptors. Trends Ecol. Evol. 30, 590–600, doi: 10.1016/j tree.2015.07.009 (2015). tree.2015.07.009 (2015). 2. Leal, W. S. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. Entomol. 58, 373–391 doi: 10 1146/annurev-ento-120811-153635 (2013) 2. Leal, W. S. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. 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Quantitative real time PCR and data analysis. qPCRs were performed for each sample using an iCycle iQ (Bio-Rad, CA, USA) according to the minimum information for publication of quantitative Real-Time PCR Experiments60. Gene-specific primers were designed by Beacon Designer 7.6 (PREMIER Biosoft International, CA, USA) and are listed in Supplementary File 1. The mRNA levels were measured in triplicate (technical rep- licates) by qPCR using TransStart Tip Green qPCR SuperMix, as described by the manufacturer (TransGen Biotech, Beijing, China). The mRNA levels were quantified using ADP-ribosylation factor (the cDNA sequence was identified from this study), actin (Genbank accession number: AY11670), and 60S ribosomal protein L17 (the cDNA sequence was identified from this study) as the reference genes. Means and standard errors were obtained based on at least two biological replicates. The relative expression level of the mRNAs for OR, IR, and GR genes Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 10 www.nature.com/scientificreports/ Large-scale candidate gene scan reveals the role of chemoreceptor genes in host plant specialization and speciation in the pea aphid. Evolution 66, 2723–2738, doi: 10.1111/j.1558-5646.2012.01612.x (2012).h p p p j ( ) 60. Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622, doi: 10.1373/clinchem.2008.112797 (2009). p p p j 0. Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 55, 611–622, doi: 10.1373/clinchem.2008.112797 (2009). Acknowledgements g We thank bachelor students, Zhi-Qiang Wang and Geng Chen (Huaibei Normal University, China), for their help in collecting the insects. This research was supported by Science and technology innovation advanced individual of Guizhou educational department (Grant no. QJHKY [2015]492 for YC), Natural Science Foundation of China (Grant Nos 31360528 and 31401750 for PH), Natural Science Foundation of Guizhou Province of China (Grant No. QKH-J [2014]2062 for PH), Scientific Research Foundation of Guizhou University, China (RJH Project Grant No. 2013–28 for PH). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 37. Croset, V. et al. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste olfaction. PLoS Genetics 6, e1001064, doi: e1001064 10.1371/journal.pgen.1001064 (2010). 38. Liu, C. et al. Distinct Olfactory Signaling Mechanisms in the Malaria Vector Mosquito Anopheles gambiae. PLoS Biol. 8 e1000467 10.1371/journal.pbio.1000467 (2010).h j p ( ) 39. Wanner, K. W. & Robertson, H. M. 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Large Gene Family Expansions and Adaptive Evolution for Odorant and Gustatory Receptors in the Pea Aphid, Acyrthosiphon pisum. Mol. Biol. Evol. 26, 2073–2086, doi: 10.1093/molbev/msp116 (2009). y y p p 9. Smadja, C. M. et al. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Chen, Y. et al. Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana. Sci. Rep. 6, 27495; doi: 10.1038/srep27495 (2016). How to cite this article: Chen, Y. et al. Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana. Sci. Rep. 6, 27495; doi: 10.1038/srep27495 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 12
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Ecoeficiencia: clave de la responsabilidad ambiental empresarial en el sector textil
Desarrollo gerencial
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Ecoeficiencia: clave de la responsabilidad ambiental empresarial en el sector textil Eco-efficiency: The Key to Corporate Environmental Responsibility in the Textile Industry Remedios Pitre-Redondo Universidad de la Guajira, Riohacha, Colombia. Santander De la Ossa-Guerra Universidad de Sucre, Sincelejo, Colombia. Hugo Hernández-Palma Universidad del Atlántico, Barranquilla, Colombia. Resumen Objetivo: Este estudio tuvo como finalidad identificar si las pequeñas y medianas empresas textiles (PYMES), ubicadas en el Departamento del Atlántico, Colombia, realizan prácticas de Ecoeficiencia para el control de residuos contaminantes. Método: se utilizó una investigación de tipo descriptivo, con un enfoque cuantitativo y un diseño de campo no experimental, en la que se aplicó una encuesta cerrada, dirigida a una muestra de 53 empresas. Resultados y discusión: Entre los principales hallazgos se encontró que el 56% de las PYMES participantes no realizan acciones para reducir el volumen o la peligrosidad de sus residuos, ya que no los consideran importantes ni rentables. Por lo tanto, estos procesos no son ecoeficientes y el compromiso con la preservación del medio ambiente y la sostenibilidad de las PYMES en estudio es insuficiente. Conclusiones: Dado que el sector textil es uno de los recursos con menor índice de reutilización del mundo, es fundamental que este vínculo con los procesos de gestión sostenible y la ecoeficiencia sea claro y constante, apuntando a una internalización de los procesos de responsabilidad social, apoyándose en el Estado, las familias, las personas y las leyes para su constante evolución y desarrollo. Palabras clave: Desarrollo sostenible, Prácticas ambientales, Responsabilidad social, Residuos sólidos. Clasificación JEL: Q01, Q50, Q56. Autor de Correspondencia hugoghernandezp@mail.uniatlant ico.edu.co Recibido: 03-03-2020 Aceptado: 08-07-2020 Publicado: 30-11-2020 Copyright © 2020 Desarrollo Gerencial Abstract Objective: This study aimed to identify whether small and medium sized textile companies (SMEs), located in the Department of Atlántico, Colombia, currently carry out Eco-efficiency practices for the control of waste, contaminating residues, among other aspects. Method: A descriptive type of research was used, with a quantitative approach and a non-experimental field design, in which a closed survey was applied, directed at a sample of 53 companies. Results and discussion: Among the main findings, it was found that 56% of the participating SMEs do not take actions to reduce the volume or hazardousness of their waste, since they do not consider it important or profitable. Therefore, these processes are not eco-efficient and the commitment to environmental preservation and sustainability of the SMEs under study is insufficient. Conclusions: Given that the textile sector is one of the resources with the lowest rate of reuse in the world, it is fundamental that this link with the processes of sustainable management and eco-efficiency be clear and constant, aiming at an internalization of the processes of social responsibility, relying on the State, families, people and laws for their constant evolution and development. Keywords: Sustainable development, Social responsibility, Environmental practices, Solid waste. JEL classification: Q01, Q50, Q56. Como citar este artículo (APA): Pitre-Redondo, R., De la Ossa-Guerra, S., & Hernández-Palma, H. (2020). Ecoeficiencia: clave de la responsabilidad ambiental empresarial en el sector textil. Desarrollo Gerencial, 12(2), 1-20. https://doi.org/10.17081/dege.12.2.3870 1 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma Introducción Con el paso de los años, el ser humano ha pasado de ser una especie nómada el cual se dedicaba a la casa y a los cultivos estacionarios a convertirse hoy en día en seres sedentarios; caracterizados por el establecimiento de comunidades y el desarrollo de industrias a gran escala para lograr suplir las necesidades de sus iguales. A partir de este punto, es innegable reconocer el actual impacto que ha tenido esta tendencia y estilo de vida en el medio ambiente; pues el cambio climático con el pasar de cada segundo está teniendo mayores connotaciones y trayendo consecuencias que podrían llegar a ser irreversibles si no se toman las acciones necesarias a tiempo. Basta con revisar los números de emisiones de CO2 (Dióxido de carbono), que al sol de hoy han alcanzado los números más altos en los últimos 800.000 años, según reportes de Oceanografía Scripps (2019) y al año 2007 según reportes de Intergovernmental Panel on Climate Change (2007), el 19% de esta cifra corresponde a la responsabilidad del sector industrial. En tal sentido, la sociedad actual está tomando conciencia acerca del impacto que tiene la industria sobre el deterioro del medio ambiente; según Suárez y Molina (2014), el desarrollo industrial si bien induce una fuerte reactivación socioeconómica, contribuye a la generación de diversas problemáticas ambientales como es el caso de la contaminación, el desequilibrio en los ecosistemas y la extinción de fauna y flora y, así mimo, da paso a la génesis de problemáticas sociales, entre ellas y quizás la más relevante, el detrimento de la salud pública principalmente, a partir de la contaminación atmosférica (Ballester, et. al., 2003; Vargas, 2005) y de la contaminación en las cuencas hídricas (Maldonado, 2009) Este hecho viene como resultado de un extenso proceso histórico por el cual ha pasado la sociedad para poco a poco ir reconociendo el cambio climático como una problemática real, la cual es resultado de la acción indiscriminada del hombre y de sus industrias que por mucho tiempo se desarrollaron obviando las necesidades y el impacto hacia los ecosistemas. Lo anterior, ha sido el punto de partida para la creación de leyes proteccionistas que impulsan estrategias direccionadas a la evaluación, prevención y control de los factores desligados de la actividad industrial, que influyen en la aparición de problemas ambientales (Torres, Barreto y Rincón, 2015). Pero así mismo, ha motivado a las empresas que desean ser competitivas a desarrollar sistemas de gestión que les ayuden a reducir su huella ambiental, por medio del mejoramiento de sus procesos, la adquisición de tecnológicas y prácticas de producción limpias, el posicionamiento de políticas de optimización de recursos e incluso, el eco-diseño de sus productos; todo ello con la finalidad de hacer un uso efectivo de sus recursos, reducir costes y eco-posicionarse en el mercado. Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 2 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma El eco-posicionamiento, según la Fundación Fórum Ambiental (2014) se ha popularizado entre las empresas por cuestiones como la calidad, el ahorro, el mercado, la imagen, las oportunidades de negocio y, las consideraciones de tipo ético y social. De acuerdo con Eryuruk (2012) las empresas de hoy deben rediseñar sus estructuras existentes, para convertirse en ambientalmente responsables, de este modo puedan aumentar su credibilidad en el medio pero, así mismo, producir más, con menos recursos, lo que evita el desperdicio de las materias primas, disminuye las emisiones y reduce los daños al medio ambiente (Mansour et. al., 2006). Uno de los sectores industriales con mayor grado de contaminación ambiental en Colombia, es el textil; de acuerdo con el informe de Colombia Productiva (2012) la mayoría de las empresas de este sector no cuentan con políticas de sostenibilidad ni de Responsabilidad Social Empresarial RES ya que el 67% de estas empresas no elabora ningún tipo de informe de sostenibilidad, el 25% no tiene gestión de aguas residuales, el 58% no cuenta con un sistema de control de emisiones atmosféricas y solo el 4,4% de las empresas recuperan o reutilizan residuos de su producción. Estas cifras son alarmantes debido a que la producción textil en Colombia sigue en aumento, mientras que la mayoría de estas empresas adolecen de iniciativas para reducir su impacto medioambiental, siendo evidentes factores de riesgo desligados de si operación, como es el caso de los residuos químicos altamente contaminantes. En atención a lo anterior, surge el concepto de Ecoeficiencia, una práctica que intenta sostener el valor ecológico de las empresas al tiempo que potencia el crecimiento económico del mercado; así pues, las empresas que implementan prácticas de Ecoeficiencia consiguen aumentar su competitividad, contribuyendo, al tiempo, al desarrollo sostenible del territorio, puesto que, añaden mayor valor a sus productos con un menor consumo de recursos naturales y disminuyen la contaminación, lo que ayuda a mitigar los riesgos ambientales que pueden llegar a afectar a sus stakeholders internos y externos. En este sentido, se destaca que las prácticas relacionadas con la preservación del ambiente por parte de las organizaciones vienen como resultados de lo que se llama la Responsabilidad Social Empresarial RSE, lo cual es un propósito importante para toda organización, así como para los grupos de interés, pues permite lograr sus objetivos de mejor manera y tener un impacto positivo en el entorno y es una serie de obligaciones y compromisos tanto legales, como éticos, nacionales e internacionales que adquieren las empresas con los llamados grupos de interés, derivados del impacto generado por la existencia, actividad, además las operaciones de las organizaciones, las cuales son producidas en el contexto social, laboral, medioambiental, así como también al relacionado a los derechos humanos, en tal aspecto, este tiene un efecto significativo en el proceso de gestión de las empresas, tanto a lo relacionado a sus diversas actividades productivas y comerciales, así como en el desarrollo de las relaciones con los mencionados grupos de interés (Duran, Fuenmayor, Sergio y Hernández, 2016). Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 3 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma Bajo estas ideas, se aporta que actualmente Colombia es una nación que dentro del marco de la sostenibilidad se encuentra atrasado con relación a otros países, pues no se toman en cuenta diversos procesos o regulaciones que permitirían asegurar la prevalencia de los ecosistemas en el país (Cuartas y Méndez, 2016). A demás, desde ciertas posturas, se aporta que brindar especial atención a las necesidades del medio ambiente sobre las necesidades de las organizaciones puede traer como resultados un declive de la calidad competitiva de dichas empresas frente a los mercados; trayendo como resultado un declive en los índices económicos y laborales de los países y regiones. Ciertamente, se debe tener en cuenta que hoy en día se vive una época caracterizada por los mercados altamente competitivos; donde los constantes avances tecnológicos por resultado de la globalización obligan a las empresas a siempre buscar la innovación y la calidad como premisa para mantener su estatus competitivo (Parra et al, 2018). A partir de este planteamiento, surge la disyuntiva de encontrar un equilibrio entre la calidad de los procesos productivos de manera que se tengan en cuenta las necesidades del medio ambiente a través de procesos de gestión sostenible; concepto direccionado a la gestión de los recursos encaminado a la calidad de los procesos cuidando la necesidad de los ecosistemas (Custodio y De Oliveira, 2016) A partir de lo anterior se considera que la Ecoeficiencia es una alternativa para alcanzar este valor de posicionamiento dentro de los mercados, tomando en consideración el cambio climático como hecho catalizador de acciones enfocadas a la sostenibilidad. De esta manera, el presente artículo de investigación se desarrolló con la finalidad de identificar si las pequeñas y medianas empresas (Pymes) textiles, ubicadas en el departamento del Atlántico, Colombia en la actualidad llevan a cabo prácticas de Ecoeficiencia para el control de despilfarros, desechos contaminantes, consumo de energía, entre otros; aspectos que apuntan al posicionamiento de prácticas ambientalmente responsables. Si bien, según la Superintendencia de Sociedades (2013), el departamento del Atlántico solo aporta el 3,62% de la producción textil nacional, se selecciona este segmento geográfico para el estudio, dada la evolución que este ha tenido en los últimos años en el sector textil, además, porque, a partir de su posición privilegiada y vocación exportadora, el departamento del Atlántico ha dado paso a dos importantes clústeres: uno de confecciones y otro diseño textil. Fundamentación Teórica Responsabilidad Social Empresarial RSE La RSE es uno de los elementos más representativos de las organizaciones en la actualidad pues ha generado una gran nivel de debate sobre su alcance e implicaciones dentro de las diversas industrias (Raufflet, Lozano, Duque y de la Torre, 2012; Padilla et al., 2017) proponiendo cambios en las prácticas Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 4 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma empresariales de manera que estas cambien direccionamiento hacia la sociedad, respondiendo a las exigencias de las comunidades que piden empresas más consientes con el impacto de sus actividades en la sociedad y en el medio ambiente (Solarte y Duque, 2017). En este sentido, se aporta que la RSE es una obligación que poseen las empresas con relación al impacto que estas llegan a generar en su entorno y como estas tienen un importante efecto sobre las dimensiones social, económica y ambiental (Ramírez et al., 2018). Del mismo modo, esta busca garantizar que el impacto positivo equitativo, sustentable y que cause el menor impacto posible a las llamadas partes interesadas. Ciertamente la RSE implica que estas sean conscientes sobre el papel que tienen dentro un todo más complejo donde las organizaciones, al ser un fenómeno humano se involucren con las comunidades para aporta a las construcciones de una nueva sociedad (Pérez, Espinoza y Peralta, 2016; Castro, et al., 2019). De esta manera, se convierte en un apunte al cambio, al desarrollo y a la calidad de las organizaciones que a la larga permite crear una percepción más humana de los consumidores, lo cual permitirá que la organización como marca tenga un reconocimiento positivo y una mayor aceptación. Asimismo, en el libro verde se hacen mención a dos importantes dimensiones de la RSE (Salazar, Hidalgo y Manríquez, 2017; Abreo, Pinzón y Chacón, 2018); los cuales se enfocan a lo interno, referido a prácticas que se llevan a cabo dentro de la organización (Ortiz y Bandeira, 2015) y la dimensión externa, referida al impacto que las acciones tomadas por la organización tienen en el entorno de esta (Chumaceiro, Hernández, Yori y Ziritt, 2013). A partir de aquí, el Centro para la Acción de la Responsabilidad Social Empresarial en Guatemala [Centra RSE] (2006) citado en De Grazia (2006) propone la pirámide de la responsabilidad social empresarial como medio de apoyo a las organizaciones que busquen implementarla; priorizando áreas de acción referidas a esta responsabilidad social empresarial. En esta pirámide se establecen los niveles de los criterios para diseñar políticas funcionales direccionadas al cambio sostenible y responsable: Figura 1. Pirámide de la responsabilidad social empresarial Diseño/ejecucion de politicas de estado Proyección comunidad Proyección familia Desarrollo de la persona Cumplimiento régimen legal Fuente: Elaboración propia de autores basado en CENTRA RSE (2006). Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 5 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma Autores como Barros Retamozo y González (2015) aportan que las políticas de estado permiten que las acciones vayan acorde a los planteamientos legales para responder a las proyecciones comunitarias. Así mismo generando un involucramiento con las familias, creando un espacio de confianza entre empresapersona para promover el desarrollo de las personas dentro y fuera de esta, siempre cumpliendo con los estándares legales requeridos, que fungen como la base de las acciones en todo momento. En este sentido, se reconoce que dentro de la responsabilidad social empresarial existen los llamados grupos de interés (Stakeholders); los cuales se direccionan a las partes interesadas o afectadas por las actividades (Freeman, 2010). Entre los grupos de interés se pueden mencionar a los trabajadores o las comunidades los cuales establecen sus intereses hacia la relación de las organizaciones son las temáticas políticas, sociales o ambientales. Dentro del presente artículo se realiza un énfasis a la relación entre la comunidad, las empresas y el medio ambiente al reconocer el impacto que se tiene sobre esta. Del cambio climático a la Ecoeficiencia Por muchos años se pensó que el cambio climático era un hecho ficticio sin soporte científico hasta que en los años 70 la comunidad científica comenzó a preocuparse por los constantes cambios de clima que sucedían para esa época, comenzando a hacer las primeras predicciones sobre el calentamiento global (Peterson, Connolley y Fleck, 2008; Deryugina, 2013). Con base a ello, ciertas décadas después los gobiernos del mundo apoyados en esta comunidad científica llevan a cabo en 1994 la Convención Marco de las Naciones Unidas sobre el Cambio Climático la cual se encaminó a estabilizar los niveles de contaminación generados a nivel mundial estableciendo un concepto de cambio climático que lo comprende desde la actividad humana que altera la composición de la atmosfera mundial (Oscullo y Haro, 2016). A partir de estos primeros pasos se generan nuevos documentos como el Protocolo De Kioto de la Convención Marco de las Naciones Unidas sobre el Cambio Climático en 1998 hasta llegar al 2019, donde no solo existen rigurosas regulaciones sobre los temas ambientales, si no que se han generado modelos y propuestas que buscan mantener la rentabilidad y competitividad de la industria sin dejar a un lado los compromisos adquiridos en años anteriores. Dentro de estos procesos se destaca la Ecoeficiencia (Deryugina, 2013), término que se atribuye a la relación existente entre el valor de un producto y/o servicio y los impactos medioambientales derivados de todo el proceso desde su comienzo hasta que este deja de ser utilizado (Durán, Calderón y Montero, 2018). Este proceso de Ecoeficiencia trae como resultado la vinculación de la conciencia ambiental dentro de las organizaciones, de manera que se logre tener el mayor margen de producción y ganancias, con el menor consumo de recursos y energía y así reducir el impacto sobre el medio ambiente (Carmelo, 2018). Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 6 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma En tal sentido y basado en Rincón y Wellens (2011) se presentan los cinco principios de la Ecoeficiencia: Figura 2. Principios de la eco-eficiencia 1. Consumo de agua por unidad de valor agregado neto 2. Requerimientos energéticos por unidad de valor agregado neto 3. Contribución al calentamiento global por unidad de valor agregado neto 4. Dependencia de sustancias que deterioran la capa de ozono por unidad de valor agregado neto 5. Residuos sólidos generados por unidad de valor agregado neto Fuente: Rincón y Wellens (2011). Se destaca que al implementar la Ecoeficiencia dentro de una organización implica plantear una iniciativa de valor pues al promover el desarrollo socioeconómico con un bajo coste de recursos (Vallejo y Cabrera, 2017), las mismas empresas se convierten en lideres dentro de su sector al ser estas las que generan una verdadera innovación (Sánchez, 2011). Aquí se plantea un interesante propuesto al afirmar que el daño ambiental proviene de los procesos para lograr los objetivos y no de los objetivos, por lo que son estos los que deben ser mejorados y trabajados (Cantú, 2015). Se aporta que el empleo de la Ecoeficiencia permite a que las organizaciones sean vistas como parte de un todo en los sistemas naturales (González y Pérez, 2011), por lo que los procesos para su desarrollo y administración deberían poseer un enfoque sobre los componentes de la industria y la biosfera, la relación del flujo de la materia con el sistema industrial y considerar la tecnología como medio de transición para el logro de la no sustentabilidad (Carrillo, 2009). Actualmente existen diversos enfoques al momento de medir la Ecoeficiencia, pero uno de los más representativos es el brindado por Desimone y Popoff (2000):  Reducción del derroche de recursos apoyándose en la mejora continua.  Disminución del volumen y toxicidad de los residuos.  Reducción del consumo energético y emisiones contaminantes.  Disminución de los riesgos de incumplimiento de leyes y se mejoran las relaciones de la comunidad y la empresa. Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 7 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma En este sentido, se considera que un punto fundamental para lograr la Ecoeficiencia se relaciona con el empleo de las llamadas Mejores Tecnologías Disponible (MTD), término que se direcciona al uso de los equipamientos tecnológicos más avanzados y que su uso permite optimizar de la manera más adecuada los procesos de una organización. Ciertamente se ha reconocido el uso de las MTD para la reducción del impacto ambiental, ligado al rol ecológico que buscan tomar las nuevas tecnologías en la actualidad (Jiang et al., 2012) Método El presente estudio se desarrolló desde un enfoque cuantitativo, el cual permitió el análisis de resultados numéricos para su interpretación como procesos humanos. Del mismo modo, se direccionó desde la investigación descriptiva y exploratoria, para lograr identificar si las pequeñas y medianas empresas (Pymes) textiles, ubicadas en el departamento del Atlántico cumplen con las claves de la Ecoeficiencia. El diseño empleado fue de campo, no experimental y transversal pues la investigación requirió de realizar una medición única en el lugar de origen del fenómeno estudiado, pero sin llevar a cabo cualquier manipulación de las variables objeto de estudio dentro de la muestra seleccionada. Este estudio se aplicó a los gerentes, administradores y jefes de producción de las PYMES textiles del departamento del Atlántico, Colombia. La muestra de la investigación es de tipo no probabilística (Otzen y Manterola, 2017), para la selección de esta, se consultaron las bases de datos de la Cámara de Comercio de Barranquilla, en donde se identificaron las pymes textiles ubicadas en el departamento del Atlántico, legalmente constituidas y, a partir de allí, se seleccionaron de manera aleatoria 53 empresas. Las pymes elegidas fueron contactadas vía telefónica y, previo consentimiento informado, se les fue aplicado el instrumento, al cual dieron respuesta de manera efectiva. Para llevar a cabo el mencionado proceso de recolección de la data, el presente artículo se apoyó en el uso de un cuestionario cerrado con opciones dicotómicas (Si o No), el cual se configuró a partir de los principios básicos de la Ecoeficiencia, estructurando de esta manera seis preguntas clave que permiten evidencias el grado de compromiso y responsabilidad ambiental de las empresas seleccionadas como muestra en este estudio. La distribución de las preguntas del cuestionario responde de la siguiente manera a las claves de la Ecoeficiencia: Tabla 1. Distribución del instrumento Indicador Reducción del derroche de recursos apoyándose en la mejora continua. Disminución del volumen y toxicidad de los residuos. Ítem Ítem 1 y 5 Ítem 2 Reducción del consumo energético y emisiones contaminantes. Ítem 3 y 4 Disminución de los riesgos de incumplimiento de leyes y se mejoran las relaciones de la comunidad y la empresa. Ítem 6 Fuente: Elaboración Propia de autores (2019) Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 8 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma Se destaca que este cuestionario se basa en los cinco principios de la Ecoeficiencia que son enumerados por Rincón y Wellens (2011). Este instrumento fue validado por un grupo de tres expertos en el área de sostenibilidad empresarial y responsabilidad social corporativa. Del mismo modo, a este instrumento se le aplico un proceso para determinar su confiabilidad a través del alfa de Cronbach la cual en este caso fue de 0,67 por lo que se considera como un valor aceptable en estudios de índole social (Oviedo y Arias, 2005). Se destaca que los datos fueron procesados por medio del programa estadístico SPSS. Resultados En esta sección se estructuran los resultados del cuestionario aplicado a los representantes de las 53 empresas seleccionadas como muestra en esta investigación, los cuales se estructuran a partir de cada una de las seis preguntas claves que les fueron formuladas durante la etapa de recolección. Una de las claves de la Ecoeficiencia es el control de los despilfarros de los recursos mediante la mejora continua; de acuerdo con el estudio el 76% de las pymes textiles participantes efectúa estos procesos de control de despilfarro; sin embargo, el 24% restante afirma no llevar a cabo tales procesos. (Gráfico 1). Gráfico 1. Control de despilfarros ¿La empresa controla el despilfarro de los recursos mediante la mejora continua? 24% 76% Si No Cuando las empresas no tienen un control preciso sobre sus desperdicios no solo corren con el riesgo de que aumenten sus costos de producción, sino de que se disminuya de manera sustancial su rendimiento y la percepción de valor que poseen sus clientes en torno a los productos y servicios que ofrece, esto último puede explicarse de la siguiente manera: si los clientes perciben que la empresa no es consciente de reciclar, y reutilizar sus residuos de producción o de establecer técnicas para disminuir sus despilfarros, podrían preferir a empresas que presten los mismos servicios o productos pero cuya producción sea más amigable con la naturaleza, en aras de apoyar a compañías ambientalmente responsables y así, disminuir la huella ambiental industrial. Por fortuna, la mayoría de las compañías objeto de estudio, afirman tener un control preciso de sus despilfarros. Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 9 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma Ahora bien, las empresas textiles utilizan diversos insumos químicos considerados como peligrosos en su proceso de producción, los cuales de no ser gestionados de la manera adecuada pueden constituirse como residuos altamente contaminantes para el medio ambiente; de allí la importancia de que las empresas textiles se esfuercen por disminuir el volumen y cambiar algunos insumos químicos que utilizan en la manufactura de sus productos por sustitutos con menor nivel de toxicidad. Se preguntó a las pymes textiles objeto de estudio si en la actualidad se esfuerzan por disminuir el volumen y el nivel de toxicidad de sus residuos, los resultados dejan en claro que la mayoría de estas entidades, representada por el 56% no llevan a cabo acciones para disminuir el volumen ni la peligrosidad de sus residuos y que, solo el 43% de estas empresas desarrolla este tipo de acciones. (Gráfico 2). Gráfico 2. Disminución volumen y toxicidad de residuos ¿La empresa se esfuerza por dismuir el volumen y toxicidad de residuos? 44% 56% Si No El hecho de que estas empresas no gestionen de la manera adecuada sus residuos, más aún si estos son peligrosos (sustancias químicas), se constituye como un riesgo evidente para el medio ambiente y para los stakeholders externos, principalmente la comunidad. A partir de esta falta de control, los residuos pueden alterar el equilibrio de los ecosistemas, contaminar las cuencas hídricas y afectar de manera profunda la salud de los animales y seres humanos. Otro de los cuestionamientos que se expusieron en el instrumento de investigación se basó en indagar si estas empresas controlaban su consumo de energía y emisiones contaminantes por medio de prácticas de producción limpia; el 72% de las pymes participantes dio una respuesta afirmativa; el 28% de las pymes restantes, expresó que no llevaban a cabo prácticas de producción limpias para el control de dichos aspectos (Gráfico 3). Gráfico 3. Prácticas de Producción limpia ¿En la empresa se controla el consumo de energía y emisiones contaminantes por medio de practicas de producción limpia? 28% 72% Si No Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 10 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma Seguidamente se preguntó a las empresas si en la actualidad incorpora MTD para optimizar su producción; el 72% niega implementar este tipo de procesos; mientras que el 28% restante asegura si ejecutarlos. (Gráfico 4). Gráfico 4. MTD para optimizar producción ¿En la actualidad la empresa incorpora mejores tecnologías disponibles para optimizar su producción? 28% 72% Si No Las MTD, además, contribuyen a disminuir el impacto ambiental a través de la prevención de la contaminación y el diseño de productos ecológicos, lo que crea valor para las partes de interés y posiciona a la empresa como ambientalmente responsable. Otro punto importante, que se estudió a partir del instrumento, se centra en el desarrollo de estrategias orientadas a reducir costos en el consumo de recursos (energéticos) y materias primas; muchas empresas no cuentan con el capital económico para incorporar prácticas de producción limpia o MTD, sin embargo, pueden diseñar e incorporar otro tipo de estrategias que les permita minimizar el consumo de recursos, más aun si se trata de aquellos categorizados como no renovables como la energía eléctrica convencional; según los resultados del estudio, el 56% de las pymes participantes afirma que no desarrolla citadas estrategias; mientras que el 44% restante asegura si ejecuta este tipo de estrategias. (Gráfico 5) Gráfico 5. Estrategias reducción consumo ¿En la actualidad la empresa desarrolla estrategias orientadas a reducir costos en el consumo de recursos/materias primas? 44% 56% Si No Finalmente, se preguntó a las empresas si consideraban que aumentando la seguridad ambiental podrían generar valor para sus partes de interés y aumentar su credibilidad en el mercado; el 84% estuvo Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 11 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma de acuerdo con esta afirmación; mientras que el 16% ignora que estas prácticas surtan beneficios para su operación. (Gráfico 6) Gráfico 6. Seguridad Ambiental para crear valor ¿Considera que aumentando la seguridad ambiental podrá generar valor para sus partes de interes y aumental su credibilidad en el mercado? 16% 84% Si No De esta manera, se observa que un gran porcentaje de las organizaciones que forman parte de la muestra reconocen el gran valor que genera la mejoría de la seguridad ambiental para las partes interesadas y así mejorar su credibilidad en el mercado. Discusiones A partir de los resultados del trabajo de campo, puede afirmarse que la mayoría de las pymes textiles ubicadas en el departamento del Atlántico no son empresas eco-eficientes; tal aseveración se sustenta en que, si bien la mayoría de las empresas asegura controlar sus despilfarros, estas no llevan a cabo procesos que les permitan disminuir el volumen y el grado de toxicidad de los insumos que utilizan en su producción, así pues, de poco sirve que una empresa regule las pérdidas de materiales, sino controla por ejemplo, el uso de sus materias primas o la cantidad de desechos peligrosos que dispone en el medio ambiente. En consonancia con lo anterior, Rincón (2001) manifiesta que las empresas no pueden ser competitivas mientras no eliminen sus desperdicios y no trabajen con parámetros de productividad; pero, así mismo, esta competitividad depende de que las compañías minimicen el tiempo y la cantidad de recursos que utilizan en su producción; en este orden de ideas, las pymes textiles objeto de estudio, tienen grandes retos para ser competitivas a nivel nacional e internacional, puesto que, según el estudio, la mayoría de estas empresas no cuentan con estrategias orientadas a reducir el consumo de recursos (energéticos) y materias primas, lo cual conlleva a altos costos de producción y, por consiguiente, a aumentar la huella ambiental de su operación. El consumo desmedido de energía, de acuerdo con Blanco y Arce (2012) produce grandes impactos ambientales, ya que, algunas centrales productoras de este recurso no renovable (Al menos en Colombia, por su plantas de generación convencional), utilizan procesos de combustión de los derivados de petróleo Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 12 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma para generarlo; así mismo, Duart (2007) manifiesta que el uso de la energía eléctrica en la industria contribuye al aumento en las emisiones de CO2, situación que tiene graves repercusiones sobre el calentamiento global y al aumento de las tasas de contaminación atmosférica. De igual modo, el consumo descontrolado de materias primas no solo genera una sobreexplotación de los recursos naturales, sino que incrementa la tasa de despilfarros y residuos que pueden contaminar el medio ambiente. A partir de lo anterior, las empresas textiles deben reconocer la importancia de reducir el consumo de energía, según Campos et al. (2008) citado en Del Pilar et. al., (2013), “Las empresas industriales pueden lograr ahorros de energía de hasta un 40%, algunos sin inversión de capital, mediante la aplicación de métodos de gestión energética” (p. 115); pero así mismo la importancia de reducir al máximo la utilización de materias primas no renovables y de incursionar en materiales sustitutos que cumplan con la misma función a un costo ambiental más bajo (Cañizares y Jurado, 2019). Dentro del cuestionario se trabajó sobre la aplicación de las MTD para reducir el consumo de recursos, donde se encontró un bajo porcentaje representativo de su aplicación a nivel organizacional. De este modo, se aporta que las prácticas de producción limpias, más allá de evitar la emisión de residuos y de minimizar el uso indiscriminado de recursos energéticos, según Van y Herrera (2007) permiten la optimización y mejoramiento de procesos empresariales, promueven la innovación tecnológica (ecodiseño), la sostenibilidad e incluyen el concepto de ciclo vida como orientación de la gestión ambiental empresarial. De la mano de las prácticas de producción limpia, surgen las MTD o Mejores Tecnologías Disponibles, las cuales, según Evrard et. al., (2016) permiten la reducción del uso de recursos finitos y, así mismo la efectividad en el uso de materias primas, para beneficiar a la empresa, el consumidor y la sociedad en general. Estos procesos de reducción y optimización, de acuerdo con Serna (2010) pueden levarse a cabo a partir de que las empresas determinen los sistemas y equipos que permitan la disminución de consumos, costos energéticos y costos ambientales; estos sistemas son reconocidos en el medio como las Mejores Tecnologías Disponibles (MTD) y su propósito es reducir al máximo los consumos, los despilfarros y los residuos de la producción. Si bien estas MTD ofrecen grandes beneficios a las empresas, en el caso de las pymes textiles del departamento del Atlántico no han sido implementadas a cabalidad, de hecho el 72% de estas empresa expresa que en la actualidad no incorpora este tipo de tecnología a su operación; las razones no se especifican en el estudio, sin embargo, se estima que estas empresas no acuden a las MTD porque no están dispuestas a invertir recursos económicos o, así mismo, porque no ven su realmente utilidad, dejando de lado los grandes ahorros que estas MTD pueden generarles. Lo mencionado se enlaza con la pregunta direccionada a saber si se reducían los índices de consumos de materia prima, donde las respuestas fueron dividas. Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 13 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma De allí la importancia de que las compañías objeto de estudio, hagan un uso consiente de sus materias primas, creen una cultura de reducción, reutilización y reciclaje, además, reemplacen los insumos nocivos para el medio ambiente, como son las tintas con base química, por elementos biodegradables, los cuales no solo les ayudarán a prevenir enfermedades de su fuerza laboral y la disminución de costes, sino que permitirán la salvaguarda del medio ambiente y la salud pública. A su vez, se percibe que las organizaciones perciben el valor de mejorar la seguridad ambiental como herramienta para su relación con los grupos de interés y su reputación en el mercado. Cuando las empresas están enajenadas de los beneficios que pueden percibir al ser ambientalmente responsables, difícilmente se direccionaran a modificar sus prácticas, procesos de producción y tecnologías, ya que esto supone para ellas más gastos que utilidades; sin embargo, según Pérez y Camacho (2013) cuando las empresas logran ser reconocidas en el medio como una entidad con alto sentido de responsabilidad social y ambiental, esta puede mantener e incrementar su rentabilidad, puesto que se apalanca su imagen, posicionamiento y trascendencia de la marca en el mercado. Es claro que, la mayoría de las empresas participantes en el estudio saben que, aumentando la seguridad ambiental, podrían generar valor para sus partes de interés y, de este modo, incrementar su credibilidad en el mercado; no obstante, son evidentes los grandes obstáculos que estas entidades tienen que sobrepasar para ser empresas ambientalmente responsables, dado que sus procesos y condiciones actuales están yendo en contravía de las crecientes exigencias en la legislación ambiental y los requerimientos de los consumidores “verdes” actuales. A partir de lo anterior, las pymes textiles deben implementar herramientas de gestión de Ecoeficiencia que les permitan diagnosticar las oportunidades de mejora ambiental, optimizar el consumo energético y de materias primas, establecer incluso medidas de ahorro de agua, tales como las auditorías ambientales, los sistemas de gestión medio ambiental ISO 14001 y programas de contabilidad ambiental. Conclusiones En primera instancia, se reconoce que la Responsabilidad Social Empresarial es un elemento de cambio dentro de las dinámicas empresariales de la actualidad; la cual brinda la oportunidad de direccionar a las organizaciones a convertirse entes más humanos, los cuales reconocen su importante papel dentro de la sociedad y de las comunidades como elemento promotor del crecimiento y del desarrollo local y regional. De este modo, la vinculación a la Ecoeficiencia como proceso de cambio se presenta como una interesante propuesta para alcanzar altos estándares de calidad y de competitividad de las empresas vinculando estos objetivos con las necesidades del medioambiente, que, si bien en el pasado parecía ideas contrarias, hoy en día se comprueba que ambas van de la mano a través de una relación de sostenibilidad e innovación. De esta manera, al ser el sector textil el generador uno de los recursos con Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870 14 Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma menor índice de reutilización en el mundo, se hace fundamental que esta vinculación con procesos de gestión sostenible y Ecoeficiencia sean claros y constantes, apuntando a una internalización de los procesos responsabilidad social, apoyándose en el estado, las familias, las personas y las leyes para su constante evolución y desarrollo. Puede visualizarse en el mercado actual un compromiso creciente por parte de las empresas por contribuir a la sostenibilidad ambiental; estos avances además de tener un impacto positivo en la naturaleza, en la medida que se disminuye la contaminación, también tiene una incidencia en la mitigación de enfermedades que perjudican a los stakeholders externos y, cuyo foco son los residuos de la actividad industrial; sin embargo, al efectuar el estudio en las pymes textiles del departamento de Atlántico, Colombia, tal compromiso por la preservación y sostenibilidad ambiental es insuficiente, en primer lugar porque sus acciones son superfluas y estas direccionadas a reparar problemáticas coyunturales. En segundo lugar, porque se centran más en desarrollar acciones para su propio beneficio que para beneficio ambiental, por ejemplo, estas empresas reducen la cantidad de despilfarros para ahorrar recursos y disminuir costos, pero, no reducen el volumen y toxicidad de los insumos que utilizan y que luego se convierten en residuos potencialmente peligrosos para el medio ambiente. Los resultados obtenidos permiten observar que si bien las empresas reconocen la importancia de emplear mecanismos de seguridad ambiental en sus procesos y buscan disminuir el derroche de recursos, aún deben trabajar de mejor manera la reducción de materia prima, contaminación y recursos energéticos que se consumen dentro de estas. Al igual en el empleo de las mejores tecnologías disponibles como base de apoyo para la consecución de estas metas y puntos clave de la Ecoeficiencia. Referencias Abreo, A., Pinzón, B., & Chacón, L. (2018). 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https://iris.unipa.it/bitstream/10447/527842/2/Crespi%20et%20al.%20-%202020%20-%20Unraveling%20Moral%20Reasoning%20in%20Amyotrophic%20Lateral%20Sclerosis%20How%20Emotional%20Detachment%20Modifies%20Moral%20Judgment.pdf
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Unraveling Moral Reasoning in Amyotrophic Lateral Sclerosis: How Emotional Detachment Modifies Moral Judgment
Frontiers in psychology
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BRIEF RESEARCH REPORT published: 21 August 2020 doi: 10.3389/fpsyg.2020.02083 Edited by: Fabio Giovannelli, University of Florence, Italy Reviewed by: Francesca Trojsi, University of Campania Luigi Vanvitelli, Italy Mattia Siciliano, Second University of Naples, Italy *Correspondence: Chiara Crespi chiara.crespi@unipv.it; chiara.crespi@iusspavia.it Specialty section: This article was submitted to Neuropsychology, a section of the journal Frontiers in Psychology Received: 19 May 2020 Accepted: 28 July 2020 Published: 21 August 2020 Unraveling Moral Reasoning in Amyotrophic Lateral Sclerosis: How Emotional Detachment Modifies Moral Judgment Chiara Crespi1*, Gaia Chiara Santi2, Alessandra Dodich3, Federica Lupo4, Lucia Catherine Greco5, Tommaso Piccoli4, Christian Lunetta5 and Chiara Cerami2,6 1 Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy, 2 Istituto Universitario di Studi Superiori, Pavia, Italy, 3 CeRiN, Center for Mind/Brain Sciences, University of Trento, Rovereto, Italy, 4 Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy, 5 NEuroMuscular Omnicentre, Niguarda Ca’ Granda Hospital, Milan, Italy, 6 IRCCS Mondino Foundation, Pavia, Italy In the last decade, scientific literature provided solid evidence of cognitive deficits in amyotrophic lateral sclerosis (ALS) patients and their effects on end-life choices. However, moral cognition and judgment are still poorly investigated in this population. Here we aimed at evaluating both socio-cognitive and socio-affective components of moral reasoning in a sample of 28 ALS patients. Patients underwent clinical and neuropsychological evaluation including basic cognitive and social cognition measures. Additionally, we administered an experimental task including moral dilemmas, with instrumental and incidental conditions. Patients’ performances were compared with a control group [healthy control (HC)], including 36 age-, gender-, and education- matched healthy subjects. Despite that the judgment pattern was comparable in ALS and HC, patients resulted less prone to carry out a moral transgression compared to HC. Additionally, ALS patients displayed higher levels of moral permissibility and lower emotional arousal, with similar levels of engagement in both instrumental and incidental conditions. Our findings expanded the current literature about cognitive deficits in ALS, showing that in judging moral actions, patients may present non-utilitarian choices and emotion flattening. Such a decision-making profile may have relevant implications in applying moral principles in real-life situations and for the judgment of end-of-life treatments and care in clinical settings. In the last decade, scientific literature provided solid evidence of cognitive deficits in amyotrophic lateral sclerosis (ALS) patients and their effects on end-life choices. However, moral cognition and judgment are still poorly investigated in this population. Here we aimed at evaluating both socio-cognitive and socio-affective components of moral reasoning in a sample of 28 ALS patients. Patients underwent clinical and neuropsychological evaluation including basic cognitive and social cognition measures. Additionally, we administered an experimental task including moral dilemmas, with instrumental and incidental conditions. Patients’ performances were compared with a control group [healthy control (HC)], including 36 age-, gender-, and education- matched healthy subjects. Unraveling Moral Reasoning in Amyotrophic Lateral Sclerosis: How Emotional Detachment Modifies Moral Judgment Despite that the judgment pattern was comparable in ALS and HC, patients resulted less prone to carry out a moral transgression compared to HC. Additionally, ALS patients displayed higher levels of moral permissibility and lower emotional arousal, with similar levels of engagement in both instrumental and incidental conditions. Our findings expanded the current literature about cognitive deficits in ALS, showing that in judging moral actions, patients may present non-utilitarian choices and emotion flattening. Such a decision-making profile may have relevant implications in applying moral principles in real-life situations and for the judgment of end-of-life treatments and care in clinical settings. In the last decade, scientific literature provided solid evidence of cognitive deficits in amyotrophic lateral sclerosis (ALS) patients and their effects on end-life choices. However, moral cognition and judgment are still poorly investigated in this population. Here we aimed at evaluating both socio-cognitive and socio-affective components of moral reasoning in a sample of 28 ALS patients. Patients underwent clinical and neuropsychological evaluation including basic cognitive and social cognition measures. Keywords: amyotrophic lateral sclerosis, emotion detachment, moral cognition, moral judgment, social cognition Subjects Twenty-eight patients with a diagnosis of either probable or definite ALS (Brooks et al., 2000) (25 males; mean age = 57.79 ± 11.72 years; mean education = 11.50 ± 4.78 years; mean disease duration from the onset = 1.84 ± 1.47 years) were enrolled (Table 1) for the present study. According to the clinical presentation, ALS patients were classified as 19 spinal and nine bulbar onsets. We excluded patients with global cognitive deficits, as revealed by a mini mental state examination (MMSE) raw score < 23 (Duchesne et al., 2005; Diniz et al., 2007; Mitchell, 2017), or comorbid psychiatric disorders potentially interfering with cognitive functioning, as well as patients with respiratory disorders (forced vital capacity < 70% of predicted capacity), severe dysarthria, and communication difficulties potentially invalidating the administration and/or interpretation of neuropsychological assessment. Morality is defined as the set of values and habits adopted by a cultural group in order to orient its social conduct (Moll et al., 2005). Tasks involving moral dilemmas—i.e., situations where an agent cannot fulfill, with his/her choice, all applicable moral requirements—classically measure the patient’s ability to evaluate actions made in respect to a set of virtues (i.e., “moral judgment”). Moral reasoning is defined as the sum of all mental conscious processes that allows the agent to achieve moral judgment (Moll et al., 2005). From the neuroscience perspective, neurocognitive processes underlying moral reasoning primarily encompass two main dimensions: the socio-cognitive and the socio-affective components (Bzdok et al., 2012). In evaluating a moral dilemma, subjects are often required to define the “moral acceptability/permissibility” of the leading character’s behavior (e.g., completely morally unacceptable or completely morally acceptable), referred to the individual’s ability to evaluate the character’s action as tolerable, according to the personal moral values and habits, and to evaluate the experience considering the relative “emotional valence” (e.g., pleasantness/unpleasantness) and “emotional arousal” (e.g., activation/calm). These emotional dimensions represent those parameters that typically account for most of the variance in the final moral judgment (Lotto et al., 2014). Thirty-six age-, gender-, and education-matched healthy controls (HCs; 25 males; mean age = 52.97 ± 14.03 years; mean education = 9.97 ± 3.17 years) were recruited at local senior community centers. They underwent a clinical interview, a neurologic examination, and a brief neuropsychological assessment in order to test cognitive efficiency. Subjects Medical history positive for neuropsychiatric disorders, positive neurologic examination, MMSE raw score <28 (Measso et al., 1993; Crespi et al., 2014, 2016; Van Patten et al., 2019), as well as verbal and visuospatial delayed memory performances (i.e., Rey Auditory Verbal Learning test, Rey Figure Recall task) below 25th percentile according to the Italian normative values, were considered as exclusion criteria for HC enrollment. A recent paper on moral judgment in ALS and behavioral variant of frontotemporal dementia (bvFTD) (Semler et al., 2019) proved comparable poor patient’s attitude in evaluating a moral situation in both neurodegenerative conditions. In this study, experimental assessment included the Moral Competence Test, the Ethics Position Questionnaire, and the Idler Index of Religiosity to respectively test moral competence, moral position, and religiosity. Impairment in moral judgment was regardless of ethic and religiosity scores and did not correlate with basic cognitive performances (Semler et al., 2019). INTRODUCTION Crespi C, Santi GC, Dodich A, Lupo F, Greco LC, Piccoli T, Lunetta C and Cerami C (2020) Unraveling Moral Reasoning in Amyotrophic Lateral Sclerosis: How Emotional Detachment Modifies Moral Judgment. Front. Psychol. 11:2083. doi: 10.3389/fpsyg.2020.02083 Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative multisystem disorder that selectively targeted the motor system (Hardiman et al., 2017). A variable range of non-motor behavioral and cognitive manifestations affects patients, with a subgroup of ALS developing a frank frontotemporal dementia (FTD) syndrome (Hardiman et al., 2017). Cognitive disorders often characterize the clinical phenotype, with a major involvement of language and executive abilities August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org Moral Cognition in ALS Crespi et al. (Consonni et al., 2013; Beeldman et al., 2016; Strong et al., 2017). Behavioral changes may occur with predominant apathy and depression (Lillo et al., 2011). (Consonni et al., 2013; Beeldman et al., 2016; Strong et al., 2017). Behavioral changes may occur with predominant apathy and depression (Lillo et al., 2011). other neurodegenerative patients (e.g., FTD) investigated the relationship between moral judgment, basic and social cognition abilities (Mendez et al., 2005; Mendez and Shapira, 2009; Gleichgerrcht et al., 2011) showing low performance, compared to control subjects, in a subtask requiring an immediate emotionally driven moral judgment (Mendez et al., 2005). The role of affective and emotional processes on moral decision-making in bvFTD is also supported by results of Gleichgerrcht et al. (2011). p ( ) In the last decade, scientific literature provided evidence of social cognition deficits in ALS, particularly, in the domains of recognition and processing of emotional materials (Lulé et al., 2005; Papps et al., 2005; Zimmerman et al., 2007; Palmieri et al., 2010; Crespi et al., 2014), affective decision- making (Meier et al., 2010; Girardi et al., 2011), and empathy (Gibbons et al., 2007; Cavallo et al., 2011; Cerami et al., 2014). Notwithstanding such a large literature on cognitive deficits and socio-emotional disorders in ALS, moral cognition has been poorly investigated yet. Research studies on the field mainly investigated ethical implications of end-of-life treatment decisions, supporting the independence between basic cognitive and behavioral impairments and patient decisions on the use of invasive medical devices (Böhm et al., 2016). In this view, the literature suggests the need for longitudinal assessment of decision-making skills to early recognize possible alterations and their effects on end-of-life choices (Khin Khin et al., 2015). INTRODUCTION In this view, our research study was aimed at investigating how affective and emotional dimensions influence the decision- making process of ALS patients in a moral dilemmas task. Frontiers in Psychology | www.frontiersin.org Basic Cognitive and Social Assessment of Amyotrophic Lateral Sclerosis Patients Basic cognitive functioning in patients was evaluated with the Italian version of the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) battery. This is a widely used multi-domain brief assessment designed to evaluate a range of cognitive functions typically affected in ALS (i.e., ALS-Specific domains: executive functions, social cognition, fluency, and language) (Poletti et al., 2016). In addition to executive (reverse digit span, The influence of affective parameters on moral judgment processes in ALS patients is a matter of debate. No detailed information about the socio-affective dimension of moral reasoning was available yet. Some studies on August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 2 Moral Cognition in ALS Crespi et al. TABLE 1 | Demographic, clinical, and neuropsychological features of the sample. (A) Demographic and clinical features ALS HC Statistics Female/Male ratio 3/25 11/25 χ2(1) = 0.057 Age in years (mean ± SD) 57.79 ± 11.72 52.97 ± 14.03 t(62) = 0.603 Education in years (mean ± SD) 11.50 ± 4.79 9.97 ± 3.17 t(62) = 0.075 Disease duration in years (mean ± SD) 1.84 ± 1.47 – – Age at the onset (mean ± SD) 55.15 ± 11.54 – – ALS-FRSr (mean ± SD) 29.80 ± 7.18 – – (B) Cognitive and behavioral assessment % out of cutoff score* % borderline score* Mini mental state examination (mean ± SD) 26.29 ± 2.44 (6/28) 21.4% – ECAS-global score (mean ± SD) 106.54 ± 12.49 (4/28) 14.28% (1/28) 3.57% ECAS-specific (mean ± SD) 77.72 ± 11.09 (4/28) 14.28% (3/28) 10.71% ECAS-non-specific (mean ± SD) 28.61 ± 3.0 (1/28) 3.57% (3/28) 10.71% SET global score (mean ± SD) 12.57 ± 3.78 (5/28) 17.9% (3/28) 10.7% SET emotion attribution condition (mean ± SD) 4.50 ± 1.36 (2/28) 7.1% (5/28) 17.9% SET intention attribution condition (mean ± SD) 4.24 ± 1.60 (4/28) 14.3% (6/28) 21.4% SET causal inference condition (mean ± SD) 3.94 ± 1.60 (5/28) 17.9% (4/28) 14.3% Ek-60F global score (mean ± SD) 46.71 ± 6.70 (4/28) 14.3% (2/28) 7.1% The table reports, (A) demographic information for patients and controls, as well as clinical characteristics of the patient group. (B) Illustrates scores obtained from the cognitive and behavioral assessment in patients, reporting mean and standard deviation (SD), the proportion of patients scoring out of the cutoff score and that of patients obtaining a borderline score. Basic Cognitive and Social Assessment of Amyotrophic Lateral Sclerosis Patients ALS, amyotrophic lateral sclerosis; HC, healthy control; ALS-FRSr, ALS functional rating scale revised; ECAS, Edinburgh cognitive and behavioral ALS screen battery; SET, story-based empathy task; Ek-60F, Ekman 60 Faces test. ∗percentage of patients obtaining an impaired/borderline performance was computed according to the Italian normative values. TABLE 1 | Demographic, clinical, and neuropsychological features of the sample. The table reports, (A) demographic information for patients and controls, as well as clinical characteristics of the patient group. (B) Illustrates scores obtained from the cognitive and behavioral assessment in patients, reporting mean and standard deviation (SD), the proportion of patients scoring out of the cutoff score and that of patients obtaining a borderline score. ALS, amyotrophic lateral sclerosis; HC, healthy control; ALS-FRSr, ALS functional rating scale revised; ECAS, Edinburgh cognitive and behavioral ALS screen battery; SET, story-based empathy task; Ek-60F, Ekman 60 Faces test. ∗percentage of patients obtaining an impaired/borderline performance was computed according to the Italian normative values. proved to be sensitive on ALS patients (Girardi et al., 2011; Crespi et al., 2014). alternation, and sentence completion tasks), social cognition (Yoni task), fluency (verbal fluency tasks for words beginning with the letter “S” and for four-letter words starting with the letter “C”), and language (naming, comprehension, and spelling) tasks, ECAS explored also cognitive domains not typically affected in ALS but common in pathological aging [ALS Non-specific domains: memory (immediate recall and delayed recognition) and visuospatial (dot counting, cube counting, and number location)] (Poletti et al., 2016). Neuropsychological assessment also included the MMSE (Folstein et al., 1975) to assess global cognitive efficiency. Primary caregivers were asked to complete the frontal behavioral inventory (FBI) (Kertesz et al., 1997; Alberici et al., 2007) to assess behavioral changes in patients. All subjects gave informed consent to the experimental procedure, which was approved by the local ethics committee. See Table 1 for details on demographic variables. Frontiers in Psychology | www.frontiersin.org Experimental Moral Task The task of moral dilemmas includes eight scenarios derived from Lotto et al. (2014). Each dilemma was presented as text in two phases. The first phase described the scenario in which different kinds of threats were going to cause death to a group of people. The second phase described a hypothetical resolution in which the participant, identifying him/herself as the main character, can choose to kill or not one individual to save the others, who otherwise would have died. Dilemmas were subgrouped into two conditions: “instrumental” (i.e., the death of one person is a mean to save more people) and “incidental” (i.e., the death of one person is a foreseen but unintended consequence of the action aimed at saving more people). Additionally, half of dilemmas involved directly the main character because its life was at risk (i.e., self-involvement). See Supplementary Table S1 for stimulus examples. g p A brief social cognition battery exploring basic emotion recognition and emotion and intention attribution in others was also administered including the Ekman-60-Faces test (Ek- 60F) (Dodich et al., 2014) and the Story-based Empathy Task (SET) (Dodich et al., 2015). The EK-60F consists of 60 b/w pictures from the Ekman and Friesen series of pictures of facial affect. Pictures depict the faces of 10 actors, each displaying one of the six basic emotions (i.e., happiness, sadness, anger, fear, surprise, and disgust). The SET is a non-verbal cartoon task that consists of two main experimental conditions, i.e., identifying intentions (SET-IA) and emotional states (SET-EA), plus a control condition entailing the inference of causality reaction based on the knowledge of the physical properties of objects and human bodies (SET-CI). These tasks have been Participants were asked to indicate whether they would do the proposed action. Then, they were asked to judge how morally acceptable was the resolution (0 = not at all, 7 = completely), to attribute an emotional valence to the moral action (0 = totally unpleasant, 8 = totally pleasant), and to rate the emotional arousal (0 = totally calm, 8 = totally involved). Outcome variables August 2020 | Volume 11 | Article 2083 3 Moral Cognition in ALS Crespi et al. TABLE 2 | Performance at moral judgment task in patients and healthy controls. Experimental Moral Task A Type of Dilemma Significant results Incidental Instrumental Group ALS HC ALS HC Affirmative response% 14.28% 88.8% 7.14% 47.22% Group difference (Incidental): χ2(4) = 36.70, p < 0.001 Group difference (Instrumental): χ2(4) = 14.65, p = 0.005 Moral permissibility 3.15 ± 1.65 2.24 ± 1.50 2.05 ± 2.31 1.07 ± 1.08 Group effect: F(1,62) = 6.09, p < 0.001 Type of dilemma effect: F(1,62) = 46.58, p < 0.001 Emotional arousal 4.93 ± 2.34 6.88 ± 1.32 5.06 ± 2.70 7.59 ± 1.28 Group effect: F(1,62) = 22.33, p < 0.001 Type of dilemma effect: F(1,62) = 13.162, p = 0.001 Interaction effect: F(1,62) = 6.40, p = 0.014 Emotional valence 2.20 ± 1.40 2.22 ± 1.06 2.00 ± 1.20 1.74 ± 0.79 Type of dilemma effect: F(1,62) = 19.96, p < 0.001 B Involvement Self No self Group ALS HC ALS HC Affirmative response% 10.71% 61.11% 10.71% 86.11% Group difference (Self): χ2(4) = 18.25, p = 0.001 Group difference (No self): χ2(4) = 38.38, p < 0.001 Moral permissibility 2.48 ± 1.84 1.73 ± 1.39 2.72 ± 2.06 1.58 ± 1.08 Group effect: F(1,62) = 6.09, p < 0.001 Emotional arousal 5.03 ± 2.60 7.12 ± 1.19 4.96 ± 2.45 7.35 ± 1.37 Group effect: F(1,62) = 22.33, p < 0.001 Emotional valence 2.16 ± 1.37 2.06 ± 0.88 2.04 ± 1.23 1.90 ± 1.02 – The table reports performances at the moral judgment task and significant results obtained from the analysis of variables of interest (i.e., proportion of affirmative responses, moral permissibility, emotional arousal, and emotional valence). While (A) shows results from mixed ANOVA considering the type of dilemma (incidental vs. instrumental) as between variable, (B) reports results from mixed ANOVA considering the personal involvement (self vs. no self-involvement) as between variables. ALS, amyotrophic lateral sclerosis; HC, healthy control. TABLE 2 | Performance at moral judgment task in patients and healthy controls. The table reports performances at the moral judgment task and significant results obtained from the analysis of variables of interest (i.e., proportion of affirmative responses, moral permissibility, emotional arousal, and emotional valence). While (A) shows results from mixed ANOVA considering the type of dilemma (incidental vs. instrumental) as between variable, (B) reports results from mixed ANOVA considering the personal involvement (self vs. no self-involvement) as between variables. ALS, amyotrophic lateral sclerosis; HC, healthy control. Experimental Moral Task Analyses were conducted using IBM SPSS Statistics for Windows v24.0 (IBM Corporation, Armonk, NY, United States). were (i) the rate of yes/no responses to the proposed resolution (i.e., moral judgment); the rating of (ii) moral acceptability, (iii) emotional valence, and (iv) emotional arousal experienced during the decision-making process. Frontiers in Psychology | www.frontiersin.org Moral Cognition Disorders in Amyotrophic Lateral Sclerosis Patients Compared to Healthy Controls The impairments of decision-making processing of moral actions ascribe ALS to a more extensive social cognition dysfunction (Lulé et al., 2005; Papps et al., 2005; Zimmerman et al., 2007; Meier et al., 2010; Palmieri et al., 2010; Cavallo et al., 2011; Girardi et al., 2011; Cerami et al., 2014; Crespi et al., 2014), as also proved by the neuropsychological performances of our sample. About a third of the patient group displayed in fact impaired or borderline performances at emotion recognition and socio-emotional processing tasks. Impairments of such a multifaceted cognitive domain may be underestimated by ECAS battery alone. According to this, an in-depth Concerning the judgment of moral permissibility, the result of a 2 × 2 (type of dilemma condition × group) mixed ANOVA showed a significant effect of both group [F(1,62) = 6.1, p = 0.016] and type of dilemma [F(1,62) = 46.58, p < 0.001] with no interaction effect, indicating significantly higher ratings in ALS patients than HC; higher ratings for incidental compared to instrumental dilemmas characterized both groups (Figure 1). Results from personal involvement × group mixed ANOVA highlighted a significant effect of group [F(1,62) = 6.1, p = 0.016], with ALS patients showing higher ratings than HC. We did not find a significant effect of personal involvement nor an interaction effect with group. About ratings of emotional valence, the result of a 2 × 2 (type of dilemma condition × group) mixed ANOVA showed a significant effect of the type of dilemma [F(1,62) = 19.96, p < 0.001], describing low emotional valence for instrumental dilemmas compared to incidental ones in both groups, while we did not observe significant group and interaction effects. We found no significant effects in personal involvement × group mixed ANOVA. About evaluation of emotional arousal, the result of a 2 × 2 (type of dilemma condition × group) mixed ANOVA showed a significant effect of both group [F(1,62) = 22.33, p < 0.001] and type of dilemma [F(1,62) = 13.16, p = 0.001], as well as a significant interaction effect [F(1,62) = 6.403, p = 0.014], revealing lower ratings in ALS patients than HC and higher ratings for instrumental than incidental dilemmas in both groups, with HC showing a greater difference between instrumental and incidental conditions than ALS (Figure 1). DISCUSSION See Table 1 for further details. Moral cognition is still a poorly investigated domain in ALS. Although previous results suggested that ALS patients presented deficits in integrating their moral values into situational conditions, even if their knowledge of rules and ethics dogma is preserved (Semler et al., 2019), the impact of changes in affective processing on decision-making, and specifically related to the moral judgment, has not been estimated yet. Moral Cognition Disorders in Amyotrophic Lateral Sclerosis Patients Compared to Healthy Controls Overall, the ALS sample showed impairments in moral judgment ability. Chi-Square Independence test highlighted that the global rate of yes/no response in resolution of moral dilemmas was significantly different in ALS compared to HC (Incidental dilemmas: χ2 = 36.70, p < 0.001; Instrumental dilemmas: χ2 = 14.65, p = 0.005; No self-involvement: χ2 = 38.39, p < 0.001; Self-involvement: χ2 = 18.25, p < 0.001). These results were confirmed by one-way ANOVA results, with ALS patients showing significantly lower percentages of utilitarian responses than HC in each condition, both factors considered [i.e., type of dilemma: incidental: F(1,62) = 38.16, p < 0.001; instrumental: F(1,62) = 3.81, p = 0.05; personal involvement: no self-involvement: F(1,62) = 20.36, p < 0.001; self-involvement: F(1,62) = 13.02, p = 0.001]. In the present study, we explored moral cognition in ALS patients using one of the most reliable moral tasks (Lotto et al., 2014) and extensively applied on neuropsychiatric populations (Pletti et al., 2017) and healthy aging (McNair et al., 2018), aiming at assessing moral judgment controlling for different variables (i.e., moral acceptability, emotional valence, and arousal). p y Our findings provided evidence of different moral judgment behaviors in patients compared to HC. In detail, ALS showed significantly lower yes/no rate of responses both in incidental and instrumental conditions, also considering dilemmas on the basis of personal involvement. At the same time, patients displayed higher levels of moral permissibility than HC, reflecting an overall tendency to judge as more acceptable actions that they would not actually perform. Such a performance may reflect a defective evaluation of the consequences of the moral transgression committed in the scenario. Indeed, patients showed less utilitarian behaviors and more emotion-based evaluations compared to healthy subjects. Although moral judgment is considered to occur quickly as an intuitive automatic response, based on deontological principles rather than being a conscious and rational reasoning process that follows a careful evaluation of the moral situation (Haidt, 2001), the definition of the acceptability of moral actions entails the engagement of specific decision-making competences to calculate the preferable solution (Bretz and Sun, 2018). Rational calculation of moral actions usually results in utilitarian solutions (the greatest good for the greatest number), while emotion-based evaluations imply taking non-utilitarian behaviors, as seen in our patient sample. Preserved social cognition skills are thus crucial to maintain the best efficiency in affective decision- making processes and in moral cognition. Statistical Analyses Basic Cognitive and Social Impairments in Amyotrophic Lateral Sclerosis Patients Six out of the 28 patients (21.4%) had an impaired performance on the MMSE, scoring under the cutoff. Four out of 28 ALS patients (14.28%) showed significant impairments in the ECAS global score, and one patient had a borderline score. Four of these five patients with impaired and/or borderline ECAS global score presented impaired and/or reduced performance at ECAS ALS-specific cognitive domains and at social cognition tests. Seven additional patients (25%) showed isolated impaired or borderline scores at the ECAS ALS-specific cognitive domains. Only one patient showed significant impairments at the ECAS ALS non-specific cognitive domains, while three others obtained a borderline performance at ALS non-specific tasks. Impairments of Ek-60F and SET occurred either isolated or combined in patients. Global EK-60F or SET scores were impaired or borderline in half of the patient sample (50%, 14/28). FBI was completed by 20/28 caregivers, showing mild-to-moderate behavioral changes in patients. Increased negative (6.42 ± 6.38) and positive (3.11 ± 3.17) symptoms were both reported, with a prevalence of symptoms as apathy, emotional flatness, loss We explored patients’ performances at the moral dilemmas task in comparison to the control group. Preliminarily, we explored the distribution for each variable with the Kolmogorov–Smirnov test. Although some variables did not show a normal distribution, we analyzed data by testing parametric models as well (one-way ANOVA, mixed ANOVA) according to the results from Blanca et al. (2017). First, Chi-Square Independence test was used to analyze group differences in rate of yes/no responses at moral dilemmas for the diverse task conditions of both type of dilemma (instrumental or incidental conditions) and personal involvement factors (self- involvement or no self-involvement conditions). We additionally performed one-way ANOVA on percentages of yes/no responses to provide a better description of the pattern of utilitarian responses in ALS vs. HC. Mixed-ANOVA models were then computed to detect differences in the rating of moral permissibility, emotional valence, and emotional arousal experienced during the decision- making process, considering the type of dilemma and the personal involvement as within-subject variables and the group (ALS and HC) as a between-subject variable. August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 4 Crespi et al. Moral Cognition in ALS of insight, and inflexibility. According to the criteria by Strong et al. Statistical Analyses (2017), six patients (21.5%) were classified as pure ALS, six (21.5%) as ALSci, four (14%) as ALSbi, and four (14%) as ALScbi. Eight patients (29%) were not classifiable as their caregivers did not complete the FBI. not find a significant effect of the personal involvement nor an interaction effect with group. See Table 2 for further details. Frontiers in Psychology | www.frontiersin.org Moral Cognition Disorders in Amyotrophic Lateral Sclerosis Patients Compared to Healthy Controls Results from personal involvement × group mixed ANOVA highlighted a significant effect of group [F(1,62) = 22.33, p < 0.001] with ALS patients showing lower ratings than HC. We did August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 5 Moral Cognition in ALS Crespi et al. FIGURE 1 | Significant differences in moral permissibility and emotional arousal in amyotrophic lateral sclerosis (ALS) patients compared to healthy controls (HCs). The figure depicts, on the left, the significant group (*) and type of dilemma (**) effects for the moral permissibility variable, showing significantly higher ratings in ALS patients than HC and higher ratings for incidental compared to instrumental dilemmas in both groups. On the right, we report significant group (*), type of dilemma (**) and group × type of dilemma interaction (***) effects for the emotional arousal variable, highlighting lower ratings in ALS patients than HC and higher ratings for instrumental than incidental dilemmas in both groups, with HC showing a greater difference between instrumental and incidental conditions than ALS patients. FIGURE 1 | Significant differences in moral permissibility and emotional arousal in amyotrophic lateral sclerosis (ALS) patients compared to healthy controls (HCs). The figure depicts, on the left, the significant group (*) and type of dilemma (**) effects for the moral permissibility variable, showing significantly higher ratings in ALS patients than HC and higher ratings for incidental compared to instrumental dilemmas in both groups. On the right, we report significant group (*), type of dilemma (**) and group × type of dilemma interaction (***) effects for the emotional arousal variable, highlighting lower ratings in ALS patients than HC and higher ratings for instrumental than incidental dilemmas in both groups, with HC showing a greater difference between instrumental and incidental conditions than ALS patients. assessment of social cognition performances in ALS should be systematically promoted. alterations in brain regions normally recruited for the elaboration of socio-emotional stimuli and thus impacting on judgments about moral permissibility of fictitious scenarios (Bzdok et al., 2012). Our evidence on the emotion arousal subtask of moral dilemmas further confirmed what was previously stated. HC showed higher emotional arousal ratings for both incidental and instrumental conditions compared to ALS patients, with a greater arousal for incidental vs. instrumental dilemmas. Patients showed similar levels of emotional engagement in both conditions. CONCLUSION Although the small sample size, the cross-sectional design, and the lack of neuroimaging markers of moral changes limit the generalizability of our findings, we expand current literature (Semler et al., 2019) suggesting for the first time that the presence of deficits in emotion recognition and mental state attribution abilities, and emotional flattening (socio-affective component of moral decision-making) may influence the attitude in evaluating a moral situation (socio-cognitive component of moral decision-making) in ALS patients. Moreover, this study contributes to define the extent and boundaries of the frontotemporal cognitive syndrome in ALS. Although the small sample size, the cross-sectional design, and the lack of neuroimaging markers of moral changes limit the generalizability of our findings, we expand current literature (Semler et al., 2019) suggesting for the first time that the presence of deficits in emotion recognition and mental state attribution abilities, and emotional flattening (socio-affective component of moral decision-making) may influence the attitude in evaluating a moral situation (socio-cognitive component of moral decision-making) in ALS patients. Moreover, this study contributes to define the extent and boundaries of the frontotemporal cognitive syndrome in ALS. From a cognitive neuroscience perspective, brain substrates underlying cognitive and affective facets of moral cognition (e.g., temporoparietal junction, medial prefrontal cortex, middle temporal gyrus) are common to nodes included in brain networks of both theory of mind (ToM) and empathy (Bzdok et al., 2012). In particular, brain atrophy and changes in white matter integrity in structures supporting socio- affective skills (i.e., ventromedial prefrontal and temporo-limbic cortex and right ventral associative bundles) have proven to occur early in ALS (Cerami et al., 2014; Crespi et al., 2014, 2016). These findings are in line with neuropsychological findings of the present and previous studies indicating reduced emotional engagement, emotion recognition, and attribution in ALS patients (Lulé et al., 2005; Papps et al., 2005; Zimmerman et al., 2007; Palmieri et al., 2010; Girardi et al., 2011; Crespi et al., 2014). Indeed, significant differences in patients compared to controls in the socio-affective component of moral reasoning might reflect anatomo-functional Besides its theoretical interest, deficits in the judgment of moral actions entail crucial practical implications for the management of neurological patients and should be appropriately taken into account in the evaluation of therapeutic approaches and critical life choices. Moral Cognition Disorders in Amyotrophic Lateral Sclerosis Patients Compared to Healthy Controls These findings are suggestive of an incipient emotional blunting, which can be clinically observed in a proportion of ALS patients, also at early stages (Phukan et al., 2007; Strong et al., 2009), and that can interfere with the ability to modulate the application of moral principles in a consistent and nuanced manner to the different social situations. REFERENCES F., Folstein, S. E., and McHug, H. P. R. 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DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. SUPPLEMENTARY MATERIAL The studies involving human participants were reviewed and approved by the San Raffaele Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2020.02083/full#supplementary-material CONCLUSION Further studies based on clinical settings are needed to confirm our data and estimate in ALS the weight of moral judgment alterations and their relationship with the quality of life and socio- affective disorders at the individual level, particularly with regard to gender effect and disease subtype. Overall, the variable vulnerability to social and non-social cognitive changes observed in ALS patients indicates that larger August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 6 Moral Cognition in ALS Crespi et al. AUTHOR CONTRIBUTIONS longitudinal studies are also needed to estimate the impact of moral cognition impairments in real-life situations (e.g., end- life decisions). CCr: conceptualization, formal analysis, writing – original draft, and visualization. GS: investigation, formal analysis, and writing – reviewing and editing. AD: investigation and writing – reviewing and editing. FL and LG: investigation and resources. TP and CL: investigation and supervision. CCe: conceptualization, project administration, supervision, investigation, data curation, and writing – reviewing and editing. All authors contributed to the article and approved the submitted version. REFERENCES Diniz, B. S., Yassuda, M. S., Nunes, P. 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Neurol. 257, 1970–1978. doi: 10.1007/s00415-010-5640-2 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Papps, B., Abrahams, S., Wicks, P., Leigh, P. N., and Goldstein, L. H. (2005). Changes in memory for emotional material in amyotrophic lateral sclerosis (ALS). Neuropsychologia 43, 1107–1114. doi: 10.1016/j.neuropsychologia.2004. 11.027 Copyright © 2020 Crespi, Santi, Dodich, Lupo, Greco, Piccoli, Lunetta and Cerami. 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. Frontiers in Psychology | www.frontiersin.org August 2020 | Volume 11 | Article 2083 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Crespi, Santi, Dodich, Lupo, Greco, Piccoli, Lunetta and Cerami. 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. Phukan, J., Pender, N. P., and Hardiman, O. (2007). Cognitive impairment in amyotrophic lateral sclerosis. Lancet Neurol. 6, 994–1003. Pletti, C., Lotto, L., Buodo, G., and Sarlo, M. (2017). It’s immoral, but I’d do it! Psychopathy traits affect decision-making in sacrificial dilemmas and in everyday moral situations. Br. J. Psychol. 108, 351–368. doi: 10.1111/bjop.12205 August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 8
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Propagation of a viscous gravity current beneath a granular mush
Journal of fluid mechanics
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10,509
J. Fluid Mech. (2024), vol. 985, A18, doi:10.1017/jfm.2024.305 J. Fluid Mech. (2024), vol. 985, A18, doi:10.1017/jfm.2024.305 ( ), , , j Propagation of a viscous gravity current beneath a granular mush Edward M. Hinton1,† and Anja C. Slim2,3 1School of Mathematics and Statistics, The University of Melbourne, Victoria 3010, Australia 2School of Mathematics, Monash University, Victoria 3800, Australia 3School of Earth, Atmosphere and Environment, Monash University, Victoria 3800, Australia (Received 8 September 2023; revised 24 March 2024; accepted 25 March 2024) Edward M. Hinton1,† and Anja C. Slim2,3 1School of Mathematics and Statistics, The University of Melbourne, Victoria 3010, Australia 2School of Mathematics, Monash University, Victoria 3800, Australia 3School of Earth, Atmosphere and Environment, Monash University, Victoria 3800, Australia (Received 8 September 2023; revised 24 March 2024; accepted 25 March 2024) (Received 8 September 2023; revised 24 March 2024; accepted 25 March 2024) The two-dimensional gravity-driven motion of a relatively dense viscous liquid at the base of a granular mush is investigated using a model that exploits the relative shallowness of the flow. The granular mush obeys a μ(I)-rheology, and we assume that the two phases are segregated throughout the motion. The viscous liquid spreads under gravity, carrying the granular mush above and transporting it outwards as levees at either end of the flow. The accumulation of granular material away from the centre of the deposit produces hydrostatic pressure gradients that retard the viscous gravity current. At later times, the granular mush is quasi-static relative to the moving liquid owing to the balance of outward granular transfer by the liquid and inward hydrostatic pressure gradients associated with the granular free surface. The viscous liquid exhibits a Poiseuille-like flow structure with negligible velocity at both the base and the granular interface. The flow of a fixed volume of viscous liquid becomes self-similar with the effective viscosity quadrupled relative to a classical viscous gravity current owing to the retarding effects of the granular mush. The case of constant input flux of viscous liquid is also analysed. The qualitative features are akin to the fixed volume case with the granular mush forming levees and slowing the viscous spreading. The case in which the upper medium is a Bingham material rather than a granular mush is also discussed, and the same features are observed, demonstrating the importance of the yield criterion in the upper medium. Key words: gravity currents, magma and lava flow © The Author(s), 2024. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/ licenses/by/4.0), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. 985 A18-1 1. Introduction Models of the gravity-driven motion of a viscous liquid have been used to describe and interpret a range of geophysical phenomena, including lava flows, magma chambers and † Email address for correspondence: edward.hinton@unimelb.edu.au † Email address for correspondence: edward.hinton@unimelb.edu.au 985 A18-1 985 A18-1 E.M. Hinton and A.C. Slim xˆ g Levee Granular mush Viscous liquid hˆu (xˆ, tˆ) Hˆ (xˆ, tˆ) hˆl (xˆ, tˆ) zˆ Figure 1. Schematic diagram illustrating two-dimensional gravity-driven flow of a viscous liquid beneath a granular mush. The red lines and arrows show a typical flow profile. E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim Levee Granular mush Figure 1. Schematic diagram illustrating two-dimensional gravity-driven flow of a viscous liquid beneath a granular mush. The red lines and arrows show a typical flow profile. glaciers (Huppert 1986). The classical model of the spreading of a viscous liquid on a horizontal or inclined plane (Huppert 1982; Lister 1992), has been extended to account for many geophysically relevant features, including cooling (Lyman, Kerr & Griffiths 2005), a yield stress (Balmforth, Craster & Sassi 2002), and the effect of a second overlying fluid that can deform during the motion (Kowal & Worster 2015). Here, we analyse the gravity-driven spreading of a viscous liquid at the base of a granular mush of finite thickness, which is transported and deformed by the viscous motion; see figure 1. This problem has fundamental fluid dynamical interest as a novel two-fluid gravity current of materials with distinct rheological properties. However, the study is also motivated by the formation of massive sulphide ores in magmatic systems (Hinton & Slim 2023), and has a secondary application to layered mafic intrusions (Maier, Barnes & Groves 2013; Liu et al. 2014). Massive sulphide ore deposits form small portions of basaltic magmatic intrusions within the Earth’s crust. They are thought to have formed from metal-sulphide-rich liquid droplets within the dominant silicate melt, coalescing and depositing in particular parts of the magmatic plumbing system (Robertson, Barnes & Le Vaillant 2016). Transport of the sulphide-rich liquid occurs as the system cools, and hence involves interaction with a mush of silicate melt and silicate crystals. Of particular interest is the final location of this sulphide-rich liquid, thus understanding the transport within the crystal-rich environment is key. 1. Introduction y To capture the dominant processes associated with a coalesced volume of dense sulphide-rich liquid interacting with a mixture of silicate crystals and liquid, we model the system as a two-dimensional shallow isothermal flow. The sulphide-rich liquid is represented by a relatively dense viscous liquid, whilst the silicate crystals within the silicate liquid are modelled by a continuum granular mush (see figure 1). Importantly, the two liquids are different, and the silicate crystals within the magma are non-wetting to the sulphide liquid (Mungall & Su 2005). Hence we assume that there is no interpenetration across the sharp interface between the viscous liquid and the granular mush. The granular mush is assumed to obey a μ(I)-rheology, which has been shown to capture accurately many experimental features of granular flows (GDR-MiDi 2004; Gray & Edwards 2014). Recently, Hinton & Slim (2023) investigated the spreading of a viscous gravity current atop a dense granular mush. This was motivated by the same magmatic system but with the sulphide liquid remaining atop the silicate mush and unable to penetrate downwards owing to the capillary entry pressure. They found that the viscous liquid initially erodes the underlying granular mush into levees. Subsequently, the top part of the levee is pushed 985 A18-2 Viscous gravity current beneath a granular mush outwards by the viscous liquid, with the remnants of the levees trapping a significant fraction of the liquid. In the present work, we consider the opposite configuration, with the relatively dense viscous liquid lying at the bottom of the granular mush. The lower boundary (at ˆz = 0) is impermeable and corresponds to the host country rock bounding the magma chamber. Both configurations are geologically relevant as the dense liquid sulphide may penetrate the mush or become trapped above, depending on its thickness (Naldrett 1973; Chung & Mungall 2009). Although this leads to substantially different dynamics, there are some analogies between the two flows, particularly the relatively fast emergence of granular levees either side of the liquid, which subsequently decay in size. The shallow model that we deploy builds upon studies of two-liquid gravity-driven flows (Kliakhandler & Sivashinsky 1997; Balmforth, Craster & Toniolo 2003), which have been used recently to investigate the lubrication of ice sheets (Kowal & Worster 2015; Kumar et al. 2021; Christy & Hinton 2023). 1. Introduction These inertialess two-liquid flows have the common feature that the volume flux in each liquid is driven by hydrostatic pressure gradients associated with the liquid thicknesses. The flux in the upper fluid also includes a term associated with the lubrication provided by the lower liquid (the lower liquid effectively carries the upper liquid along with it), which is key in the present work. Two-liquid flows with different rheology or densities of the two media can give rise to various instabilities (Balmforth et al. 2003; Leung & Kowal 2022), but here we neglect any such behaviour, noting that our solutions could be used as the base state for future stability analysis. g y y The model is presented in § 2. In § 3, we analyse the evolution of a fixed volume of viscous liquid, and show that the behaviour is self-similar at late times, with the fluid spreading at a quarter of the rate of a classical viscous gravity current (Huppert 1982). The case of constant input of viscous liquid is explored in § 4, and conclusions are given in § 5. Appendix A provides a scaling analysis for the model, and Appendix B discusses the case of an overlying Bingham material (rather than a granular mush), for which almost identical dynamics occurs. 2. Model formulation We analyse the two-dimensional motion of a viscous liquid of density ˆρl and viscosity ˆη displacing a granular mush with μ(I)-rheology; see figure 1. There is no interpenetration of the viscous liquid into the granular mush. The granular mush consists of ambient liquid of density ˆρa and grains of density ˆρg > ˆρa. The solids fraction is denoted by φ so that the bulk density ˆρu of the granular mush is ˆρu = ˆρa(1 −φ) + ˆρgφ, (2.1) (2.1) and we assume that ˆρu < ˆρl (i.e. the granular mush is lighter than the viscous liquid). There is an impermeable horizontal boundary at ˆz = 0. The viscous liquid occupies 0 < ˆz < ˆhl(ˆx,ˆt), whilst the granular mush occupies ˆhl(ˆx,ˆt) < ˆz < ˆH(ˆx,ˆt). The vertical extent of the granular mush is ˆhu(ˆx,ˆt) = ˆH(ˆx,ˆt) −ˆhl(ˆx,ˆt). Throughout the paper, we use subscripts u and l to refer to quantities related to the upper and lower media, respectively, and we use ˆ· to indicate dimensional or unscaled quantities. The flow is assumed to be shallow (a scaling analysis is given in Appendix A). The pressure in each medium is thus hydrostatic and given by (Huppert 1982) ˆpu = ˆρuˆg( ˆH −ˆz), for ˆhl < ˆz < ˆH, (2.2) ˆpl = ˆρuˆgˆhu + ˆρlg(ˆhl −ˆz), for0 < ˆz < ˆhl. (2.3) (2.2) (2.3) 985 A18-3 E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim The leading-order momentum balance (∂ˆτ/∂ˆz = ∂ˆp/∂ˆx) then furnishes the shear stress ˆτ in each medium: ˆτu = −ˆρuˆg( ˆH −ˆz) ∂ˆH ∂ˆx , ˆhl < ˆz < ˆH, (2.4) ˆτl = ˆη ∂ˆul ∂ˆz = −ˆρuˆg  ˆhu ∂ˆH ∂ˆx +  D ∂ˆhl ∂ˆx + ∂ˆH ∂ˆx  (ˆhl −ˆz)  , 0 < ˆz < ˆhl, (2.5) (2.4) (2.5) where we have used continuity of the shear stress at the interface between the media, ˆz = ˆhl, and vanishing shear stress at the free surface, ˆz = ˆH, and introduced the density ratio D = ˆρl −ˆρu ˆρu > 0. (2.6) (2.6) Integrating (2.5) with respect to ˆz furnishes the velocity within the viscous liquid (Kowal & Worster 2015): ˆul = −ˆρuˆg ˆη  ∂ˆH ∂ˆx ˆhuˆz +  D ∂ˆhl ∂ˆx + ∂ˆH ∂ˆx  ˆz  ˆhl −1 2 ˆz  , (2.7) (2.7) where we have used no slip at ˆz = 0. 2. Model formulation The constitutive model for the granular mush is provided by the μ(I)-rheology with (GDR-MiDi 2004; Gray & Edwards 2014) The constitutive model for the granular mush is provided by the μ(I)-rheology with (GDR-MiDi 2004; Gray & Edwards 2014) ˆτu = μ(I) ˆpu sgn ∂ˆuu ∂ˆz  . (2.8) (2.8) For the relatively slow flows considered in the present work, the relationship between the inertial number I and the effective friction coefficient μ is linearised to obtain (Da Cruz et al. 2005; Kamrin & Koval 2012) For the relatively slow flows considered in the present work, the relationship between the inertial number I and the effective friction coefficient μ is linearised to obtain (Da Cruz et al. 2005; Kamrin & Koval 2012) I = max(0, μ −μs) b , where I =  ∂ˆuu ∂ˆz  ˆm/ˆpu, (2.9) (2.9) and μs is the minimum friction coefficient for which the granular mush deforms; μs is assumed to be small (for further discussion, see Appendix A). The parameter b is a dimensionless constant, and ˆm is the grain mass (per unit length in the third dimension). Substituting (2.2) and (2.4) into (2.8), we find that μ(I) is independent of the ˆz coordinate and given by (Gray & Edwards 2014) μ(I) =  ∂ˆH ∂ˆx  . (2.10) (2.10) The definition of I in (2.9) then furnishes the relation The definition of I in (2.9) then furnishes the relation  ∂ˆuu ∂ˆz  ˆm ˆpu = max  0,  ∂ˆH ∂ˆx  −μs  b . (2.11) 985 A18-4 (2.11) 985 A18-4 Viscous gravity current beneath a granular mush Equation (2.11) is integrated with respect to ˆz to obtain the velocity profile in the granular mush: Equation (2.11) is integrated with respect to ˆz to obtain the velocity profile in the granular mush: ˆuu = −2 ˆρuˆg 3b √ ˆm ˆh3/2 u −( ˆH−ˆz)3/2 ˆF  ∂ˆH ∂ˆx  −ˆρuˆg ˆη  ∂ˆH ∂ˆx ˆhuˆhl +  D ∂ˆhl ∂ˆx + ∂ˆH ∂ˆx  1 2 ˆh2 l  , (2 12) where we have used continuity of velocity at the interface, ˆz = ˆhl, utilising (2.7) (there is no interpenetration of viscous liquid into the granular medium), and we have introduced the function ˆF  ∂ˆH ∂ˆx  = sgn  ∂ˆH ∂ˆx  max  0,  ∂ˆH ∂ˆx  −μs  . 2.1. Non-dimensionalisation 2.1. Non-dimensionalisation Initially, the two media combined have thickness ˆa, which motivates the non- dimensionalisation Initially, the two media combined have thickness ˆa, which motivates the non- dimensionalisation (z, hl, hu, H) = (ˆz, ˆhl, ˆhu, ˆH)/ˆa, x = ˆxμs/ˆa, t = ˆt ˆρuˆgˆaμ2 s/ˆη, (2.18a–c (2.18a–c) (2.18a–c) where ˆt has been scaled with the time scale for viscous deformation. The horizontal length scale is chosen to remove the parameter μs from the dimensionless model, and thus make the critical slope for yielding of the granular layer equal to unity. The dimensionless velocities in each medium ((2.7) and (2.12)) take the forms ul = −1 2  2 ∂H ∂x huz +  D ∂hl ∂x + ∂H ∂x  z (2hl −z)  , (2.19) uu = −5 3 K h3/2 u −(H −z)3/2 F ∂H ∂x  −1 2  2 ∂H ∂x huhl +  D ∂hl ∂x + ∂H ∂x  h2 l  , (2.20) (2.19) 3  ∂x  2  ∂x  ∂x ∂x   (2.20)       (2.20) where where where F ∂H ∂x  = sgn ∂H ∂x  max  0,  ∂H ∂x  −1  (2.21) and and K = 2ˆη 5b ˆm ˆρuˆgˆa , (2.22) (2.22) which is the ratio of the viscosity of the Newtonian liquid, ˆη, to the effective ‘viscosity’ of the granular mush (which quantifies the resistance of the granular mush to deformation when the yield criterion is exceeded, i.e. |∂H/∂x| > 1). The dimensionless volume fluxes ((2.14) and (2.15)) become Ql = −  ∂H ∂x huh2 l 2 +  D ∂hl ∂x + ∂H ∂x  h3 l 3  , (2.23) Ql = −  ∂H ∂x huh2 l 2 +  D ∂hl ∂x + ∂H ∂x  h3 l 3  , (2.23) Qu = −Kh5/2 u F1 ∂H ∂x  −  ∂H ∂x h2 uhl +  D ∂hl ∂x + ∂H ∂x  huh2 l 2  . (2.24) (2.23) Qu = −Kh5/2 u F1 ∂H ∂x  −  ∂H ∂x h2 uhl +  D ∂hl ∂x + ∂H ∂x  huh2 l 2  . (2.24) (2.24) This is combined with dimensionless mass conservation (2.16): ∂hi ∂t + ∂Qi ∂x = 0, for i = u, l. 2. Model formulation (2.13) (2.13) The volume flux in each medium is calculated by integrating the velocity over the thickness: ˆQl = −ˆρuˆg 6ˆη  3 ∂ˆH ∂ˆx ˆhuˆh2 l + 2  D ∂ˆhl ∂ˆx + ∂ˆH ∂ˆx  ˆh3 l  , (2.14) ˆQu = −2 ˆρuˆg 5b √ ˆm ˆh5/2 u ˆF  ∂ˆH ∂ˆx  −ˆρuˆg 2ˆη  2 ∂ˆH ∂ˆx ˆh2 uˆhl +  D ∂ˆhl ∂ˆx + ∂ˆH ∂ˆx  ˆhuˆh2 l  . (2.15) (2.14) The flux in the viscous liquid, ˆQl, is driven by viscous shearing arising from hydrostatic pressure gradients associated with the thickness variations of the two media. The flux in the granular mush, ˆQu, consists of two terms: the first term is associated with shearing of the granular mush, whilst the second term arises from ‘lubrication’ by the viscous liquid (motion in the liquid can carry the granular mush) (cf. Kowal & Worster 2015). Hence the granular mush can move even when |∂ˆH/∂ˆx| does not exceed μs owing to the motion of the underlying viscous liquid; see figure 1. For further discussion regarding motion of the quasi-rigid mush, see Appendix A. Mass conservation in each medium is written as ∂ˆhi ∂ˆt + ∂ˆQi ∂ˆx = 0, for i = u, l. (2.16) (2.16) The model is completed with appropriate initial and boundary conditions; we generally take a constant total thickness ˆH(x, 0) ≡ˆa and consider different starting shapes of the viscous deposit. The model is completed with appropriate initial and boundary conditions; we generally take a constant total thickness ˆH(x, 0) ≡ˆa and consider different starting shapes of the viscous deposit. p We analyse two distinct cases: (i) a fixed volume of viscous liquid (§ 3), and (ii) a constant input flux of viscous liquid (§ 4). In each case, global mass conservation of the viscous liquid takes the form https://doi.org/10.1017/jfm.2024.305 Pu ˆxf −ˆxf ˆhl dˆx = ˆA0 (fixed volume), ˆq0ˆt (constant input flux), (2.17) (2.17) where ˆxf (ˆt) is the distance of the tip of the viscous layer from the origin. where ˆxf (ˆt) is the distance of the tip of the viscous layer from the origin. 985 A18-5 985 A18-5 E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim 2.1. Non-dimensionalisation Viscous gravity current beneath a granular mush –1 0 x 1 –1 0 x 1 –1 0 x 1 –1 0 1 –1 0 1 –1 0 1 –1 0 1 –1 0 1 –1 0 1 1.0 (a) (b) (c) (d) (e) ( f ) (g) (h) (i) 0.5 Granular mush Granular mush z 0 Liquid 1.0 0.5 z 0 1.0 0.5 z 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 Figure 2. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K = 1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.1). (b–i) The interface shapes at t = 1, 5, 20, 30, 35, 40, 100, 250. The red arrows in (e,h) show the flow directions schematically; see also figure 3 and the discussion in the text. –1 0 1 (b) (e) 1.0 0.5 0 –1 0 1 (c) ( f ) 1.0 0.5 0 ( f ) –1 0 1 ( f ) (i) 1.0 0.5 0 (i) –1 0 x 1 (g) 1.0 0.5 z 0 –1 0 x 1 1.0 0.5 0 Figure 2. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K = 1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.1). (b–i) The interface shapes at t = 1, 5, 20, 30, 35, 40, 100, 250. The red arrows in (e,h) show the flow directions schematically; see also figure 3 and the discussion in the text. 2.1. Non-dimensionalisation (2.25) (2.25) Equation (2.25) is integrated numerically using finite differences with an appropriate initial condition; the method is described in the appendix of Hinton & Slim (2023). Equation (2.25) is integrated numerically using finite differences with an appropriate initial condition; the method is described in the appendix of Hinton & Slim (2023). Finally, global mass conservation (2.17) becomes Finally, global mass conservation (2.17) becomes xf −xf hl dx =  A (fixed volume), see § 3, Qt (constant input flux), see § 4, (2.26) (2.26) where A = ˆA0μs ˆa2 , Q = ˆq0 ˆη ˆa3 ˆρuˆgμs . (2.27a,b) (2.27a,b) 985 A18-6 Viscous gravity current beneath a granular mush 3. Fixed volume of viscous liquid Parameter values and initial condition are as in figure 2, with (a) t = 5 corresponding to figure 2(c), (b) t = 40 corresponding to figure 2(g), and (c) t = 250 corresponding to figure 2(i). the origin and the crest of the levee in the combined height profile. This provides an adverse contribution to the hydrostatic pressure gradient, decelerating the viscous flow and in particular reducing the outward flow velocity near the viscous–granular interface; see figure 3. Eventually, the slumping liquid detaches from the upper free surface as the central granular material moves slowly inwards; see figures 2 and 3. (This was handled numerically by initially adding an artificial film of granular material of thickness 10−4 over the entire region of the viscous liquid, which ensures that in the numerical method, there is no transition from two-layer flow to one-layer flow; the resulting solution was not sensitive to the thickness of this granular film.) Subsequently, the viscous liquid continues to thin and spread laterally, pushing the levees outwards. The perturbation to the upper free surface diminishes in time owing to the lateral spreading, and the levees are also reduced in size. The small gradients in the upper free surface continue to retard the viscous deformation, even at late times. The system becomes self-similar at late times, which is analysed in § 3.1. To investigate the sensitivity to the details of the initial condition, figure 4 shows the flow evolution with a semicircular initial condition (and D = 0.5, K = 1) given by hl(x, 0) =  √ 1 −x2, |x| < 1, 0, otherwise, (3.2) (3.2) which is shown in figure 4(a). Comparison of figures 2 and 4 demonstrates that the system generally passes through the same stages regardless of the initial shape. Indeed, figures 2(e) and 4(e) are very similar, and the evolution is almost identical at later times. (This is associated with late-time self-similar behaviour that ‘forgets’ the initial conditions; see § 3.1.) The effect of varying the density ratio D is demonstrated in figure 5. The qualitative behaviour of the flow is unchanged with different values of D. Larger D is associated with faster flow because the motion of both media is driven primarily by the gravity-driven spreading of the liquid. However, there is no simple quantitative rescaling of time with D. 3. Fixed volume of viscous liquid Numerical results for the spreading of a fixed volume of relatively dense viscous liquid beneath a granular mush are shown in figure 2 with D = 0.5, K = 1. The initial shape takes the form hl(x, 0) = 1 2 [1 −tanh(50(|x| −0.5))] , (3.1) (3.1) which is shown in figure 2(a). This is an approximation to the Heaviside function. (The Heaviside function is slightly smoothed for efficient numerical integration, but we note that steeper initial profiles lead to imperceptible changes in the results.) The initial shape of the upper free surface is H(x, 0) = hl(x, 0) + hu(x, 0) = 1. Figure 2 shows that initially, the viscous liquid spreads outwards driven by hydrostatic pressure gradients associated with the thickness of the viscous liquid, D ∂hl/∂x. Although there is negligible deformation in the granular mush initially (with |∂H/∂x| < 1), the outward motion of the liquid carries the granular mush outwards; see figures 2(a,b). Granular material is piled into levees above the edges of the viscous liquid; see figure 2(b). These levees grow initially with their outer slope being quasi-rigid, |∂H/∂x| ≈1. Figure 3 shows the horizontal velocity fields for figures 2(c,g,i). The outward carrying of the crest of the levees by the liquid can be seen in the thin vertical slices of high velocity at edges of the liquid in figure 3. The combination of outward transfer of granular material in the levee and inward collapse of granular material above the viscous liquid creates a positive slope between 985 A18-7 E.M. Hinton and A.C. Slim 1.0 (a) 0.02 0 –0.02 0.5 z 0 –0.5 0x 0.5 1.0 (a) (b) (c) 0.02 0 –0.02 0.02 0 –0.02 2 0 u –2 0.5 z 0 –0.5 0x 0.5 –1 0x 1 –1 0x 1 1.0 0.5 0 1.0 0.5 0 (×10–3) Figure 3. Horizontal flow velocity u in each medium obtained using (2.19) and (2.20). Parameter values and initial condition are as in figure 2, with (a) t = 5 corresponding to figure 2(c), (b) t = 40 corresponding to figure 2(g), and (c) t = 250 corresponding to figure 2(i). (c) 2 0 u –2 –1 0x 1 1.0 0.5 0 (×10–3) (b) 0.02 0 –0.02 –1 0x 1 1.0 0.5 0 (b) (c) (a) Figure 3. Horizontal flow velocity u in each medium obtained using (2.19) and (2.20). 3. Fixed volume of viscous liquid Changes in the relative granular viscosity K have negligible influence on the results because the granular material is rigid or quasi-rigid everywhere. Viscous gravity current beneath a granular mush 1.0 (a) (d) 0.5 z 0 –2 0 2 1.0 (a) (d) (g) (b) (e) (c) ( f ) (h) (i) 0.5 z 0 1.0 0.5 z 0 1.0 0.5 z 0 –2 0 x 2 –2 0 x 2 –2 0 x 2 –2 0 2 –2 0 2 –2 0 2 –2 0 2 –2 0 2 –2 0 2 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 Figure 4. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K = 1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.2). (b–i) The interface shapes at t = 1, 5, 20, 40, 60, 80, 150, 300. The red arrows in (a,d,g) show the flow directions schematically. (b) (e) (c) ( f ) 2 –2 0 2 –2 0 2 1.0 0.5 0 1.0 0.5 0 (a) (d) (e) ( ) ( f ) (h) (i) 2 –2 0 2 –2 0 2 1.0 0.5 0 1.0 0.5 0 (h) (i) (g) (i) –2 0 x 2 1.0 0.5 0 (g) 1.0 0.5 z 0 –2 0 x 2 ) –2 0 x 2 1.0 0.5 0 Figure 4. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K = 1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.2). (b–i) The interface shapes at t = 1, 5, 20, 40, 60, 80, 150, 300. The red arrows in (a,d,g) show the flow directions schematically. 1.0 (a) (b) (c) 0.5 z 0 –1 0 x 1 –1 0 x 1 –1 0 x 1 1.0 0.5 0 1.0 0.5 0 Figure 5. Flow evolution for three different values of the density ratio D with K = 1: D = 0.25 (green lines), D = 0.5 (black lines) and D = 1 (red lines). The initial condition is (3.1) (as in figure 2). Times are (a) t = 1, (b) t = 10, and (c) t = 100. 1.0 (a) ( 0.5 z 0 –1 0 x 1 (c) –1 0 x 1 1.0 0.5 0 b) –1 0 x 1 1.0 0.5 0 (a) (c) (b) Figure 5. 3.1. Late-time self-similar behaviour For a wide range of initial conditions and parameter values, the late-time evolution becomes self-similar, with the viscous liquid spreading outwards and the perturbation 985 A18-8 For a wide range of initial conditions and parameter values, the late-time evolution becomes self-similar, with the viscous liquid spreading outwards and the perturbation Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush Thus the velocity at the top of the viscous liquid is approximately zero. y p q pp y Using (2.19), the flow velocity at the interface is Using (2.19), the flow velocity at the interface is ul(z = hl) = −∂H ∂x huhl −  D ∂hl ∂x + ∂H ∂x  h2 l 2 ≈0. (3.3) (3.3) This condition provides a relation between the two thickness gradients, ∂hl/∂x and ∂H/∂x, which ensures that the granular mush is quasi-static. Equation (3.3) can be used to rewrite the velocity in the viscous liquid (2.19) as This condition provides a relation between the two thickness gradients, ∂hl/∂x and ∂H/∂x, which ensures that the granular mush is quasi-static. Equation (3.3) can be used to rewrite the velocity in the viscous liquid (2.19) as ul = −D ∂hl ∂x 1 2 z(hl −z), (3.4) (3.4) (3.4) where we have also assumed that |∂H/∂x| ≪D |∂hl/∂x|, which we confirm a posteriori (this assumption also implies that hl ≪1; see (3.3)). Equation (3.4) is analogous to a Poiseuille flow with no-slip at both the top and bottom of the viscous liquid; see figure 6. The Poiseuille flow structure contrasts with an ‘unconfined’ viscous gravity current with no overlying granular mush, which has a velocity field with no-slip at z = 0, and zero vertical gradient at z = hl. The flux in the viscous liquid is obtained by integrating (3.4) across its thickness to obtain ∂hl ∂t + ∂Ql ∂x = 0, where Ql = −D ∂hl ∂x h3 l 12. (3.5) (3.5) The flux in the viscous liquid is driven by the same hydrostatic pressure gradients as a classical (or ‘unconfined’) viscous gravity current (Huppert 1982), but the flux is reduced by a factor of four owing to the different boundary condition at the top of the viscous liquid (arising from the retarding influence of the overlying granular mush). The flux in the viscous liquid is driven by the same hydrostatic pressure gradients as a classical (or ‘unconfined’) viscous gravity current (Huppert 1982), but the flux is reduced by a factor of four owing to the different boundary condition at the top of the viscous liquid (arising from the retarding influence of the overlying granular mush). Viscous gravity current beneath a granular mush Flow evolution for three different values of the density ratio D with K = 1: D = 0.25 (green lines), D = 0.5 (black lines) and D = 1 (red lines). The initial condition is (3.1) (as in figure 2). Times are (a) t = 1, (b) t = 10, and (c) t = 100. to the upper free surface thinning and extending laterally; see figures 2(i) and 4(i). At first sight, it appears that ∂H/∂x ≈0 at late times, so the viscous liquid should spread independently of the overlying granular mush as the classical self-similar gravity current of Huppert (1982). As we show below, this description is incorrect; the flow is self-similar, but the small gradients in the upper free surface retard the outward propagation, and this leads to the rate of spreading being exactly a quarter of that of a classical (or ‘unconfined’) viscous gravity current. The hydrostatic pressure gradient associated with ∂H/∂x competes with the outwards carrying of the granular mush by the gravity-driven spreading liquid as discussed in § 3. 985 A18-9 E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim 1.0 0.5 z 0 1.10 1.15 1.20 x 1.25 15 10 5 u 0 (×10–4) Figure 6. A slice 1.1 ≤x ≤1.25 from the horizontal velocity field at t = 250 shown in figure 3(c). The granular mush is quasi-static, and the viscous liquid moves with a parabolic profile for the horizontal velocity (Poiseuille flow). 1.0 0.5 z 0 1.10 1.15 1.20 x 1.25 15 10 5 u 0 (×10–4) Figure 6. A slice 1.1 ≤x ≤1.25 from the horizontal velocity field at t = 250 shown in figure 3(c). The granular mush is quasi-static, and the viscous liquid moves with a parabolic profile for the horizontal velocity (Poiseuille flow). At late times, the granular mush is in a quasi-static balance; any substantial velocity at the top of the viscous liquid drives significant movement of granular material, which then immediately suppresses the motion through the increased reverse gradient of the upper free surface. Thus the velocity at the top of the viscous liquid is approximately zero. At late times, the granular mush is in a quasi-static balance; any substantial velocity at the top of the viscous liquid drives significant movement of granular material, which then immediately suppresses the motion through the increased reverse gradient of the upper free surface. Viscous gravity current beneath a granular mush The self-similar flow of the viscous liquid is calculated by combining (3.5) with global mass conservation (2.26), which motivates the similarity solution hl = A2/5D−1/5t−1/5 fl(ξ), ξ = x (A3Dt)1/5 . (3.6) (3.6) 985 A18-10 Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush –0.6 0.4 0.2 0 F(ξ) –0.2 1.0 (a) (b) 0.5 fl (ξ) 0 –0.4 –0.2 0 ξ 0.2 0.4 0.6 –0.6 –0.4 –0.2 0 0.2 0.4 0.6 Figure 7. Late-time self-similar interface shapes. (a) Comparison of the numerical solutions for hl(x, t) at t = 100, 250, 1000, 5000, 50 000 (solid lines) with the similarity solution (3.7) (dot-dashed magenta line). Parameters and initial conditions are as in figure 2. (b) Comparison for the upper free surface H(x, t). The similarity solution is given by (3.11). –0.6 0.4 0.2 0 F(ξ) –0.2 1.0 (a) (b) 0.5 fl (ξ) 0 –0.4 –0.2 0 ξ 0.2 0.4 0.6 –0.6 –0.4 –0.2 0 0.2 0.4 0.6 (a) 1.0 0.5 (ξ) 0 –0.6 –0.4 –0.2 0 0.2 0.4 0.6 Figure 7. Late-time self-similar interface shapes. (a) Comparison of the numerical solutions for hl(x, t) at t = 100, 250, 1000, 5000, 50 000 (solid lines) with the similarity solution (3.7) (dot-dashed magenta line). Parameters and initial conditions are as in figure 2. (b) Comparison for the upper free surface H(x, t). The similarity solution is given by (3.11). The self-similar shape function is given by The self-similar shape function is given by The self-similar shape function is given by The self-similar shape function is given by The self-similar shape function is given by fl(ξ) = 18 5 1/3  ξ2 0 −ξ21/3 , ξ0 = 1 2 ⎡ ⎣5 π1/2 10 9 1/3 Γ  5 6  Γ  1 3  ⎤ ⎦ 3/5 ≈0.566, (3.7) where we have used global mass conservation of viscous liquid (2.26) to determine ξ0. The solution (3.7) is compared to the numerical results at various late times in figure 7(a). where we have used global mass conservation of viscous liquid (2.26) to determine ξ0. The solution (3.7) is compared to the numerical results at various late times in figure 7(a). Once hl(x, t) has been determined, the evolution of the upper free surface H(x, t) is obtained via (3 3) which we rewrite as where we have used global mass conservation of viscous liquid (2.26) to determine ξ0. The solution (3.7) is compared to the numerical results at various late times in figure 7(a). Once hl(x, t) has been determined, the evolution of the upper free surface H(x, t) is obtained via (3.3), which we rewrite as Once hl(x, t) has been determined, the evolution of the upper free surface H(x, t) is obtained via (3.3), which we rewrite as −∂H ∂x hl −D h2 l 2 ∂hl ∂x ≈0, (3.8) (3.8) since hl ≪1, hu ≈1 and |∂H/∂x| ≪D |∂hl/∂x|. Equation (3.8) reflects the balance of the competing hydrostatic pressure gradients associated with the two interfaces. Given the self-similar scalings for hl and x (3.6), we obtain the following solution for H(x, t): since hl ≪1, hu ≈1 and |∂H/∂x| ≪D |∂hl/∂x|. Equation (3.8) reflects the balance of the competing hydrostatic pressure gradients associated with the two interfaces. Given the self-similar scalings for hl and x (3.6), we obtain the following solution for H(x, t): H(x, t) = 1 + A4/5D3/5t−2/5 F(ξ). (3.9) (3.9) Equation (3.8) is recast in the similarity variables as Equation (3.8) is recast in the similarity variables as −fl dF dξ = 1 2 f 2 l dfl dξ , (3.10) (3.10) https://doi.org/10.1017/jfm.2024.305 Pu which we integrate to obtain which we integrate to obtain F(ξ) =  9 20 2/3  F0 −  ξ2 0 −ξ22/3 , F0 = 10 7 25π3 12 1/30 ⎡ ⎣ Γ  5 6  Γ  1 3  ⎤ ⎦ 9/5 ≈0.346, (3 11) 985 A18-11 E.M. Hinton and A.C. Slim E.M. The self-similar shape function is given by The key feature that gives rise to the self-similar behaviour is a yield criterion in the upper layer. y pp y Finally, we observe that since hl ∼t−1/5 and H −1 ∼t−2/5, our assumptions that |∂H/∂x| ≪D |∂hl/∂x| and hl ≪1 are valid. It is worth noting that although ∂H/∂x is relatively small, it is non-zero and cannot be neglected when determining the leading-order behaviour. The self-similar shape function is given by Hinton and A.C. Slim 1.0 (a) (b) (c) (d) (e) ( f ) 0.01 0 –0.01 0.5 z 0 –0.5 0 0.5 1.0 0.05 0 –0.05 0.5 0 –1 0 1 1.5 0.05 0 u –0.05 0.5 1.0 0 –2 0 2 2 0.05 0 –0.05 1 z 0 –2 0 x 2 2 0.1 0 –0.1 1 0 –2 –4 0 x 4 2 2 0.1 0 u –0.1 1 0 –5 0 x 5 Figure 8. Interface evolution and horizontal flow velocity u of the granular mush and viscous liquid for a constant input flux Q = 0.1, of viscous liquid with K = 1 and D = 0.5, for (a) t = 1, (b) t = 5, (c) t = 15, (d) t = 30, (e) t = 50, and ( f) t = 80. The red dashed lines show the numerical integration of (3.5) with the same input flux condition as for the two-media flow. (c) 5 1.5 0.05 0 u –0.05 0.5 1.0 0 –2 0 2 1.0 (a) (d) 0.01 0 –0.01 0.5 z 0 –0.5 0 0.5 (b) ( ) 1.0 0.05 0 –0.05 0.5 0 –1 0 1 (b) (c) (a) (d) ( f ) (e) (d) ( 2 0.05 0 –0.05 1 z 0 –2 0 x 2 f ) 2 0.1 0 u –0.1 1 0 –5 0 x 5 ) 2 0.1 0 –0.1 1 0 –2 –4 0 x 4 2 Figure 8. Interface evolution and horizontal flow velocity u of the granular mush and viscous liquid for a constant input flux Q = 0.1, of viscous liquid with K = 1 and D = 0.5, for (a) t = 1, (b) t = 5, (c) t = 15, (d) t = 30, (e) t = 50, and ( f) t = 80. The red dashed lines show the numerical integration of (3.5) with the same input flux condition as for the two-media flow. where we have used global mass conservation of the combined media,  ∞ −∞(H −1) dx = 0, to determine F0. The solution (3.11) is compared to the numerical results at various late times in figure 7(b). We note that this similarity solution also applies to the gravity-driven spreading of a viscous liquid beneath a viscoplastic material; see Appendix B. Viscous gravity current beneath a granular mush 1.5 (a) (b) (c) (d) (e) ( f ) (g) (h) (i) 1.0 0.5 z 0 2 5.0 2.5 0 5.0 2.5 0 5.0 2.5 0 1 0 2 1 0 2 1 0 1.5 1.0 0.5 z 0 1.5 1.0 0.5 z 0 –1 0 x 1 –4 –2 0 2 x 4 –10 0 10 –1 0 1 –4 –2 0 2 4 –10 0 10 –1 0 1 –4 –2 0 2 4 –10 0 10 x Figure 9. Constant input flux of viscous liquid with K = 1. (a–c) Thicknesses at t = 0.5, 5, 50 for Q = 0.5 and D = 0.25. (d–f) Corresponding plots for Q = 0.5 and D = 1. (g–i) Corresponding plots for Q = 1.5 and D = 1. 1.5 (a) (d) 1.0 0.5 z 0 –1 0 1 (b) ( ) 2 1 0–4 –2 0 2 4 (e) (i) (g) 1.5 1.0 0.5 z 0 –1 0 x 1 i) 5.0 2.5 0 –10 0 10 x (h) 2 1 0 –4 –2 0 2 x 4 Figure 9. Constant input flux of viscous liquid with K = 1. (a–c) Thicknesses at t = 0.5, 5, 50 for Q = 0.5 and D = 0.25. (d–f) Corresponding plots for Q = 0.5 and D = 1. (g–i) Corresponding plots for Q = 1.5 and D = 1. figure 8 show the numerical integration of (3.5) with the same input flux condition as for the two-media flow. There is good agreement at early and intermediate times. At later times, the viscous liquid penetrates the upper free surface owing to continued input of viscous liquid; see figure 8( f). The viscous gravity current becomes segregated into different regions separated by the contact points with the granular mush: (i) an ‘unconfined’ region in the centre where the liquid is not bounded above by granular mush and the flow is driven solely by the liquid’s weight, and (ii) outer regions on either side in which the gravity-driven spreading of the liquid is retarded by the hydrostatic pressure gradients associated with the upper granular interface; see figure 8( f). This resistance to the liquid motion is evidenced in the discrete change in the gradient of the liquid interface across the contact point with the granular mush. The influence of varying the parameters D and Q is indicated in figure 9. 4. Constant input flux of viscous liquid In this section, we analyse the evolution in the case that the volume of viscous liquid increases linearly in time at a rate Q; see (2.26). The viscous liquid is injected uniformly in the interval −0.5 ≤x ≤0.5. Initially, hl = 0 and hu = 1 everywhere. y l u y Numerical results for the interface shapes (black lines) and the velocity field are shown in figure 8 at six different times, with K = 1, Q = 0.1 and D = 0.5. At early times, the viscous liquid is entirely enclosed by the granular mush and the upper free surface develops an ‘M’ shape (e.g. figure 8c). This is because the early-time input of viscous liquid has two key effects: (i) the upper free surface is lifted around x = 0, and (ii) granular material is transferred horizontally outwards from x = 0 into two levees as it is carried by the liquid; see the horizontal velocity fields in figure 8. The ‘M’ shape of the upper free surface leads to hydrostatic pressure gradients that retard the viscous gravity current, in a fashion identical to the evolution of a fixed volume release; see § 3. Indeed, the horizontal velocity has an approximately Poiseuille-like flow structure in the liquid until the liquid penetrates the upper free surface; see figure 8(c). This suggests that the viscous liquid evolves as a gravity current with a quarter of the flux of an ‘unconfined’ viscous gravity current (so (3.5) applies). The red dashed lines in Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush Figures 9(a–c) and 9(d–f) show identical results but with two values of the density difference D. At early times, the density difference has little effect on the motion because the input flux dominates the evolution. At later times, a larger density difference is associated with greater gravity-driven spreading of the viscous liquid in the lateral direction. g g y p g q Figures 9(g–i) demonstrate the effect of a larger input flux Q, which is predominantly to reduce the time scale of the flow (with the qualitative features unchanged). Varying K leads to negligible change in the results. In the absence of a granular mush, the constant input of viscous liquid leads to a self-similar gravity current with increasing thickness (h ∼t1/5) and extent (x ∼t4/5) (Huppert 1982). This scaling does not apply to the present configuration at intermediate times as demonstrated in figure 10, which shows the solution from figures 9(d–f) in rescaled coordinates. If the ‘unconfined’ scalings applied, then the volume of displaced granular material either side of the liquid would increase in proportion to t4/5 because 985 A18-13 E.M. Hinton and A.C. Slim –0.3 –0.2 –0.1 0 0.1 0.2 0.3 0 0.5 z/t1/5 x/t4/5 1.0 1.5 Figure 10. Thicknesses for constant input flux in rescaled coordinates at t = 10 (blue), t = 100 (red) and t = 1000 (black). Parameter values as in figures 9(d–f). Although the gradients appear to be getting steeper in terms of these scaled coordinates, in (x, z) coordinates, the interface gradients are diminishing so the flow remains shallow. E.M. Hinton and A.C. Slim –0.3 –0.2 –0.1 0 0.1 0.2 0.3 0 0.5 z/t1/5 x/t4/5 1.0 1.5 Figure 10. Thicknesses for constant input flux in rescaled coordinates at t = 10 (blue), t = 100 (red) and t = 1000 (black). Parameter values as in figures 9(d–f). Although the gradients appear to be getting steeper in terms of these scaled coordinates, in (x, z) coordinates, the interface gradients are diminishing so the flow remains shallow. initially hu = 1, but the volume of viscous liquid is proportional to t so it is not possible to have the same length and thickness scalings in the granular mush and the viscous liquid. At very late times, the granular volume and thickness will become negligible in comparison to the viscous liquid, so the ‘unconfined’ similarity solution will apply (results not shown). Declaration of interests. The authors report no conflict of interest. Author ORCIDs. Edward M. Hinton https://orcid.org/0000-0002-2204-1204. Declaration of interests. The authors report no conflict of interest. Author ORCIDs. Edward M. Hinton https://orcid.org/0000-0002-2204-1204. 5. Discussion and conclusion This contribution has analysed the gravity-driven spreading of viscous liquid at the base of a dense granular mush. Numerical integration of a lubrication model has revealed the key features of the flow of a fixed volume of viscous liquid. At early times, there is a relatively fast outward transfer of granular material owing to the viscous spreading, which carries the overlying granular mush. This leads to the formation of levees of granular material at either side of the liquid. In addition, it produces a hydrostatic pressure gradient against the viscous flow because the granular mush is thicker away from the centre of the liquid deposit. The counter-pressure gradient retards the flow. At late times, the flow becomes self-similar, with the spreading rate quartered relative to a classical viscous gravity current due to the evolution of the upper free surface (which is also self-similar). We have also analysed the case in which viscous liquid is injected at a constant rate at the base of the granular mush. At early times, the liquid is contained entirely by the granular mush, and the mush is carried into levees. A Poiseuille velocity field develops in the liquid, and the flux is a quarter of that for an unconfined viscous gravity current. At later times, the liquid penetrates the upper free surface, but the granular levees continue to be pushed outwards. We have used a thin-film model for the flow, but we note that if the system is initially not shallow, then it will still spread under gravity and eventually become shallow owing to the outward spreading. Given the insensitive nature of the flow to the initial shape, the dynamics at later times will be qualitatively unchanged. Although we focused on a two-dimensional geometry, the qualitative flow features would be identical for an axisymmetric configuration. This includes the quartering of the flux relative to a classical viscous gravity current and the late-time self-similar evolution. Finally, we have shown that the dynamics is unchanged if the overlying medium is a Bingham material rather than a granular mush; see Appendix B. 985 A18-14 Viscous gravity current beneath a granular mush Appendix A. Scaling analysis In this appendix, we analyse the validity of the shallow-flow approximation. We assume that μs ≪1. 5. Discussion and conclusion μ The governing equations in each medium are conservation of mass and conservation of momentum in the ˆx and ˆz directions, given by 0 = ∂ˆui ∂ˆx + ∂ˆwi ∂ˆz , (A1) 0 = −∂ˆpi ∂ˆx + ∂ˆτxx,i ∂ˆx + ∂ˆτxz,i ∂ˆz , (A2) 0 = −∂ˆpi ∂ˆz + ∂ˆτxz,i ∂ˆx + ∂ˆτzz,i ∂ˆz −ˆρiˆg, (A3) (A1) (A2) (A3) where i = u, l. These equations are combined with the Newtonian constitutive law in the viscous liquid and the μ(I)-rheology in the granular mush. where i = u, l. These equations are combined with the Newtonian constitutive law in the viscous liquid and the μ(I)-rheology in the granular mush. We follow a standard thin-film analysis (Ockendon & Ockendon 1995) and scale thicknesses (ˆz, ˆh) with ˆH and lengths ˆx with ˆL, where we assume that ϵ = ˆH/ ˆL ≪1. Horizontal velocities are scaled with ˆu ∼ˆU, where this scale is determined later (see (A9)). Continuity (A1) furnishes the vertical velocity scale: ˆw ∼ϵ ˆU. The ˆz-momentum equation (A3) motivates the pressure scale ˆp ∼ρug ˆH in both materials, where we have assumed that the two densities ˆρu and ˆρl have the same order of magnitude. In the (Newtonian) viscous liquid, the constitutive law furnishes the following scalings for the stresses: ˆτxx ∼ˆη ∂ˆu ∂ˆx ∼ˆη ϵ ˆU ˆH , (A4) ˆτxz ∼ˆη ∂ˆu ∂ˆz ∼ˆη ˆU ˆH , (A5) ˆτzz ∼ˆη ∂ˆw ∂ˆz ∼ˆη ϵ ˆU ˆH . (A6) (A4) (A5) (A6) The leading-order terms in the momentum equations in the viscous liquid ((A2) and (A3)) are (Huppert 1982) 0 = −∂ˆp ∂ˆx + ∂ˆτxz ∂ˆz , (A7) 0 = −∂ˆp ∂ˆz −ˆρlˆg, (A8) (A7) (A8) and for a balance in (A7), the horizontal velocity scale is set to and for a balance in (A7), the horizontal velocity scale is set to ˆU = ϵ ˆρuˆg ˆH2 ˆη . (A9) ˆU = ϵ ˆρuˆg ˆH2 ˆη . (A9) This furnishes a viscous shear stress scale This furnishes a viscous shear stress scale This furnishes a viscous shear stress scale ˆτxz ∼ϵ ˆρuˆg ˆH. (A10) ˆτxz ∼ϵ ˆρuˆg ˆH. (A10) 985 A18-15 ˆτxz ∼ϵ ˆρuˆg ˆH. 985 A18-15 E.M. Hinton and A.C. Slim We next consider the stresses in the granular mush, and we assume that μ(I) ∼μs ∼ϵ. 5. Discussion and conclusion The dominant contribution to the second strain-rate invariant ˙γ is ˙γ ∼∂ˆu ∂ˆz , (A11) (A11) provided that there is deformation of the granular material; the case of quasi-rigid material (∂ˆu/∂ˆz ≈0) is discussed in § A.1. The μ(I)-rheology furnishes the following scaling for the stresses: ˆτxx ∼μ(I) ˆp ∂ˆu/∂ˆx ˙γ ∼μ(I) ϵ ˆρuˆg ˆH, (A12) ˆτxz ∼μ(I) ˆp ∂ˆu/∂ˆz ˙γ ∼μ(I) ˆρuˆg ˆH, (A13) ˆτzz ∼μ(I) ˆp ∂ˆw/∂ˆz ˙γ ∼μ(I) ϵ ˆρuˆg ˆH. (A14) (A12) (A14) Since μ(I) ∼ϵ, the shear stress in the granular mush has the same order of magnitude as in the Newtonian liquid (A10). The stresses τxx and τzz also have the same magnitude as in the Newtonian liquid. Hence the momentum equations for the granular mush reduce to a shallow form identical to that in the Newtonian liquid, given by (A7) and (A8). Since μ(I) ∼ϵ, the shear stress in the granular mush has the same order of magnitude as in the Newtonian liquid (A10). The stresses τxx and τzz also have the same magnitude as in the Newtonian liquid. Hence the momentum equations for the granular mush reduce to a shallow form identical to that in the Newtonian liquid, given by (A7) and (A8). A.1. Pseudo-rigid granular material For shallow flows of viscoplastic fluids, the reduction of the governing equations to the leading-order shallow form leads to an apparent inconsistency; there are regions that appear rigid at leading order, but they can deform in the streamwise direction (Lipscomb & Denn 1984; Putz, Frigaard & Martinez 2009). For free-surface flows, this so-called ‘lubrication paradox’ was resolved by Balmforth & Craster (1999), who showed that the regions in which ∂u/∂z vanishes at leading order are in fact ‘pseudo-plugs’ that are weakly yielded owing to the importance of extensional variations in the velocity. A similar feature occurs for a granular mush carried by an underlying viscous liquid. The granular mush can appear to be rigid at leading order but is in fact deforming owing to streamwise variations as it is carried by the spreading viscous liquid below. As for viscoplastic pseudo-plugs, the velocity in these regions can be written as u = u0(x, t) + ϵ u1(x, z, t) + · · ·, (A15) (A15) u = u0(x, t) + ϵ u1(x, z, t) + · · ·, so the leading-order velocity appears plugged since ∂u0/∂z = 0. The dominant contributions to ˙γ then arise from both ϵ ∂u1/∂z and ∂u0/∂x, which are of the same order of magnitude, and (A11) must be adjusted. The key conclusion is that the leading-order velocity profiles from the shallow theory remain correct. Hence, for μs ∼ϵ ≪1, the model (and the velocity profiles) in § 2 are justified. The non-dimensionalisation with the horizontal length scale being order 1/μs times the thickness scale (2.18) is also valid. 985 A18-16 Viscous gravity current beneath a granular mush Appendix B. Viscous gravity current beneath a Bingham material The flux in each medium is (Balmforth et al. 2002; Christy & Hinton 2023) Appendix B. Viscous gravity current beneath a Bingham material The flux in each medium is (Balmforth et al. A.1. Pseudo-rigid granular material 2002; Christy & Hinton 2023) ˆQl = −ˆρuˆg 6ˆη  3 ∂ˆH ∂ˆx ˆhuˆh2 l + 2  D ∂ˆhl ∂ˆx + ∂ˆH ∂ˆx  ˆh3 l  , (B1) (B1) 6η  ∂x  ∂x ∂x   ˆQu = −ˆρuˆg 6ˆν ˆY2(3ˆhu −ˆY) ∂ˆH ∂ˆx −ˆρuˆg 2ˆη  2 ∂ˆH ∂ˆx ˆh2 uˆhl +  D ∂ˆhl ∂ˆx + ∂ˆH ∂ˆx  ˆhuˆh2 l  , (B2) ˆY = max ⎛ ⎜⎜⎜⎜⎝ 0, ˆhu − ˆτY ˆρuˆg  ∂ˆH ∂ˆx  ⎞ ⎟⎟⎟⎟⎠ , (B3) (B2) (B3) where ˆτY is the yield stress of the Bingham material, and ˆν is the plastic shear viscosity. These forms of the flux are identical to (2.14) and (2.15), except for the different first term in the Bingham flux, associated with its plastic behaviour. g p We non-dimensionalise using the following scalings (cf. (2.18)): (z, hl, hu, H) = (ˆz, ˆhl, ˆhu, ˆH)/ˆa, x = ˆxˆτY ˆρuˆgˆa2 , t = ˆt ˆρuˆgˆa ˆη  ˆτY ˆρuˆgˆa 2 . (B4a–c) (B4a–c) The shallow approximation requires the vertical length scale to be much smaller than the horizontal length scale, which becomes ˆτY ˆρuˆgˆa ≪1, (B5) (B5) which is equivalent to the Bingham number being small. This condition is analogous to μs ≪1 for the granular mush; the yield criterion must be triggered at relatively small gradients in the free surface so that the outer slopes of the levees are small. The dimensionless fluxes in each medium are Ql = −  ∂H ∂x huh2 l 2 +  D ∂hl ∂x + ∂H ∂x  h3 l 3  , (B6) (B6) Qu = −M 6 Y2(3hu −Y) ∂H ∂x −  ∂H ∂x h2 uhl +  D ∂hl ∂x + ∂H ∂x  huh2 l 2  , (B7 Qu = −M 6 Y2(3hu −Y) ∂H ∂x −  ∂H ∂x h2 uhl +  D ∂hl ∂x + ∂H ∂x  huh2 l 2  , (B7) Y = max ⎛ ⎜⎜⎝0, hu − 1  ∂H ∂x  ⎞ ⎟⎟⎠, (B8) (B7) Y = max ⎛ ⎜⎜⎝0, hu − 1  ∂H ∂x  ⎞ ⎟⎟⎠, (B8) (B8) where M = ˆη ˆν (B9) (B9) is the ‘viscosity’ ratio, analogous to K; see (2.22). Mass conservation is given by (2.16). A.1. Pseudo-rigid granular material The system is integrated numerically, and results are shown in figure 11 for M = 1, 985 A18-17 is the ‘viscosity’ ratio, analogous to K; see (2.22). Mass conservation is given by (2.16). The system is integrated numerically, and results are shown in figure 11 for M = 1, 985 A18-17 is the ‘viscosity’ ratio, analogous to K; see (2.22). Mass conservation is given by (2.16). The system is integrated numerically, and results are shown in figure 11 for M = 1, E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim 1.0 (a) (b) (c) (d) (e) ( f ) 0.5 z 0 –1 0 1 1.0 0.5 0 –1 0 1 1.0 0.5 0 1.0 0.5 z 0 1.0 0.5 0 1.0 0.5 0 –1 0 1 –1 0 x x x 1 –2 0 2 –2 0 2 Figure 11. Gravity-driven flow of a fixed volume of viscous liquid beneath a Bingham material with M = 1 and D = 0.5. The initial condition is given by (3.1). Times are (a) t = 1, (b) t = 5, (c) t = 20, (d) t = 100, (e) t = 1000, and ( f) t = 10 000. In (a–c), the blue dashed lines correspond to the granular case from figure 2. In (e, f), the red dashed line shows the similarity solution ((3.6) and (3.7)) for the lower layer. 1.0 (a) 0.5 z 0 –1 0 1 (b) 1.0 0.5 0 –1 0 1 (c) 1.0 0.5 0 –1 0 1 (b) (a) (e) 1.0 0.5 0 x –2 0 2 (d) 1.0 0.5 z 0 –1 0 x 1 (d) (e) ( f ) 1.0 0.5 0 x –2 0 2 Figure 11. Gravity-driven flow of a fixed volume of viscous liquid beneath a Bingham material with M = 1 and D = 0.5. The initial condition is given by (3.1). Times are (a) t = 1, (b) t = 5, (c) t = 20, (d) t = 100, (e) t = 1000, and ( f) t = 10 000. In (a–c), the blue dashed lines correspond to the granular case from figure 2. In (e, f), the red dashed line shows the similarity solution ((3.6) and (3.7)) for the lower layer. D = 0.5, and a fixed volume release of viscous liquid with initial shape given by (3.1). A.1. Pseudo-rigid granular material The evolution is qualitatively similar to the case of an overlying granular mush. Indeed, the results from figure 2 for a granular mush with the same density difference are overlain as blue dotted lines in figures 11(a–c) with excellent agreement. At late times, an identical similarity solution, given by ((3.6) and (3.7)), applies, and it is shown as a red dashed line in figure 11(e, f). This demonstrates that the key feature that controls the dynamics is the yield criterion in the overlying material. Viscous gravity current beneath a granular mush N, K. & KOVAL, G. 2012 Nonlocal constitutive relation for steady granular flow. Phys. Rev. Lett. 108 178301. KLIAKHANDLER, I.L. & SIVASHINSKY, G.I. 1997 Viscous damping and instabilities in stratified liquid film flowing down a slightly inclined plane. Phys. Fluids 9 (1), 23–30. KOWAL, K.N. & WORSTER, M.G. 2015 Lubricated viscous gravity currents. J. Fluid Mech. 766, 626–655. S G & S 2021 b i d i f KOWAL, K.N. & WORSTER, M.G. 2015 Lubricated viscous gravity currents. J. Fluid Mech. 766, 626–655. KUMAR, P., ZURI, S., KOGAN, D., GOTTLIEB, M. & SAYAG, R. 2021 Lubricated gravity currents of power-law fluids. J. Fluid Mech. 916, A33. L.T. & KOWAL, K.N. 2022 Lubricated viscous gravity currents of power-law fluids. Part 2. Stability ysis. J. Fluid Mech. 940, A27. LIPSCOMB, G.G. & DENN, M.M. 1984 Flow of Bingham fluids in complex geometries. J. Non-Newtonian Fluid Mech. 14, 337–346. , LISTER, J.R. 1992 Viscous flows down an inclined plane from point and line sources. J. Fluid Mech. 242, 631–653. LIU, P.-P., ZHOU, M.-F., WANG, C.Y., XING, C.-M. & GAO, J.-F. 2014 Open magma chamber processes in the formation of the Permian Baima mafic–ultramafic layered intrusion, SW China. Lithos 184, 194–208. the formation of the Permian Baima mafic–ultramafic layered intrusion, SW China. Lithos 184, 194–208. LYMAN, A.W., KERR, R.C. & GRIFFITHS, R.W. 2005 Effects of internal rheology and surface cooling on the emplacement of lava flows. J. Geophys. Res. 110, B08207. LYMAN, A.W., KERR, R.C. & GRIFFITHS, R.W. 2005 Effects of internal rheology and surface cooling on the emplacement of lava flows. J. Geophys. Res. 110, B08207. MAIER, W.D., BARNES, S.-J. & GROVES, D.I. 2013 The Bushveld Complex, South Africa: formation of platinum–palladium, chrome- and vanadium-rich layers via hydrodynamic sorting of a mobilized cumulate slurry in a large, relatively slowly cooling, subsiding magma chamber. Miner. Deposita 48, 1–56. MUNGALL, J.E. & SU, S. 2005 Interfacial tension between magmatic sulfide and silicate liquids: constraints on kinetics of sulfide liquation and sulfide migration through silicate rocks. Earth. Planet. Sci. Lett. 234 (1–2), 135–149. ( ) NALDRETT, A.J. 1973 Nickel sulfide deposits – their classification and genesis with special emphasis on deposits of volcanic association. Trans. Can. Inst. Mining Metallurgy 76, 183–201. p g gy OCKENDON, H. & OCKENDON, J.R. 1995 Viscous Flow, vol. 13. Cambridge Texts in Applied Mathematics, Cambridge University Press. PUTZ, A., FRIGAARD, I.A. & MARTINEZ, D.M. REFERENCES BALMFORTH, N.J. & CRASTER, R.V. 1999 A consistent thin-layer theory for Bingham plastics. J. Non-Newtonian Fluid Mech. 84 (1), 65–81. BALMFORTH, N.J., CRASTER, R.V. & SASSI, R. 2002 Shallow viscoplastic flow on an inclined plane. J. Fluid Mech. 470, 1–29. BALMFORTH, N.J., CRASTER, R.V. & TONIOLO, C. 2003 Interfacial instability in non-Newtonian fluid layers. Phys. Fluids 15 (11), 3370–3384. CHRISTY, I. & HINTON, E.M. 2023 Two-layer gravity currents of generalized Newtonian fluids. Proc. R. Soc. A 479 (2279), 20230429. CHUNG, H.-Y. & MUNGALL, J.E. 2009 Physical constraints on the migration of immiscible fluids through partially molten silicates, with special reference to magmatic sulfide ores. Earth Planet. Sci. Lett. 286 (1–2), 14–22. DA CRUZ, F., EMAM, S., PROCHNOW, M., ROUX, J.-N. & CHEVOIR, F. 2005 Rheophysics of dense granular materials: discrete simulation of plane shear flows. Phys. Rev. E 72 (2), 021309. p GDR-MIDI 2004 On dense granular flows. Eur. Phys. J. E 14, 341–365. g y GRAY, J.M.N.T. & EDWARDS, A.N. 2014 A depth-averaged μ(I)-rheology for shallow granular free-surface flows. J. Fluid Mech. 755, 503–534. HINTON, E.M. & SLIM, A.C. 2023 Dynamics and containment of a vicous liquid atop a granular bed. J. Fluid Mech. 969, R3. HUPPERT, H.E. 1982 The propagation of two-dimensional and axisymmetric viscous gravity currents over a rigid horizontal surface. J. Fluid Mech. 121, 43–58. g HUPPERT, H.E. 1986 The intrusion of fluid mechanics into geology. J. Fluid Mech. 173, 557–594. 985 A18-18 985 A18-18 Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush 2009 On the lubrication paradox and the use of regularisation methods for lubrication flows. J. Non-Newtonian Fluid Mech. 163 (1–3), 62–77. ROBERTSON, J.C., BARNES, S.J. & LE VAILLANT, M. 2016 Dynamics of magmatic sulphide droplets during transport in silicate melts and implications for magmatic sulphide ore formation. J. Petrol. 56 (12), 2445–2472. 985 A18-19 985 A18-19
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https://europepmc.org/articles/pmc3835131?pdf=render
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Serum Biomarkers for Early Detection of Gynecologic Cancers
Cancers
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cancers ISSN 2072-6694 www.mdpi.com/journal/cancers OPEN ACCESS cancers ISSN 2072-6694 www.mdpi.com/journal/cancers OPEN ACCESS cancers ISSN 2072-6694 www.mdpi.com/journal/cancers Yutaka Ueda, Takayuki Enomoto *, Toshihiro Kimura, Takashi Miyatake, Kiyoshi Yoshino, Masami Fujita and Tadashi Kimura Yutaka Ueda, Takayuki Enomoto *, Toshihiro Kimura, Takashi Miyatake, Kiyoshi Yoshino, Masami Fujita and Tadashi Kimura Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka, 565-0871, Japan; E-Mails: ZVF03563@nifty.com (Y.U.); kimbinbin@gmail.com (T.K.); tmiyatake@aim.com (T.M.); yosino-ki@mc.pref.osaka.jp (K.Y.); fujita@gyne.med.osaka-u.ac.jp (M.F.); tadashi@gyne.med.osaka-u.ac.jp (T.K.) * Author to whom correspondence should be addressed; E-Mail: enomoto@gyne.med.osaka-u.ac.jp; Tel: +81-66879-3351; Fax: +81-66879-3359. Received: 8 March 2010; in revised form: 31 May 2010 / Accepted: 3 June 2010 / Published: 14 June 2010 Received: 8 March 2010; in revised form: 31 May 2010 / Accepted: 3 June 2010 / Published: 14 June 2010 Received: 8 March 2010; in revised form: 31 May 2010 / Accepted: 3 June 2010 / Published: 14 June 2010 Abstract: Ovarian, endometrial, and cervical cancers are three of the most common malignancies of the female reproductive organs. CA 125, historically the most reliable serum marker for ovarian cancer, is elevated in 50% of early-stage ovarian tumors. For endometrial cancers, there are no established serum markers. SCC, which is the best studied serum marker for squamous cell carcinomas, has been unreliable; SCC is elevated in cervical squamous cell carcinomas ranging from 28–85% of the time. Recent proteomics-based analyses show great promise for the discovery of new and more useful biomarkers. In this review, we will discuss the currently utilized serum tumor markers for gynecologic cancers and the novel biomarkers that are now under investigation. Keywords: ovarian; endometrial; cervical; cancer; serum; tumor marker; CA 125; SCC; proteomics; biomarkers Cancers 2010, 2, 1312-1327; doi:10.3390/cancers2021312 Cancers 2010, 2, 1312-1327; doi:10.3390/cancers2021312 1. Introduction Endometrial, cervical, and ovarian cancers are the three most common malignancies of the female reproductive tract. In the United States alone, roughly 12,000 women are diagnosed with uterine cervical cancer annually, and 4,000 will die from the disease [1]. The relatively low incidence of cervical cancer in the US is largely attributable to the effectiveness of Papanicolaou’s cytological cervical screening test (the Pap test). The importance of screening for early tumor markers is supported by the finding that 60% of cervical cancers occur in women who have never received a Pap test, or who have not been tested in the past five years [1]. For comparison of effectiveness, the serum marker SCC, which is the best studied serum marker for squamous cell carcinomas, is elevated in only 22–60% of early-stage cervical squamous cell carcinomas [2,3]. Endometrial cancer is even more common than cervical cancer in the US, with 37,000 diagnosed with it and 7,000 deaths attributable to it in 2005, making it the fourth most common cancer in women [1]. In approximately 75% of endometrial cancer cases, the tumor remains confined to the uterus (FIGO stage I) and has a favorable prognosis. The prognosis, however, worsens dramatically as the disease progresses [4]. Despite this, screening for endometrial cancer is not currently done because of the lack of an appropriate, cost-effective, and acceptable test [5]. Intermediate in frequency between cervical and endometrial cancers, but by far the most fatal, is ovarian cancer. In 2005, 20,000 women in the US were diagnosed with ovarian cancer, but a heartbreaking 15,000 (75%) of these women died from the disease [1]. Roughly three-quarters of ovarian cancer cases present at an advanced disease stage, with the disease spread well beyond the ovaries [6]. In advanced-stage disease, patients most often have first symptoms related to the presence of an enlarging tumor and ascites fluid. However, in early- and mid-stage disease, most patients are asymptomatic for a prolonged period [5]. Serum cancer antigen-125 (CA 125) levels and transvaginal ultrasonography (TV-USG) can contribute to the early detection of ovarian cancer. Unfortunately, these tests are not currently cost-effective; they are thus not used routinely to screen for ovarian cancer [5]. For ovarian, endometrial, and cervical cancers, it is critical to detect the disease at the earliest possible stage. The discovery of useful serum biomarkers for the early detection of gynecologic cancers has thus been a high priority. Keywords: ovarian; endometrial; cervical; cancer; serum; tumor marker; CA 125; SCC; proteomics; biomarkers Abbreviations: ApoA-1: apolipoprotein A-1; CA 15-3: cancer antigen-15-3; CA 19-9: cancer antigen-19-9; CA 72-4: cancer antigen-72-4; CA 125: cancer antigen-125; CEA: cancer embryonic antigen; CIN: cervical intraepithelial neoplasia; CYFRA 21-1: cytokeratin 19 fragments; HE4: (WFDC2), human epididymis-specific 4-disulfide core protein; H4: inter-α-trypsin inhibitor heavy chain fragment; IAP: immunosuppressive acidic protein; IGFII: insulin-like growth factor II; MIF: macrophage inhibitory factor; LPA: lysophosphatidic acid; MLRM: multiple logistic regression model; M-CSF: macrophage 1313 Cancers 2010, 2 colony-stimulating factor; Pap test: Papanicolaou’s test; SAA: human serum amyloid A; SCC: squamous cell carcinoma antigen; SELDI-TOF-MS: surface-enhanced laser desorption-ionization time-of-flight mass spectrometry; sFas: serum soluble Fas; TF: transferrin; TK: thymidine kinase; TTR: transthyretin; TV-USG: transvaginal ultrasonography; USG: ultrasonography; VEGF-C: serum isoform of vascular endothelial growth factor; YKL-40: (aka CHI3L1) human cartilage glycoprotein-39 Cancers 2010, 2 Cancers 2010, 2 Cancers 2010, 2 silenced genes or as an alternative mRNA splicing expression of an already expressed gene product. Some glycoproteins produced by cancer cells have altered glycan structures, although the proteins themselves are ubiquitous [7]. Tumor markers might be unique extracellular matrix or cell adhesion molecules, receptors, growth factors, cytokines, or products of abnormal metabolism. Rarely, the marker molecules can be released by other tissues and organs in response to signals from the tumor. Even the body’s own antibodies against tumor markers can be markers. Tumor markers are secreted, released, or leaked into the interstitial fluids, and thus into the lymph, and finally (or directly) into the bloodstream, where they become detectable in serum samples. To be able to enter the bloodstream directly, larger molecules, often proteins, are cleaved into truncated forms or fragments, which are sometimes specific to the protease micro-environment of the tumor. Tumor markers can be associated with patient diagnosis, prognosis, clinical management, and follow-up. Ideally, a serum marker would only appear in the blood of patients with a true malignancy; the marker would correlate with tumor stage and response to treatment, and it could be easily, cheaply, and reproducibly measured. The serum marker would be used for the screening of healthy populations and of specific groups with higher risk factors. The marker would enable a diagnosis for a specific type of cancer, help determine prognostic factors, and be used to monitor the course of treatment, remission, and recurrence, while receiving surgery, radiation, chemical, and immunological treatments. Recent advances in clinical proteomics have propelled us into an exciting period of discovery of new cancer biomarkers, although the available proteomic technologies have their limitations. The principles of proteomic technology require stringent guidelines for the collection of clinical material, the application of analytical techniques, and for our interpretation of the data. In this review, we present an overview of the serum tumor markers in current use. A lack of sensitivity and specificity has, so far, given most of the tumor markers in current use an unsatisfactory predictive value. We will discuss the novel biomarkers of the future, where there is great hope for the better detection and management of gynecologic cancers, including ovarian, endometrial, and cervical cancers. 1. Introduction Such tumor markers will be molecules arising from the presence of a tumor, which can appear in the surrounding tissue, and then within the blood and excretions. Tumor markers can be secreted or shed by the tumor in excess of the normal tissue or cell phenotype. Sometimes, the molecule is uniquely specific to the tumor phenotype, often as embryonic, fetal (i.e., AFP), undifferentiated, or stem-cell phenotypes. These can occur as re-expression of 1314 Cancers 2010, 2 Cancers 2010, 2 1315 Table 1. Diagnostic serum markers for cervical cancer in clinical use. Table 1. Diagnostic serum markers for cervical cancer in clinical use. Serum markers SCC CYFRA 21-1 CA 125 CA 19-9 CEA IAP Positive rates squamous squamous adeno adeno adeno 43–51% 28–85% 42–52% 27–75% 35–42% 26–48% Positive rates (elevated serum levels) detected for the indicated serum markers, in cases of squamous cell carcinoma (squamous), adenocarcinoma (adeno), or for all histological types. squamous cell carcinoma (squamous), adenocarcinoma (adeno), or for all histological types. Serum SCC levels have also been shown to parallel the responses to radiotherapy and chemotherapy [13,19–21]. Elevated pre-radiotherapy SCC levels were detected in 60% of 72 squamous cell carcinoma patients, whereas post-treatment SCC levels were above the cut-off value in only 2% of the patients whose disease was considered completely treated [13]. The serum SCC level also proved to be an independent predictor of response to neoadjuvant chemotherapy in locally advanced cervical cancer patients who received neoadjuvant chemotherapy and radical surgery [19]. SCC has also been used in the follow-up examination of cervical cancer patients. Increased serum SCC was shown to precede the clinical detection of recurrence of the disease [12,13,22]. The marker CYFRA 21-1 (serum fragments of cytokeratin 19) is also being used as a serum tumor marker for cervical cancer, especially for squamous cell carcinoma. Elevated CYFRA 21-1 levels have been detected in 42–52% of patients with squamous cell carcinoma of the uterine cervix [18,23,24] (Table 1). Similar to the usefulness of SCC, pre-treatment levels of CYFRA 21-1 are related to stage of the disease, size of the tumor, depth of the stromal invasion, the lymph-vascular space involvement, and lymph node metastasis [25–27]. However, raised CYFRA 21-1 levels were not demonstrated to be of predictive value for prognosis in some studies [18]. Serum CYFRA 21-1 levels were reported to be useful for monitoring the response to radiotherapy and chemotherapy [12,24]. The CYFRA 21-1 assay was also used in the follow-up examination of cervical cancer patients. Additionally, increased serum CYFRA 21-1 was shown to precede the clinical detection of recurrence of the disease [12]. Adenocarcinoma accounts for 10–15% of cervical cancers [4]. SCC, CYFRA 21-1, CA 125, CA 19-9 and CEA are positive in 20–25%, 33–63%, 27–75%, 34–42% and 26–48%, respectively, of such tumors [9–11,20,23,28–34] (Table 1). 2. Serum Markers for Cervical Cancer Screening for cervical cancer with cervical cytology reduced the incidence of cervical cancer by more than 50% over the past 30 years in the United States [8]. However, it is estimated that 50% of the women in whom cervical cancer is diagnosed each year will have never had cervical cytology testing [8]. One approach for further reducing the incidence and the mortality of cervical cancer would be to increase the screening rates among groups of women at highest risk, who currently are not being screened. Another would be the establishment of appropriate serum testing for the early detection of cervical cancer. The squamous cell carcinoma antigen, SCC, is the most commonly used serum marker for squamous cell cervical carcinoma, which makes up 85–90% of all cervical carcinomas [4]. Elevated serum SCC levels have been detected in 28–88% of cervical squamous cell carcinomas [9–15] (Table 1). Pre-treatment levels of SCC have been shown to be related to the stage of the disease, size of the tumor, depth of the stromal invasion, the lymph-vascular space involvement, and lymph node metastasis [9,11,13,14,16–19]. Elevated SCC levels were also demonstrated to have predictive value for prognosis in some studies [16,17,19]. Cancers 2010, 2 Cancers 2010, 2 cell carcinomas when compared to that of healthy women (p < 0.0001) [36]. Vascular endothelial growth factor (VEGF), especially the VEGF-C isoform, was revealed to be elevated in the serum of patients with squamous cell carcinoma and cervical intraepithelial neoplasia (CIN) when compared to that of healthy women [37,38]. In cervical squamous cell carcinoma patients, serum VEGF levels were associated with the stage of the disease, but not with prognosis [38]. The serum level of thymidine kinase (TK) was demonstrated to be significantly higher in patients with cervical carcinoma than in normal women and patients with carcinoma in situ (p < 0.01 and p < 0.05, respectively) [39]. Table 2. Diagnostic serum markers for cervical cancer currently under investigation. Serum markers M-CSF YKL-40 sFas VEGF TK Positive rates 27% squamous adeno squamous squamos N/A 75% 78% N/A N/A Positive rates (elevated serum levels) detected for the indicated serum markers, in cases of squamous cell carcinoma (squamous), adenocarcinoma (adeno), or for all histological types. able 2. Diagnostic serum markers for cervical cancer currently under investigation. ostic serum markers for cervical cancer currently under investigation. Cancers 2010, 2 Raised serum CA 125 is associated with the stage of the cervical disease and is of some prognostic significance [29]. Another novel marker (that is also a target for new drug development) is immunosuppressive acidic protein (IAP), which is elevated in 43–51% of cervical carcinomas [33,34] (Table 1). Battaglia et al. found pre-treatment-elevation of serum IAP in 53% of squamous cell carcinoma cases, and in 40% of adenocarcinoma patients [34]. IAP level is related to disease stage and lymph node metastasis, and is of predictive value for prognosis [34]. There are some newer serum markers still under investigation (Table 2). Suzuki et al. showed that serum M-SCF was elevated in 27% of cervical cancer cases [35]. YKL-40, also known as CHI3L1 or human cartilage glycoprotein-39, was shown to be a potential biomarker in the detection and management of cervical cancer [28]. Elevated serum YKL-40 was found in 75% of squamous cell carcinoma patients and 78% of adenocarcinoma patients. Moreover, elevated pre-treatment-levels of YKL-40 were shown to predict an unfavorable prognosis, independent of the stage of the disease. Significantly elevated serum levels of circulating soluble Fas (sFas) were demonstrated in squamous 1316 Cancers 2010, 2 Cancers 2010, 2 demonstrated to provide 46% sensitivity for endometrioid adenocarcinoma of the endometrium in all stages at 95% specificity [54]. For stage I disease, HE4 yielded a 17% improvement in sensitivity compared with that of CA 125. Significantly elevated levels of serum sFas were demonstrated in endometrioid adenocarcinoma of the endometrium compared to that of healthy women (p < 0.0001) [36]. Human serum amyloid A (SAA) is a high-density lipoprotein that has recently been proposed as a useful biomarker for several kinds of tumors, including gastric, lung, pancreatic, and nasopharyngeal cancers [55–59]. Cocco et al. showed that SAA was overexpressed and actively secreted by Grade-3 endometrioid adenocarcinoma and serous papillary carcinoma of the endometrium, and that SAA was present at high concentration in the serum of these patients [55,60]. Table 4. Diagnostic serum markers for endometrial cancer under investigation. Serum markers M-CSF HE4 sFas SAA Positive rates 25–73% E E G3-E/S 46% N/A N/A Positive rates (elevated serum levels) of each serum marker detected in cases of squamous cell carcinoma, adenocarcinoma, and for all histological types, are shown. E: endometrioid adenocarcinoma; G3-E: endometrioid adenocarcinoma Grade-3; S: serous adenocarcinoma; N/A: not assessed Table 4. Diagnostic serum markers for endometrial cancer under investigation. Recently, panels of novel biomarkers have been developed to better detect cancers, including endometrial tumors (Table 5). Farias-Eisner et al. constructed a multiple logistic regression model (MLRM) with the use of the values for ApoA-1 (apolipoprotein A-1), TF (transferrin), and TTR (transthyretin) for the detection of endometrial cancer [61]. This panel distinguished normal samples from early-stage endometrial cancer with a sensitivity of 71% and a specificity of 88% Additionally, the panel distinguished normal samples from late stage endometrial cancer with a sensitivity of 82% and a specificity of 86%. Table 5. Novel biomarker panels for detection of endometrial cancer. Reference Biomarkers Sensitivity Specificity Zhu et al. (2006)[63] 13 proteins 93% 100% Farias-Eisner et al. (2009) [61] 3 proteins Early Late Early Late 71% 82% 88% 86% Takano et al. (2009) [62] 2 proteins 82% 86% Shown here is the sensitivity and specificity of each serum biomarker panel used in the detection of endometrial cancer; Early: early-stage cases; Late: late-stage cases Table 5. Novel biomarker panels for detection of endometrial cancer. Recent proteomic techniques, which can identify differentially expressed proteins in a large set of samples, have been applied to the discovery of new biomarkers in many diseases [62]. 3. Serum Markers for Endometrial Cancer In current practice, screening for endometrial cancer is not undertaken because of the lack of an appropriate, cost-effective, and acceptable test that actually reduces mortality [5]. Routine use of an endometrial cytological test, comparable to the Pap test for cervical cancer, is too insensitive and nonspecific to be useful in screening for endometrial cancer [5]. Currently used serum markers and novel biomarkers under investigation for endometrial cancer are discussed below. Elevated serum CA 125 levels have been detected in 11–43% of endometrial cancers [40–46] (Table 3). Pre-treatment CA 125 levels were shown to be related to the stage of the disease, the depth of myometrial invasion, peritoneal cytology, and lymph node metastasis [42–48]. Raised CA 125 levels were also demonstrated to be of predictive value for prognosis in some studies [42,47]. The serum CA 125 level usually parallels the clinical course of the disease [43,45,49]; however, the fact that serum CA 125 levels are often elevated in disease-free endometrial cancer patients who have undergone abdominal radiation should be kept in mind [50]. Table 3. Diagnostic serum markers for endometrial cancer in clinical use. Serum markers CA 125 CA 19-9 CA 15-3 CA 72-4 CEA IAP Positive rates 11–43% 22–24% 24–32% 22–32% 14–22% 55–76% Positive rates (elevated serum levels) detected for the indicated serum marker in cases of endometrial cancer are shown. Table 3. Diagnostic serum markers for endometrial cancer in clinical use. Table 3. Diagnostic serum markers for endometrial cancer in clinical use. The serum markers CA 19-9, CA 15-3, CA 72-4, and CEA levels are raised in endometrial cancer patients in 22–24%, 24–32%, 22–32% and 14–22% of the cases, respectively [11,33,40,42,45,51,52] (Table 3). Serum CA 15-3 levels were shown to be associated with prognosis [42,53]. Elevated serum IAP levels have been detected in 55–76% of endometrial cancer patients [17,33] (Table 3). The serum markers CA 19-9, CA 15-3, CA 72-4, and CEA levels are raised in endometrial cancer patients in 22–24%, 24–32%, 22–32% and 14–22% of the cases, respectively [11,33,40,42,45,51,52] (Table 3). Serum CA 15-3 levels were shown to be associated with prognosis [42,53]. Elevated serum IAP levels have been detected in 55–76% of endometrial cancer patients [17,33] (Table 3). There are other serum markers for endometrial cancer now under investigation (Table 4). Raised serum M-SCF levels were detected in 25–73% of endometrial cancer cases [35,43]. HE4 was 1317 4. Serum Markers for Ovarian Cancer Establishment of an appropriate screening test for ovarian cancer has long been sought. This disease is the leading cause of death from gynecologic malignancies in the US, with its poor prognosis resulting from the lack of reliable, sensitive screening tests and our limited understanding of the mechanisms of chemoresistance and relapse. More than two-thirds of cases of ovarian cancers are diagnosed only after the disease has progressed to stage III or IV and involve the peritoneal cavity or other organs. Symptoms that are associated with ovarian cancer are typically nonspecific and the association is often not recognized until the disease is advanced [64]. Previous studies showed that ultrasonography (USG), with or without power Doppler, provided high sensitivity. However, ultrasonography’s specificity and positive predictive values were unsatisfactory [65,66]. Serum markers and novel biomarkers for early detection of ovarian cancer that are currently used or under investigation are discussed below. Elevated serum CA 125 levels have been detected in 50% and 92% of ovarian cancers in early and late stages, respectively [67] (Table 6). According to a review by Nossov et al., the positive predictive value of the CA 125 assay for the early detection of ovarian cancer is 57% [68]. Elevated CA 125 occurs in other cancers, such as in endometrial, breast, pancreatic, gastrointestinal, and lung cancers. Raised CA 125 levels are sometimes found in patients with benign gynecologic conditions, such as menstruation, pregnancy, endometriosis, and pelvic inflammatory disease, and even in non-gynecologic conditions, such as hepatitis and pancreatitis [49]. The predictive value of pre-treatment CA 125 levels for prognosis is controversial; however, changes in CA 125 levels correlate with the regression, stability, and progression of the disease in 87–94% of instances [49]. Table 6. Diagnostic serum markers for ovarian cancer in clinical use. Serum markers CA 125 CA 19-9 CA 15-3 CA 72-4 IAP Positive rates Early Late M non-M 50–56% 63–71% 70–93% 50% 92% 68–83% 28–29% Positive rates detected for each serum marker in cases of ovarian cancer are shown. Early: early-stage cases; Late: late-stage cases; M: mucinous adenocarcinoma; Non-M: histological types of ovarian carcinoma other than mucinous adenocarcinoma. Table 6. Diagnostic serum markers for ovarian cancer in clinical use. Serum levels of CA 19-9 (a monosialoganglioside antigen widely used in GI adenocarcinoma diagnostics) are elevated in 68–83% of mucinous ovarian cancers, and only in 28–29% of non-mucinous types. Cancers 2010, 2 Cancers 2010, 2 proportion of cancer patients than would be found in a general population, and thus a significantly lower proportion of healthy controls, had methodological limitations, as previously described [64]. The prevalence of endometrial cancer was 50% in the study by Takano et al. [62] and 57% in the study by Zhu et al. [63]. Thus, these results might have overstated the sensitivity of the tests. However, because one of the candidate proteins discovered in a study by Takano et al. [62], apolipoprotein A-1, was also a constituent of the biomarker panel constructed by Farias-Eisner et al. [61], this protein may be a very promising biomarker for endometrial cancer. Cancers 2010, 2 Zhu et al. has established a diagnostic system with 13 novel potential biomarkers, using surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to differentiate endometrial cancer patients from healthy women. The technique had a sensitivity of 93% and a specificity of 100% [63]. Takano et al. also used SELDI-TOF-MS to identify candidate markers for endometrial cancer [62]. Two of the candidates turned out to be apolipoprotein A-1 and apolipoprotein C-1. Dual-biomarker analysis for the detection of endometrial cancer yielded a sensitivity of 82% and a specificity of 86%. These studies, which analyzed test samples consisting of a significantly higher 1318 Cancers 2010, 2 Serum CA 15-3, CA 72-4, and CEA levels are raised in 50–56%, 24–32%, 63–71% and 70–93% of the ovarian cancer patients, respectively [71,73–78] (Table 6). According to Gadducci et al., CA 19-9, CA 15-3, and CA 72-4 correlated worse than CA 125 with the clinical course of the disease. Additionally, these markers did not offer additional clinical benefit for monitoring ovarian cancer, suggesting that the serial measurement of these markers may play a role only in the management of patients with a normal CA 125 assay [49]. There are serum markers for ovarian cancer that are under active investigation (Table 7). In a review by Li et al., HE4 displayed the highest sensitivity among single markers, including CA 125, in the detection of ovarian cancer in both the early (62–83%) and late (75–93%) stages, respectively [79]. Elevated serum lysophosphatidic acid (LPA) levels were found in 90% and 98% of ovarian cancer in early and late stages, respectively. However, serum levels of LPA do not correlate with the stage of the disease, and nonspecific elevation of LPA was detected in healthy and benign gynecologic conditions [68,80,81]. Significantly elevated sFas levels were detected in ovarian cancer patients when compared to that of healthy women. Additionally, the serum sFas level was demonstrated to be a statistically significant indication factor for survival, as well as for the histological grading of ovarian carcinomas [36]. Table 7. Diagnostic serum markers for ovarian cancer under investigation. Serum markers HE4 LPA sFas Positive rates Early Late Early Late N/A 62–83% 75–93% 90% 98% Positive rates detected for each serum marker in cases of ovarian cancer are shown. Early: early-stage cases; Late: late-stage cases; N/A: not assessed Table 7. Diagnostic serum markers for ovarian cancer under investigation. Novel biomarker panels have also been investigated for the early detection of ovarian cancers (Table 8). Zhang et al. identified a panel that consisted of three proteins, including ApoA-1, a truncated form of TTR, and a cleavage fragment of H4 (inter-α-trypsin inhibitor heavy chain), to detect early-stage ovarian cancer with a sensitivity of 83% and a specificity of 94% [82]. Table 8. Novel biomarker panels for the detection of ovarian cancer. Reference Biomarkers Sensitivity Specificity Zhang et al. (2004) [82] 3 proteins early early 83% 94% Su et al. (2007) [83] 4 proteins early, M early, all early, M early, all 95% 89% 92% 97% Nosov et al. 4. Serum Markers for Ovarian Cancer In contrast, whereas CA 125 is elevated in 80% of non-mucinous ovarian tumors [69–72]. 1319 The sensitivity and specificity of each serum biomarker panel used for the detection of ovarian cancer are shown; Early: early-staged cases; Late: late-staged cases; All: cases in all stages; S: serous adenocarcinoma; M: mucinous adenocarcinoma. 5. Conclusions For gynecologic cervical, endometrial, and ovarian cancers, only a small handful of tumor-associated antigens, such as SCC and CA 125, have been routinely used as tumor markers. Some markers are useful, not only as diagnostic tools, but also as a predictive marker for the prognosis and the clinical course after treatment. Some recently investigated new serum markers seem to be clinically useful, such as YKL-40 for cervical cancer and HE4 for endometrial and ovarian cancers. Recent breakthroughs in proteomics and bioinformatics technology will expand our understanding of tumor-specific biomarkers. Such investigations will establish newer and more useful biomarkers for the more accurate detection and management of ovarian, endometrial, and cervical cancers. Cancers 2010, 2 1320 Su et al. utilized an MLRM with values for CA 125, ApoA-I, TF, and TTR for the early detection of ovarian cancer [83]. This model provided a sensitivity of 89% and a specificity of 97% for the detection of early stage ovarian cancer. Furthermore, the sensitivity and the specificity of this panel in distinguishing between normal and mucinous ovarian cancer samples were 95% and 92%, respectively. Nosov et al. applied this same MLRM model and marker panel to serous and endometrioid histological types of ovarian carcinomas and demonstrated a sensitivity of 94% and a specificity of 94% for serous ovarian carcinoma in its early stage, and a sensitivity of 98% and a specificity of 98% for endometrioid ovarian carcinoma in its early stage [84]. Visintin et al. proposed a set of serum biomarkers that consisted of leptin, prolactin, osteopontin, insulin-like growth factor II (IGFII), macrophage inhibitory factor (MIF), and CA 125 to discriminate between ovarian cancer patients and healthy women. The panel had a sensitivity of 95% and a specificity of 99% [85]. Not surprisingly, this panel provided a significant improvement over CA 125 alone. However, these studies had the same methodological limitations, of excessive numbers of tumor cases versus matched population controls, described above in the endometrial cancer section. With that being said, novel proteomics-based investigations and bioinformatics analyses still provide the greatest promise of using existing approaches to find ever more accurate and useable biomarkers for gynecological cancers. One important question remains: how long before the diagnosis of an ovarian cancer does the serum level of various markers begin to rise above background levels as a tumor grows? Anderson et al. [86] studied concentrations of CA125, HE4, and mesothelin in serum samples collected from 0–18 years before the diagnosis of a tumor, as part of an unrelated study. They found that the markers may provide some evidence of ovarian cancer up to 3 years before clinical diagnosis, but the more likely lead time for the detection of a change associated with these markers appears to be less than 1 year. Cancers 2010, 2 (2009) [84] 4 proteins early, S early, E early, S early, E 94% 98% 94% 98% Visintin et al. (2008) [85] 6 proteins 95% 99% The sensitivity and specificity of each serum biomarker panel used for the detection of ovarian cancer are shown; Early: early-staged cases; Late: late-staged cases; All: cases in all stages; S: serous adenocarcinoma; M: mucinous adenocarcinoma. Table 8. Novel biomarker panels for the detection of ovarian cancer. 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Cancer Inst. 2010, 102, 26–38. © 2010 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 license (http://creativecommons.org/licenses/by/3.0/). © 2010 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 license (http://creativecommons.org/licenses/by/3.0/).
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Somatotype of Top-Level Serbian Rhythmic Gymnasts
Journal of Human Kinetics
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by Tijana Purenović-Ivanović1, Ružena Popović1 Body size and build influence performance in many sports, especially in those belonging to the group of female aesthetic sports (rhythmic gymnastics, artistic gymnastics, and figure skating). These sports pose high specific demands upon the functional, energy, motor and psychological capacities of athletes, but also upon the size, body build and composition of the performers, particularly of the top-level female athletes. The study of the top athletes (rhythmic gymnasts, in this case) may provide valuable information on the morphological requirements for achieving success in this sport. Therefore, the main objective of this research was to analyze the somatotype of 40 Serbian top-level rhythmic gymnasts, aged 13.04±2.79, and to form the five age group categories. The anthropometric variables included body height, body mass, the selected diameters, girths and skinfolds, and the Heath-Carter anthropometric somatotype. All of the anthropometric data were collected according to International Biological Programme, and then processed in the Somatotype 1.2. The applied analysis of variance indicated an increase in endomorphic component with age. The obtained results show that the balanced ectomorph is a dominant somatotype, being similar for all of the athletes that took part in the research (3.54-3.24-4.5). These results are in line with the ones obtained in previous studies. Key words: rhythmic gymnastics, selection, kinanthropometry, constitution, elite athletes. 181 Journal of Human Kinetics volume 40/2014, 181-187 DOI: 10.2478/hukin-2014-0020 Section III – Sports Training 1 - University of Niš, Faculty of Sport and Physical Education, Serbia. Authors submitted their contribution of the article to the editorial board. Accepted for printing in Journal of Human Kinetics vol. 40/2014 on March 2014. Somatotype of Top-Level Serbian Rhythmic Gymnasts by Tijana Purenović-Ivanović1, Ružena Popović1 Introduction Athletes are characterized by combination of body composition/body size traits which are believed to influence the chance of success in any given sport. Athletes are characterized by combination of body composition/body size traits which are believed to influence the chance of success in any given sport. Therefore, it is suggested that the measurement of kinanthropometry is a crucial tool in the search for information that would assist coaches and athletes in the quest for success at the highest level in sport, i.e. in talent identification (Sánchez- Muñoz et al., 2012). This discipline imposes high demands upon body size, body build and composition of the performers, particularly of elite female athletes. A vast majority of the authors (Lapieza et al., 1993; Menezes and Filho, 2006; Amigo et al., 2009; Poliszczuk and Broda, 2010; Quintero et al., 2011) have proposed one, the most common model of the somatotype that rhythmic gymnasts (RGs) pertain to - the balanced ectomorph - which implies that the ectomorphic component is the dominant one and the remaining two have equal prevalence. However, there are some other models which should not be overlooked, such as the mesoectomorph (Lopéz-Benedicto et al., 1991; Amigo et al., 2009), the mesomorph-ectomorph (Vernetta et al., 2011), as well as the central somatotype (Quintero et al., 2011; Vernetta et al., 2011), and even the balanced endomorph (Quintero et al., 2011). Therefore, it is suggested that the measurement of kinanthropometry is a crucial tool in the search for information that would assist coaches and athletes in the quest for success at the highest level in sport, i.e. in talent identification (Sánchez- Muñoz et al., 2012). Literature describes desirable model characteristics of the elite athletes in the form of basic anthropometric dimensions, their interrelations, body composition components, and somatotypes. Body size and body build contribute significantly to performance in many sports, particularly in aesthetic sports (Misigoj- Durakovic, 2012). Within the group of the so- called female aesthetic sports, the rhythmic gymnastics (RG) is one of the most demanding. Authors submitted their contribution of the article to the editorial board. Accepted for printing in Journal of Human Kinetics vol. 40/2014 on March 2014. 182 Somatotype of top-level Serbian rhythmic gymnasts The body mass was measured with a digital scale Omron BF511 (Kyoto, Japan). Measures and Procedures The measures were taken during 2012 National Championships held in Belgrade (Serbia) on December 16th 2012, and testing was conducted in agreement with the principles stated in the Declaration of Helsinki (WMA, 2002). An anthropometric method was used for obtaining the RGs’ somatic type and it included 10 following variables: body height (in cm), body mass (in kg), four skinfolds (over triceps, subscapular, supraspinale, and calf; in mm), and biceps girth (flexed 90° and tensed; in cm), standing calf girth (in cm), humerus breadth (in cm) and femur breadth (in cm). Analysis The somatotype was determined according to the methodology of Heath-Carter (Carter and Heath, 1990), applying the statistical data analysis (Descriptive statistics and one-way ANOVA) using the computer program Somatotype 1.2. Results The obtained data are presented in tables and graphs (by somatoplots). The sample of 40 top-level Serbian RGs showed that the mean somatotype was: 3.54 - 3.24 - 4.5 (values for the endomorphy, mesomorphy and ectomorphy, respectively; Table 2). The descriptive statistics of all the measurements are presented in Table 2, and all the forty profiles (squares) with the mean somatotype (circle) are presented in Figure 2. Participants Forty rhythmic gymnasts (age: 13.04±2.79 years, body height: 153.64±13.16 cm, body mass: 40.55±11.31 kg) volunteered to participate in the study. A written request was promptly sent to the Expert Committee of Gymnastics Federation of Serbia, and, after being informed about the study, its scientific value and multiple benefits, the approval was given for the testing to be conducted during the 2012 National Championships. All clubs participating in the National Championships were informed about the conducted research and four out of ten RG clubs have given their consent. All participants are an “A” program, group routines competitors. The baseline characteristics of the sample in total, and sub-samples (age categories) are presented in Table 1. Analyzing the somatoplots presented in Figure 1, a variety of somatotype categories can be noticed, with one major category (the ectomorphy) prevailing. It is obvious that an increase in age entails an increase in the value of the endomorphic component while the other two components remain rather stable. This is confirmed by ANOVA due to the presence of statistically significant differences (F=3.6, p=0.015) among the five age categories. The discriminatory factor (apart from the age, height and body mass) was one somatotype component (endomorphy, p<0.001). Such results are common in many other sports. What can be said about the range of RGs’ somatotype components is that they are moderate. Introduction Taking into account the fact that the relevant literature has not established one morphological prototype of rhythmic gymnasts as the dominant one and the fact that there are no relevant data on the somatotype of Serbian RGs, the purpose of the present study was to assess the body constitution of the top-level rhythmic gymnasts in Serbia. The main objective of the study was to determine somatic characteristics of Serbian girls of different age who were competing at the top-level RG and thus provide valuable information for the future selection process. Discussion Apart from talent, the adequate body constitution is a prerequisite for achieving success in sports. Body build is, to a large extent, determined by the human genotype, but within the defined limits it is also subject to environmental influence. The extent of sensitivity to the external environment is also hereditary conditioned. The type of body constitution in the observed RGs was mainly characterized by the prevalence of the ectomorphic component, with moderate values of the other two components (3.54-3.24-4.5). This balanced ectomorph constitution is seen in other RG studies. For All of the measurements were taken by both authors in the optimal climatic conditions, with the participants in underwear, and according to the methods proposed by the International Biological Programme (Weiner and Lourie, 1969). Journal of Human Kinetics volume 40/2014 http://www.johk.pl by Purenović-Ivanović T. and Popović R. 183 example, Lapieza et al. (1993) analyzed the somatotype of 18 female rhythmic gymnasts from Spain, aged 12 to 16 (belonging to cadet and junior teams) and achieved the following mean profile: 2.28–2.45–3.7. What can be noticed are the low values of the first and the second component, which are, at the same time, lower than the values we obtained from our cadet team gymnasts (2.93- 3.63-4.4), as well as the junior team gymnasts (3.91-3.06-4.38). Menezes and Filho (2006) analyzed the somatotype of 24 female Brazilian rhythmic gymnasts (seven from the National RG Team, 10 participants from the 2003 National Championships, and seven participants from the Regional Championships from Rio de Janeiro), and obtained the following profiles: 2.33-2.83-4.17, 2.72-2.65-4.17 and 2.88-3.16-3.51. Even here the low values of the first two components are noticeable as was the case with the previously mentioned study. Poliszczuk and Broda (2010) analyzed 19 female rhythmic gymnasts, aged 8 to 11 and found the following somatotype: 2.65-2.45- 3.95, which, when compared to the somatotype of our novices (2.76-3.6-4.74), shows the low values of the mesomorphic and the ectomorphic components. Lopéz-Benedicto et al. (1991) in the sample of 21 Spanish junior RGs of a regional and national level (11.1 to 15.8 ages), found the meso- ectomorph somatotype profile (2.3-3.1-4.5) as the result of their study. Vernetta et al. (2011) did their research with 20 Andalusian RGs, 9 to 15 years of age, and divided them into two sub- samples: A(N=12): 9-11 years of age (1.804-3.694- 3.701, mesomorphic ectomorph) and B(N=8): 12- 15 years of age (2.059-3.161-4.115, central somatotype). N – number of study participants, SD – standard deviation. N number of study participants, SD standard deviation. Age is presented in years, body height in cm, and body mass in kg. Discussion The total sample showed a central mean somatotype (2.906-3.481-3.866). Table 1 The baseline characteristics of top-level Serbian RGs Age Categories Variable Mean±SD Median Range Novices (N=5) Age 8.99±1.16 8.93 8.07 – 10.92 Body Height 133.32±6.17 133 127.2 – 142.5 Body Mass 25.3±3.4 24.7 22.3 – 30.8 Young juniors (N=12) Age 11.07±0.73 11.17 9.94 – 11.92 Body Height 145.64±7.81 145.9 134 – 158.4 Body Mass 32.37±5.65 31.3 25.9 – 44.5 Cadets (N=6) Age 12.84±0.65 12.9 12 – 13.57 Body Height 152.97±7.36 151.25 143.7 – 164 Body Mass 39.27±4.23 39.35 33.7 – 44.3 Juniors (N=12) Age 14.51±0.78 14.24 13.48 – 16.59 Body Height 164.09±4.27 163.1 158.5 - 171 Body Mass 48.63±4.64 48.05 41.3 – 56.3 Seniors (N=5) Age 18.13±1.18 17.57 16.93 – 19.45 Body Height 168.88±6.22 168.2 161 – 175.1 Body Mass 57.6±3.69 56.2 54 – 61.6 Total (N=40) Age 13.04±2.79 12.9 8.07 – 19.45 Body Height 153.64±13.16 156.75 127.2 – 175.1 Body Mass 40.55±11.31 40.4 22.3 – 61.6 N – number of study participants, SD – standard deviation. Age is presented in years, body height in cm, and body mass in kg. © Editorial Committee of Journal of Human Kinetics © Editorial Committee of Journal of Human Kinetics Somatotype of top-level Serbian rhythmic gymnasts 184 Figure 1 Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profile. 1. Novices (N=5) 2. Young juniors (N=12) 3. Cadets (N=6) 2.76 - 3.6 - 4.74 (mesomorphic ectomorph) 3.33 - 2.96 - 4.99 (balanced ectomorph) 2.93 - 3.63 - 4.4 (mesomorphic ectomorph) 4. Juniors (N=12) 5. Seniors (N=5) 6. Sample in total (all five age categories) 3.91 - 3.06 - 4.38 (endomorph-ectomorph) 4.64 - 3.5 - 3.46 (balanced endomorph) 3.54 - 3.24 - 4.5 (balanced ectomorph) Figure 1 Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profile. 1. Novices (N=5) 2. Young juniors (N=12) 3. Cadets (N=6) 2.76 - 3.6 - 4.74 (mesomorphic ectomorph) 3.33 - 2.96 - 4.99 (balanced ectomorph) 2.93 - 3.63 - 4.4 (mesomorphic ectomorph) 4. Juniors (N=12) 5. Seniors (N=5) 6. Discussion i i l C i f J l f H Ki i Table 2 The descriptive statistics of the measured anthropometric variables of the sample in total (N=40) Variable Mean±SD Median Range Triceps SF 12.74±3.87 11.9 6 – 21.2 Subscapular SF 9±2.48 8.5 4.2 – 14.2 Supraspinale SF 10.03±3.74 9.6 4.2 – 18.8 Calf SF 8.79±3.81 8.1 3 – 17.8 Flexed arm G 22.95±2.68 23.1 17.5 – 28.2 Calf G 30.91±3.62 30.8 24 – 38.1 Humerus B 6.11±0.6 6.17 4.75 – 7.02 Femur B 7.84±0.7 8 6.53 – 8.82 HWR 45.17±1.23 45.05 42.41 – 48 SAD 1.32±0.68 1.32 0.08 – 2.89 Endomorphy 3.54±0.82 3.5 2.1 – 5.3 Mesomorphy 3.24±0.86 3.2 1.8 – 5.5 Ectomorphy 4.5±0.91 4.4 2.5 – 6.6 SF – skinfold, G – girth, B – breadth, HWR – height-weight ratio, SAD – somatotype attitudinal distance, SD – standard deviation. Table 3 The evolution of somatotype with age (Spanish RGs) Study Age N Endo Meso Ecto Amigo et al. (2009) 10 12 1.4 2.6 5.5 11 11 1.4 2.5 5.4 12 15 1.4 2.4 5.5 13 23 1.5 2.4 5.5 14 27 1.6 2.7 5.2 15 25 1.7 2.5 5.1 16 14 1.7 2.6 4.8 17 14 1.9 2.7 4.8 18 10 1.8 2.6 4.9 Quintero et al. (2011) 8 - 11 12 4.25 3.28 4.8 11 - 13 11 4.13 3.01 4.64 13 - 14 23 4.59 3.06 3.89 15 - 19 25 4.45 3.33 3.57 N – number of study participants, Endo – endomorphy, Meso – mesomorphy, Ecto – ectomorphy. Age is presented in years. Table 2 The descriptive statistics of the measured anthropometric variables of the sample in total (N=40) Variable Mean±SD Median Range Triceps SF 12.74±3.87 11.9 6 – 21.2 Subscapular SF 9±2.48 8.5 4.2 – 14.2 Supraspinale SF 10.03±3.74 9.6 4.2 – 18.8 Calf SF 8.79±3.81 8.1 3 – 17.8 Flexed arm G 22.95±2.68 23.1 17.5 – 28.2 Calf G 30.91±3.62 30.8 24 – 38.1 Humerus B 6.11±0.6 6.17 4.75 – 7.02 Femur B 7.84±0.7 8 6.53 – 8.82 HWR 45.17±1.23 45.05 42.41 – 48 SAD 1.32±0.68 1.32 0.08 – 2.89 Endomorphy 3.54±0.82 3.5 2.1 – 5.3 Mesomorphy 3.24±0.86 3.2 1.8 – 5.5 Ectomorphy 4.5±0.91 4.4 2.5 – 6.6 SF – skinfold, G – girth, B – breadth, HWR – height-weight ratio, SAD – somatotype attitudinal distance, SD – standard deviation. Discussion Sample in total (all five age categories) 3.91 - 3.06 - 4.38 (endomorph-ectomorph) 4.64 - 3.5 - 3.46 (balanced endomorph) 3.54 - 3.24 - 4.5 (balanced ectomorph) Journal of Human Kinetics volume 40/2014 http://www.johk.pl Figure 1 Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profile. Figure 2 Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. 1. Novices (N=5) 2. Young juniors (N=12) 3. Cadets (N=6) 2.76 - 3.6 - 4.74 (mesomorphic ectomorph) 3.33 - 2.96 - 4.99 (balanced ectomorph) 2.93 - 3.63 - 4.4 (mesomorphic ectomorph) 4. Juniors (N=12) 5. Seniors (N=5) 6. Sample in total (all five age categories) 3.91 - 3.06 - 4.38 (endomorph-ectomorph) 4.64 - 3.5 - 3.46 (balanced endomorph) 3.54 - 3.24 - 4.5 (balanced ectomorph) Figure 1 Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profil Figure 2 Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. igure 2 Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. Journal of Human Kinetics volume 40/2014 Journal of Human Kinetics volume 40/2014 http://www.johk.pl by Purenović-Ivanović T. and Popović R. 185 urenović-Ivanović T. and Popović R. Discussion Table 3 The evolution of somatotype with age (Spanish RGs) Study Age N Endo Meso Ecto Amigo et al. (2009) 10 12 1.4 2.6 5.5 11 11 1.4 2.5 5.4 12 15 1.4 2.4 5.5 13 23 1.5 2.4 5.5 14 27 1.6 2.7 5.2 15 25 1.7 2.5 5.1 16 14 1.7 2.6 4.8 17 14 1.9 2.7 4.8 18 10 1.8 2.6 4.9 Quintero et al. (2011) 8 - 11 12 4.25 3.28 4.8 11 - 13 11 4.13 3.01 4.64 13 - 14 23 4.59 3.06 3.89 15 - 19 25 4.45 3.33 3.57 N – number of study participants, Endo – endomorphy, Meso – mesomorphy, Ecto – ectomorphy. Age is presented in years. Table 2 The descriptive statistics of the measured anthropometric variables of the sample in total (N=40) Variable Mean±SD Median Range Triceps SF 12.74±3.87 11.9 6 – 21.2 Subscapular SF 9±2.48 8.5 4.2 – 14.2 Supraspinale SF 10.03±3.74 9.6 4.2 – 18.8 Calf SF 8.79±3.81 8.1 3 – 17.8 Flexed arm G 22.95±2.68 23.1 17.5 – 28.2 Calf G 30.91±3.62 30.8 24 – 38.1 Humerus B 6.11±0.6 6.17 4.75 – 7.02 Femur B 7.84±0.7 8 6.53 – 8.82 HWR 45.17±1.23 45.05 42.41 – 48 SAD 1.32±0.68 1.32 0.08 – 2.89 Endomorphy 3.54±0.82 3.5 2.1 – 5.3 Mesomorphy 3.24±0.86 3.2 1.8 – 5.5 Ectomorphy 4.5±0.91 4.4 2.5 – 6.6 SF – skinfold, G – girth, B – breadth, HWR – height-weight ratio, SAD – somatotype attitudinal distance, SD – standard deviation. f g g g SAD – somatotype attitudinal distance, SD – standard deviation. Table 3 The evolution of somatotype with age (Spanish RGs) Study Age N Endo Meso Ecto Amigo et al. (2009) 10 12 1.4 2.6 5.5 11 11 1.4 2.5 5.4 12 15 1.4 2.4 5.5 13 23 1.5 2.4 5.5 14 27 1.6 2.7 5.2 15 25 1.7 2.5 5.1 16 14 1.7 2.6 4.8 17 14 1.9 2.7 4.8 18 10 1.8 2.6 4.9 Quintero et al. (2011) 8 - 11 12 4.25 3.28 4.8 11 - 13 11 4.13 3.01 4.64 13 - 14 23 4.59 3.06 3.89 15 - 19 25 4.45 3.33 3.57 N – number of study participants, Endo – endomorphy, Meso – mesomorphy, Ecto – ectomorphy. Age is presented in years. © Editorial Committee of Journal of Human Kinetics Somatotype of top-level Serbian rhythmic gymnasts 186 Amigo et al. Discussion (2009) (N=151 RGs, aged 10- 18) and Quintero et al. (2011) (2008 Tenerife Championships, N=70 RGs, aged 8-19) conducted longitudinal studies with national and international level Spanish RGs (results are presented in Table 3) and obtained quite different results, which can be noted when the somatotype components’ values of the gymnasts of the same age are compared. Namely, Spanish gymnasts from the study by Amigo et al. (2009) are of great somatotype stability across ages, and they have the meso-ectomorphic and balanced ectomorph body constitution, i.e. very low endomorphic and mesomorphic component values and very high ectomorphic values. In the second study (Quintero et al., 2011), one can notice the moderate values for all of the three somatotype components, but with an endomorphic value as the highest one (an increase of the endomorphic component with age). However, in the study by Quintero et al. (2011), authors did not specify the competition level of studied RGs, which could be possible explanation for the discrepancies between the obtained results of these two studies. amongst the five age group categories of the top- level Serbian RGs were established, and they indicate that the endomorphic component increases with age, while the other two components are rather stable (this could be due to maturation, or even genetics and nutrition factor). Having also in mind the fact that there is no evidence of this two-way causality between the body constitution and sport (is the somatic type a cause or a consequence?), this interpretation of our findings still does not represent sufficient information for the talent identification process in RG. It could, however, inform about where the Serbian RG was positioned in relation to more successful RG countries. Study Limitations Having in mind the fact that results in sports do not depend solely on the physique of an athlete, at this point we can detect the main flaw of the study: it does not take into consideration other abilities that influence performance in sports (such as motor abilities, psychological state, functional abilities, etc.). Another area that could be improved would be the sample of participants and a study design type, which hinders the authors from more significant conclusions. Maturity, of which the tempo and the period of time of its occurrence varies and is associated with an increase of the body height and body mass, could also represent the key points for a variety of body constitution types. Therefore, it is recommended that the status of the Serbian rhythmic gymnasts should undergo multidisciplinary analysis on the basis of a larger and more representative sample, monitored over a period of few years. Conclusions All of the previous research results mostly indicate graceful and thin (ectomorphic) body build of rhythmic gymnasts, characterized by long, slim and thin limbs, with small circumferences of muscles and thin and light bones, made for subtle and graceful movements. However, top-level Serbian RGs can be characterized as balanced ectomorph, with not so low values for endomorphic and mesomorphic components, which is not seen as consistent with the somatotype of RGs from other countries (the ones that have higher ranking in rhythmic gymnastics). Apart from that, the differences Acknowledgements: The authors would like to thank the Gymnastics Federation of Serbia, the organizer of the 2012 National Championships, the rhythmic gymnasts and the coaches of RG Serbian clubs: “Ritam-Pinki”, “Ritam”, “Radnički” and “Paraćin”, as well as the coach and Junior RGs Group routines national team of Serbia, who made this study possible. This research is a part of the project of the Ministry of Science and Technological Development of the Republic of Serbia (No: 179019, Head researcher: Prof. R. Stanković). References Amigo A, Sala V, Faciabén A, Evrard M, Marginet M, Zamora L. Height, weight, somatotype and body composition trends in rhythmic Spanish elite gymnasts from childhood to adulthood. Apunts. Journal of Human Kinetics volume 40/2014 http://www.johk.pl 187 by Purenović-Ivanović T. and Popović R. Educacíon Física y Deportes, 2009; 95(1): 64-74 Educacíon Física y Deportes, 2009; 95(1): 64-74 Carter JEL, Heath BH. Somatotyping. Development and Applications. Cambridge: Cambridge University Press; 1990 Lapieza MG, Nuviala RJ, Castillo MC, Giner A. Morphological characteristics in adolescent rhythmic gymnasts and swimmers. Apunts. Medicina de l'Esport, 1993; 30(118): 255-263 Lopéz-Benedicto A, Franco L, Terreros J. Rhythmic gymnastics. Physiological and anthropometrical evolution in the season. Archivos de medicina del deporte, 1991; 8(30): 127-133 Menezes LS, Filho JF. Identification and comparison of dermatoglyfics, somatotype and basic physical aptitude characteristics of rhythmic gymnasts of different qualification levels. Fitness & Performance Journal, 2006; 5(6): 393-401 Misigoj-Durakovic M. Anthropometry in Premenarcheal Female Esthetic Sports Athletes and Ballerinas. In V. R. Preedy (Ed.), Handbook of Anthropometry (pp. 1817-1836). New York: Springer; 2012 Poliszczuk T, Broda D. Somatic constitution and the ability to maintain dynamic body equilibrium in girls practicing rhythmic gymnastics. Pediatric Endocrinology, Diabetes and Metabolism, 2010; 16(2): 94-99 Quintero B, Martín A, Henríquez J. The anthropometric profile of rhythmic gymnasts. Apunts. Educación Física y Deportes, 2011; 103(1): 48-55 Sánchez-Muñoz C, Zabala M, Williams K. Anthropometric Variables and its Usage to Characterise Elite Youth Athletes. In V. R. Preedy (Ed.), Handbook of Anthropometry (pp. 1865-1888). New York: Springer; 2012 Vernetta M, Fernández E, López-Bedoya J, Gómez-Landero A, Oña A. The relations between morphological profile and body esteem of Andalusian rhythmic gymnastics team. Motricidad. European Journal of Human Movement, 2011; 26: 77-92 Weiner JS, Lourie JA. Human Biology, A guide to Field methods. International Biological Programme. Edinburgh: Blackwell Scientific Publications; 1969 World Medical Association. World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects, 2002. Available at http://www.fda.gov/ohrms/dockets/dockets/06d0331/06D-0331-EC20-Attach-1.pdf; accessed on 01.05.2013 © Editorial Committee of Journal of Human Kinetics © Editorial Committee of Journal of Human Kinetics
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Kapasiteitsbou van informele gemeenskapsgebaseerde organisasies in die Suid-Afrikaanse welsynsektor: Die bydrae deur gevestigde maatskaplike diensorganisasies
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Kapasiteitsbou van informele gemeenskapsge­baseerde organisasies in die Suid-Afrikaanse welsyn­sektor: Die bydrae deur gevestigde maatskap­like diensorganisasies Capacity-building of informal community-based organisations in the South African welfare sector: The contribution of established social service organisations Shanie Boshoff PhD-gegradueerde Departement Maatskaplike Werk Stellenbosch Universiteit E-pos: headoffice@acvv.org.za Lambert K. Engelbrecht Departement Maatskaplike Werk Stellenbosch Universiteit E-pos: lke@sun.ac.za Shanie Boshoff PhD-gegradueerde Departement Maatskaplike Werk Stellenbosch Universiteit E-pos: headoffice@acvv.org.za Lambert Engelbrecht Shanie Boshoff Lambert Engelbrecht Shanie Boshoff Lambert K. Engelbrecht Departement Maatskaplike Werk Stellenbosch Universiteit E-pos: lke@sun.ac.za Shanie Boshoff Lambert Engelbrecht Lambert Engelbrecht Shanie Boshoff is ’n PhD-gegradueerde van die Departement van Maatskaplike Werk aan die Universiteit van Stellenbosch. Sy het haar Meesters­ graad in Maatskaplike Werk in Maart 2006 aan die genoemde universiteit behaal. Sy is die hoof- uitvoerende beampte van die ACVV, die oudste nasionale welsynsorganisasie in Suid-Afrika wat uitgebreide ontwikkelings- en beskermings­dienste aan kinders, hul gesinne en aan ouer persone lewer. Sy het ’n besondere belangstelling in organisasie­ ontwikkeling, die ontwikkeling van vrywillger- besture en die benutting van sterktes in bestuur en dienslewering. Sy dien op verskeie nasionale en provinsiale gespreksforums vir maatskaplike diens- verskaffers. Tans dien sy as visevoorsitter van NACOSS (National Coalition of Social Services), ’n gespreksforum vir nasionale gevestigde maat­ skaplike diensverskaffers in Suid-Afrika, en op die Uitvoerende Komitee van die Wes-Kaapse Direk­ teureforum. Shanie Boshoff is a PhD graduate at the Depart­ ment of Social Work at the University of Stellen­ bosch. In March 2006 she obtained her MA Degree in Social Work at the said university. She is the chief executive officer of the ACVV, the oldest national welfare organisation in South Africa, rendering extensive development and protection services to children, their families and older persons. Her interests lie especially in the fields of organisational development, the development of volunteer- management boards, and the use of strengths in management and service delivery. She serves on several national and provincial discussion forums of social service providers. She is currently serving as vice-chairperson of NACOSS (National Coalition of Social Services), a discussion forum of established social service providers in South Africa, and on the Executive Committee of the Western Cape Director’s Forum. Lambert Engelbrecht teaches in the Depart­ ment of Social Work at the University of Stellen­ bosch. He has extensive experience as a social work prac­titioner and community developer in vulnerable communities. His interests lie in the fields of social work management and supervision, as well as strategies for poverty alleviation in social work Lambert Engelbrecht is verbonde aan die Departement Maatskaplike Werk van die Univer­ siteit van Stellenbosch. 592 592 ABSTRACT S C Capacity-building of informal community-based organisations in the South African welfare sector: The contribution by established social service organisations Capacity-building of informal community-based organisations in the South African welfare sector: The contribution by established social service organisations Large-scale poverty and increasing needs prevail in South Africa. The National Department of Social Development has the constitutional mandate to provide sector-specific national leadership in social development. Despite the Department of Social Development’s intention to restructure the social service delivery system since 1997 and although established social service organisations, as well as informal community-based organisations (CBOs), are regarded as being valuable resources of service delivery to communities at risk, all these organisations are threatened by serious challenges. Established social service organisations that provide and maintain a significant portion of the social welfare services in South Africa, are at present – despite their efficient management – engulfed in a grim battle for survival. CBOs which form an integral part of communities at risk are still excluded from state financing because they do not comply with the basic requirements of management. The Policy on Financial Awards for Social Service Providers (2012a) of the Department of Social Development indicates that the State aims to redirect its financing from established social service organisations to CBOs and to demand that established organisations build the capacity of CBOs in addition to their own direct service delivery to those at risk. y An investigation into the capacity-building of CBOs within the South African context is therefore at the same time both relevant and important. The purpose of this study is to develop an understanding of the contribution that established social service organisations in South Africa already make to the capacity-building of CBOs. The investigation not only provides information on the nature of this capacity-building, but also focuses attention on the successes and many challenges experienced by established social service organisations in their capacity-building of CBOs. Qualitative research was chosen as the approach to explore the contributions of established social service organisations to the capacity-building of CBOs in the social welfare sector and to establish the link with the social development approach as it is applied in South Africa. At the same time capacity-building of CBOs by established social service organisations is analysed and described. Kapasiteitsbou van informele gemeenskapsge­baseerde organisasies in die Suid-Afrikaanse welsyn­sektor: Die bydrae deur gevestigde maatskap­like diensorganisasies Capacity-building of informal community-based organisations in the South African welfare sector: The contribution of established social service organisations Hy beskik oor uitgebreide praktykervaring as maatskaplike werker en gemeen­ skapsontwikkelaar in kwesbare gemeen­skappe. Sy belangstellingsvelde is maatskaplike­werkbestuur en supervisie, sowel as armoedeverligting­strategieë in Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 593 service delivery. His role as lecturer, his engagement in research and interaction with the community find common purpose in his involvement in the management of non-governmental organisations and the supervision of social workers. Several of his books, book chapters, articles and papers on this subject have been published and presented nationally and internationally. He is involved in several inter­ national research projects on the impact of neolibe­ ralism on social welfare services and the manage­ ment of social work organisations. service delivery. His role as lecturer, his engagement in research and interaction with the community find common purpose in his involvement in the management of non-governmental organisations and the supervision of social workers. Several of his books, book chapters, articles and papers on this subject have been published and presented nationally and internationally. He is involved in several inter­ national research projects on the impact of neolibe­ ralism on social welfare services and the manage­ ment of social work organisations. maatskaplikewerk-dienslewering. Hy integreer akademiese onderrig, sy navorsingsfunksie en gemeenskapsinteraksie in sy betrokkenheid by die bestuur van nieregeringsorganisasies en supervisie van maatskaplike werkers. Verskeie boeke, boekhoofstukke, artikels en referate oor hierdie onderwerp is reeds deur hom nasionaal en inter­ nasionaal gepubliseer en aangebied. Hy is betrokke by verskeie internasionale navorsings­projekte oor die impak van neoliberalisme op maatskaplike welsynsdienste en die bestuur van maatskaplikewerk- organisasies. Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 ABSTRACT An instrumental case study was used as the research strategy by using NACOSS (National Coalition of Social Services) as a discussion forum of established social service providers in South Africa. A purposive non-probability sample selection was used to select participants, Nine NACOSS members indicated in a survey that they are already involved in the capacity- building of CBOs and agreed to participate in the study. Data was collected on three levels: (a) Semi-structured interviews were conducted with 9 chief executive officers of 9 established social Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 594 service organisations, (b) 15 social workers who are already building CBOs’ capacities, and (c) 9 representatives of the CBOs whose capacities were being built by the participating service organisations. service organisations, (b) 15 social workers who are already building CBOs’ capacities, and (c) 9 representatives of the CBOs whose capacities were being built by the participating service organisations. i g Based on this study, the following main findings and recommendations are made: • Capacity-building of CBOs, as it is currently operationalised in developmental social work by established service organisations, is an inherent part of social development. Therefore, capacity-building of CBOs is rather more a social work intervention than exclusively one of organisational development and is currently being interpreted and applied in this way by established service organisations. The focus of capacity-building of CBOs as a social work intervention is therefore on informal systems or bodies within communities at risk. • Capacity-building in practice is far more elementary than what is assumed to be the case in theory and policy. • Capacity-building in practice is far more elementary than what is assumed to be the case in theory and policy. • CBOs’ urgent need for continuous practice-oriented support from service organisations is evident, thus confirming the necessity for this kind of support to form an integral part of the capacity-building process. In this respect established social service organisations are excellently positioned to make a valuable contribution to the capacity-building of CBOs. • Due to the challenges of service delivery, the State should rather view the service organisations as resources in the restructuring of the South African social service delivery system than considering the redirection of financing from essential services by established service organisations to the informal sector. Urgent consideration should be given to providing state funding for the capacity-building of CBOs. TREFWOORDE: DE: Kapasiteitsbou, gevestigde maatskaplike diensorganisasies, informele gemeenskapsgebaseerde organisasies, maatskaplike ontwikkeling, maatskaplike werk KEY CONCEPTS: TREFWOORDE: Kapasiteitsbou, gevestigde maatskaplike diensorganisasies, informele gemeenskapsgebaseerde organisasies, maatskaplike ontwikkeling, maatskaplike werk ABSTRACT Constructive discussion between the Department of Social Development and established social service organisations is crucial for the advancement of an effective social service delivery system. KEY CONCEPTS: Capacity building, established social service organisations, informal community-based organisations, social development, social work S: Capacity building, established social service organisations, informal community-based organisations, social development, social work Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 OPSOMMING Ná twintig jaar van demokrasie is Suid-Afrika steeds ’n verdeelde samelewing met grootskaalse armoede en hoë vlakke van kwesbaarheid. Ontwikkeling van alle sektore van die samelewing word deur die Regering as oplossing gesien. Deur middel van die Departement Maatskaplike Ontwikkeling se finansieringsbeleid (Departement van Maatskaplike Ontwikkeling 2012a) plaas die Staat druk op gevestigde maatskaplike diensorganisasies om tot kapasiteitsbou van informele gemeen­ skapsgebaseerde organisasies by te dra. Die doel van hierdie artikel is om begrip te ontwikkel vir die bydrae van gevestigde maatskaplike diensorganisasies tot die kapasiteitsbou van informele gemeenskapsgebaseerde organisasies (voortaan in hierdie artikel na verwys as “IGO’s”) ten einde grondige bevindinge en aanbevelings daarvoor te kan maak. Om hierdie doel te bereik, word die agtergrond van die onderwerp in meer detail belig, word die navorsingsmeto­dologie wat gevolg is, verduidelik en word die bevindinge aangebied deur middel van ʼn geïntegreerde sintese van deelnemers se beskrywings van gevestigde organisasies en IGO’s, hulle beskouings van suksesse behaal en hulle reflektering van verdere uitdagings in kapasiteitsbou­pogings. Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 595 INLEIDING Maatskaplike welsynsdienste aan kwesbare mense in Suid-Afrika word gelewer deur die regeringsektor sowel as die niewinsgerigte nieregeringsektor (Departement van Maatskaplike Ontwikkeling 2012a). Daar is egter twee hoofkategorieë van niewinsgerigte maatskaplike diens­ verskaffers wat in die burgerlike samelewing bestaan, naamlik gevestigde of formele maatskaplike diensorganisasies (in die spreektaal dikwels na verwys as “welsynsorganisasies”) en IGO’s (Departement van Maatskaplike Ontwikkeling 2006; Patel 2005). Beide van hierdie groeperinge van niewinsgerigte maatskaplike diensverskaffers word as waardevolle hulpbronne vir kwesbare mense beskou en kan ’n besondere bydrae tot maatskaplike ontwikkeling lewer, maar beide organisasiegroeperinge ervaar ernstige uitdagings ten opsigte van volhoubare dienslewering. Desondanks blyk dit dat die Departement Maatskaplike Ontwikkeling (2012a) aanvoer dat gevestigde maatskaplike diensorganisasies ʼn addisionele verantwoordelikheid het om die kapasiteit van IGO’s te bou ten einde vir voortgesette staatsfinansiering te kan kwalifiseer. Geen empiriese studie is egter tans beskikbaar oor die werklike bydrae wat gevestigde maatskaplike diensorgani­ sasie tot die kapasiteitsbou van IGO’s lewer nie. AGTERGROND Naas IGO’s is gevestigde maatskaplike diensorganisasies wat nieregeringsorganisasies is, een van die grootste niewinsgerigte maatskaplike diensverskaffers in Suid-Afrika wat beduidende uitgebreide maatskaplike dienste aan kwesbare individue, gesinne en gemeenskappe lewer (Departement van Maatskaplike Ontwikkeling 2012a, 2012b; Ministerie van Welsyn en Bevolkingsontwikkeling 1997; Patel 2005). Die Tesourie Uitgawe Oorsig-dokument vir 2005/2006 tot 2011/2012 toon dat niewinsgerigte organisasies meer as 98% van die maatskaplike welsyns­ fasiliteite in Suid-Afrika bedryf en aan meer as 71% van maatskaplike verbruikers dienste lewer (Departement van Maatskaplike Ontwikkeling 2012b). Oorhoofs onderskei gevestigde welsynsorganisasies hulself van IGO’s ten opsigte van die volgende aspekte: hulle het professionele personeel in diens waarvan maatskaplike werkers ’n beduidende rol speel; beskik oor gevestigde organisasiestrukture; volg bepaalde skriftelike beleide en praktyke; beskik oor ’n eie organisasiekul­ tuur; streef bepaalde doelwitte van die organisasie na en ontvang staatsubsidie van die Departement van Maatskaplike Ontwikkeling (Habib & Kotzé 2002; Hanson 1995; Patel1998; Republiek van Suid-Afrika 1997; Swilling & Russel 2001). Hierdie eienskappe en gestruktureerd­heid bied juis aan gevestigde welsynsorganisasies of maatskaplike diensorganisasies die vermoë om aan die vereistes vir die verkryging van staatsfinansiering te kan voldoen. Tog ervaar gevestigde maatskaplike diensorganisasies – ten spyte van effektiewe bestuur – tans ’n bestaanskrisis om as maatskaplike diensverskaffers finansieel te oorleef (Davids 2012; Swart 2012; NACOSS 2012a; 2013; NAWONGO 2011). IGO’s is in teenstelling met gevestigde maatskaplike diensorganisasies slegs in dié mate georganiseerd dat hulle integraal deel van kwesbare gemeenskappe vorm, maar tog hulself in die gemeenskap as maatskaplike entiteite onderskei wat sekere dienste aan armes en kwesbares lewer. Hierdie dienste word op ’n vrywillige basis gelewer (Govender 2001; Molefe 1996; Patel 1998; 2005; Republiek van Suid-Afrika 1997; Swilling & Russel 2001). Alhoewel IGO’s soms as projekte van gevestigde maatskaplike diensorganisasies ontstaan, kom daar ’n punt wanneer hul uit vrye keuse óf amptelik affilieer by die diensorganisasie, óf kies om as onafhanklike entiteite verder te funksioneer. Ten spyte van die Departement van Maatskaplike Ontwikkeling se fokus om veral IGO’s te ondersteun in die verkryging van toegang tot staatsfinansiering, is die IGO’s tans steeds Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 596 grootliks uitgesluit van staatsfinansiering omdat hulle nie aan die nodige basiese staatsvereistes voldoen vir die kwalifisering van staatsfinansiering nie (Departement van Maatskaplike Ontwikkeling 2012a; 2012b; Ministerie van Welsyn en Bevolkingsontwikkeling 1997; Patel 2005). g ; ; y g g ; ) Die Staat se beskouing van niewinsgerigte maatskaplike diensverskaffers blyk merendeels polities-georiënteerd te wees. AGTERGROND Gevestigde maatskaplike diensorganisasies word beskou as daardie organisasies wat juis in staat is om uitgebreide dienste te lewer, omdat hulle bevoordeel is deur die staatsondersteuning wat hulle voor 1994 in die apartheidsjare ontvang het (Departement van Maatskaplike Ontwikkeling 2012a; 2012b). Daarteenoor word IGO’s deur die Staat beskou as daardie organisasies wat tot 1994 uitgesluit was van staatsfinansiering, maar ’n onontbeerlike bydrae in die apartheidsjare gelewer het tot die aanspreek van die direkte nood van kwesbare gemeenskappe (Govender 2001; Ministerie van Welsyn en Bevolkingsontwikkeling 1997; Patel 2005; Swilling & Russel 2001). Die Departement se mees resente Beleid op Finansiële Toekennings aan Maatskaplike Diensverskaffers (Departement Maatskaplike Ontwikkeling 2012a) (voortaan in hierdie artikel slegs na verwys as “Finansieringsbeleid”) voer egter die onderskeid tussen gevestigde maatskaplike diensorganisasies en IGO’s verder en beklemtoon die verantwoordelikheid van gevestigde maatskaplike diensorganisasies om die kapasiteit van IGO’s te bou sodat hulle vir voortgesette staatsfinansiering kan kwalifiseer. In teenstelling met die Suid-Afrikaanse Nasionale Ontwikkelingsplan (National Planning Commission 2011) se voorstel dat befondsing aan gevestigde maatskaplike diensverskaffers verhoog word, dui die Departement van Maatskaplike Ontwikkeling se praktyke en Finansieringsbeleid daarop dat die Staat inderwaarheid ten doel het om hul finansiering van die gevestigde diensverskaffers na IGO’s te herlei. Alhoewel hierdie herleiding nog nie plaasgevind het nie, sal dit ernstige gevolge vir maatskaplike gevestigde diensorganisasies inhou wat kan lei tot die ineenstorting van dienslewering aan kwesbares deur die gevestigde maatskaplike diensorganisasies. g g p g Die Staat se beskouing van diensverskaffers, sy siening oor die herstrukturering van die maatskaplike diensleweringsisteem en gepaardgaande finansieringsbeleid spruit voort uit die Staat se benadering tot maatskaplike dienslewering. Die Witskrif vir Welsyn (Ministerie vir Welsyn en Bevolkingsontwikkeling 1997) stel maatskaplike ontwikkeling as die verkose benadering vanaf 1997. Hierdie maatskaplike ontwikkelingsgerigte benadering is gefundeer in reaksie op die land se vorige apartheidsbestel en fokus onder andere op toeganklikheid van maatskaplike welsynsdienste vir spesifiek voorheen benadeelde en kwesbare mense (Departement van Maatskaplike Ontwikkeling 2005, 2012a; Gray 2006; Ministerie van Welsyn en Bevolkings­ ontwikkeling 1997; Lombard 2011; Midgley 1997; Patel 2005). Deur hierdie beleidsraamwerk het die Departement van Maatskaplike Ontwikkeling sy benadering tot maatskaplike dienslewering van ’n maatskaplike behandelingsmodel na ’n ontwikkelingsgerigte diensleweringsmodel verskuif en klem geplaas op die daadwerklike herstrukturering van die bestaande maatskaplike diensleweringsisteem (Departement van Maatskaplike Ontwikkeling 2006; Patel 2005; Ministerie vir Welsyn en Bevolkingsontwikkeling 1997). AGTERGROND Deur die Wet op Organisasies Sonder Winsoogmerk (Republiek van Suid-Afrika, 1997) poog die Staat om die minimum vereistes te stel waaraan organisasies moet voldoen om finansiering te bekom. Registrasie in terme van hierdie Wet sal dus as bewys aan die Staat, ander befondsers en die publiek dien dat die betrokke organisasies maatskaplike welsynsdienste verantwoordelik en verantwoordbaar bestuur (Republiek van Suid-Afrika 1997). Die Staat het in die vooruitsig gestel dat dié Wet die meganisme aan IGO’s sou bied om toegang te verkry tot staatsfinansiering, wat weer sou lei tot groter volhoubaarheid van IGO’s. Ten spyte van insette deur die Departement van Maatskaplike Ontwikkeling om die kapasiteit van IGO’s sodanig te ontwikkel dat hulle kwalifiseer vir staatsfinansiering, erken die Departement van Maatskaplike Ontwikkeling onom­ Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 597 wonde dat, alhoewel IGO’s 53% van die beraamde totaal van niewinsgerigte nieregeringsorga­ nisasies in die Suid-Afrikaanse welsynsektor uitmaak (Swilling & Russel 2001), hierdie organisasies steeds nie in status gelyk is aan gevestigde maatskaplike diensorganisasies nie en steeds uitgesluit word van staatsfinansiering juis omdat hulle nie aan die nodige staatsvereistes voldoen nie (Departement van Maatskaplike Ontwikkeling 2012b). Dit is egter onduidelik waarom daar nog nie genoegsame vordering gemaak is om IGO’s in staat te stel om aan staatsvereistes vir staatsfinansiering te voldoen nie. Een van die redes blyk te wees dat IGO’s dikwels nie oor die nodige finansiële stelsels beskik om staatsfinansiering op ’n verantwoordbare en voorgeskrewe wyse te hanteer en daarvan boek te hou nie (Departement van Maatskaplike Ontwikkeling 2012a). Hieruit word afgelei dat die kapasiteit van IGO’s nie genoegsaam ontwikkel of versterk word deur die Wet op Organisasies Sonder Winsoogmerk (Republiek van Suid-Afrika 1997) nie. Meer deurlopende plaaslike aksies en ondersteuning blyk dus nodig te wees om die kapasiteit van IGO’s te versterk en dit is juis hiérin waar gevestigde maatskaplike diensorganisasies, wat reeds uitge­ breide dienste op grondvlak lewer, ’n bydrae kan lewer. ’n Opname by ’n gespreksforum van nasionale gevestigde maatskaplike diensorganisasies in 2012 (NACOSS 2012b) het aangedui dat daar reeds ’n beduidende aantal nasionale organisasies is wat vrywillig die kapasiteit van IGO’s bou. Uit die opname blyk dit duidelik dat kapasiteitsbou van IGO’s as een van die vorme van maatskaplikewerk-intervensie deur gevestigde maatskaplike diensorganisasies toegepas word en dat die fokus van maatskaplike ontwikkeling in kapasiteitsbou op organisasievlak of die vlak van die institusionele instelling lê. AGTERGROND Gevestigde maatskaplike diensorganisasies het die ervaring ten opsigte van die nakoming van staatsvereistes en staatsfinansiering. Hulle lewer saam met IGO’s dienste in kwesbare gemeenskappe, wat hulle gunstig plaas om in bepaalde konteks praktykgerigte ondersteuning aan IGO’s te bied. Teoretiese en empiriese studies oor die bydrae van nasionale gevestigde maatskaplike diensorganisasies wat reeds die kapasiteit van IGO’s bou, is egter beperk. Studies wat wél beskikbaar is, soos dié deur Symes (2007) en Yachakaschi (1995), fokus op die kapasiteitsbou van IGO’s deur spesialiteitsorganisasies en verwys nie na gevestigde maatskaplike diensorgani­sasies waarvan die kernbesigheid direkte dienslewering aan kwesbare individue, groepe en gemeenskappe behels nie. Ten spyte daarvan dat die kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensorganisasies as vereiste in die Departement van Maatskaplike Ontwikkeling se Finansieringsbeleid (Departement van Maatskaplike Ontwikkeling 2012a) gestel word, is daar tans nie inligting beskikbaar oor die bydrae wat gevestigde maatskaplike diensorganisasies alrééds lewer tot die kapasiteitsbou van IGO’s of die uitdagings wat hulle ten opsigte daarvan ervaar nie. Daar is ook nie inligting beskikbaar oor die wyse waarop die kapasiteit van IGO’s binne die Suid-Afrikaanse konteks en binne die maatskaplike ontwikkelingsgerigte benadering toegepas word nie. Empiriese navorsing is dus nodig om die bydrae van kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensorganisasies binne die konteks van die Suid-Afrikaanse maatskaplike ontwikkelingsgerigte benadering te verken. Sodanige verkenning kan ’n waardevolle perspektief bied op die rol en bydrae deur die gevestigde maatskaplike diensorganisasies in die kapasiteitsbou van IGO’s. Navorsingsresultate kan lei tot die ontwikkeling van ’n teoretiese raamwerk vir kapasiteitsbou van Suid-Afrikaanse IGO’s deur gevestigde maatskaplike diensorganisasies wat verder in die praktyk getoets kan word. NAVORSINGSMETODOLOGIE In hierdie studie is ’n kwalitatiewe navorsingsbenadering gebruik om kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensorganisasies as maatskaplike beginsel te verstaan en te ondersoek, aangesien min inligting daaroor bekend is (Bless, Higson-Smith & Kagee 2006; Fouché Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 598 & Delport 2014; Mouton 2006). ’n Instrumentele gevallestudie is as navorsingstrategie gekies omdat dit fokus op die bestudering van kapasiteitsbou van IGO’s as fenomeen en hoe gevestigde diensorganisasies hierdie fenomeen vanuit ’n maatskaplike ontwikkelingsgerigte benadering ervaar (Creswell 2013; Fouché & Delport 2014; Fouché & Schurink 2014). Beide beskrywende en verkennende navorsingsontwerpe is in kombinasie gebruik omdat inligting oor die navorsingsonderwerp beperk is en ’n beduidende insig in ’n bepaalde fenomeen sal bied. Beskrywende vrae verwys na vrae soos “wat is die verhouding tussen gevestigde maatskaplike diensorganisasies en IGO’s?” en verkennende vrae na vrae soos “watter uitdagings word aan gevestigde organisasies vir kapasiteitsbou van IGO’s gestel?”. Die 19 gevestigde diensorganisasies wat lede van NACOSS is (“National Coalition of Social Services”, ’n gespreksliggaam van gevestigde maatskaplike diensorganisasies in Suid-Afrika, wat as sodanig deur die Departement van Maatskaplike Ontwikkeling erken word), vorm die universum van die studie (Strydom & Delport 2014; NACOSS 2012b). Daar sal voortaan in hierdie artikel slegs verwys word na “NACOSS”. Deur middel van ’n opname is bepaal dat 11 van die 19 lede (organisasies) reeds betrokke is by die kapasiteitsbou van IGO’s en word sodanige lede as die populasie in hierdie studie geneem (Strydom & Delport 2014; Tutty, Rothery & Grinnell 1996). ’n Doelbewuste steekproef (Strydom & Delport 2014; Monette, Sullivan & De Jongh 2008; Oliver 2010) is uitgevoer en het bestaan uit nege NACOSS-lede (diensorganisasies) wat aangedui het dat hulle betrokke is by kapasiteitsbou van IGO’s en bereid is om aan die studie deel te neem. ’n Semi- gestruktureerde onderhoudskedule is as ’n navorsingsinstrument benut sodat ’n meer omvattende beskrywing van deelnemers se persepsies en gewaarwordinge oor die kapasiteitsbou van IGO’s verkry kon word (Bless et al. 2006; Greeff 2014; Gochros 2005; Tutty et al. 1996). 1 Bless et al. (2006); Cherry (2000); Darlington & Scott (2002); Goodman (2001); Grinnell & Unrau (1995); Monette et al. (2008); Strydom (2014); Williams, Tutty & Grinnell (1995). NAVORSINGSMETODOLOGIE y ( y ) Data-insameling het op drie vlakke deur middel van semi-gestruktureerde onderhoude plaasgevind, naamlik met hoofuitvoerende beamptes en maatskaplike werkers van gevestigde diensorganisasies en ook met verteenwoordigers van IGO’s wie se kapasiteit hulle bou: (a) Onderhoude is met nege hoofuitvoerende beamptes gevoer (een van elke deelnemende organisasie); (b) vyftien onderhoude is met maatskaplike werkers gevoer (verteenwoordigend van elkeen van die deelnemende organisasies wat regstreeks by kapasiteitsbou van IGO’s betrokke is); en (c) nege onderhoude is met verteenwoordigers van IGO’s gevoer (waarvan die kapasiteit deur die nege deelnemende organisasies gebou word). Die onderhoude het elk tussen 60 en 75 minute geduur. g g ) g In hierdie studie het die navorser geloofwaardigheid, oordraagbaarheid en afhanklikheid as strategieë vir ʼn geldigheidsverklaring gebruik om die proses van data-insameling en data-analise se geldigheid te staaf (Barusch, Gringeri & George 2011; Creswell 2013; Strydom 2014; Whittemore, Chase & Mandle 2001). Die volgende etiese oorwegings het in die studie gegeld en word kortliks genoem:1 Etiese risiko’s is geëvalueer; etiese klaring is deur die Universiteit van Stellenbosch toegeken; die inligting verkry, is professioneel en vertroulik hanteer ingevolge die SARMD se Etiese Kode; ingeligte toestemming is van elke deelnemer aan die studie verkry; die gevolge van deelname aan die studie is deeglik met deelnemers deurgewerk; die skending van privaatheid is teengewerk; en daar is gelet op deelnemers se behoefte aan ontlonting. 2 Vergelyk Departement van Maatskaplike Ontwikkeling (2006), (2012a) en (2012b); Hanson (1995); Hepworth, Rooney, Dewberry Rooney & Strom-Gottfried (2013); Ministerie vir Welsyn en Bevolkingsontwikkeling (1997); Republiek van Suid-Afrika (1997); Zastrow (2013). Omskrywing van gevestigde maatskaplike diensorganisasies Uit die ondersoek is dit duidelik dat die organisatoriese kenmerke wat gevestigde diensorganisasies gestruktureerd maak, inherent deel van die funksionering van sodanige organisasies is. Hoof­ uitvoerende beamptes wat op ’n beleidsvlak betrokke is by gevestigde diensorganisasies, sowel as maatskaplike werkers wat direkte dienste lewer, was tydens die studie in staat om die kenmerke wat diensorganisasies as gevestigde entiteite kategoriseer, te identifiseer en te omskryf.2 Dit beklemtoon dat die kenmerke wat gevestigde diensorganisasies van informele diensverskaffers onderskei, integraal deel van die organisasies se funksionering is en dat dit op verskeie vlakke in die organisasies na vore tree. Die aard van dienste wat gelewer word, beïnvloed die fokus en beskikbaarheid van gevestigde organisasies se interne kapasiteit vir kapasiteitsbou van IGO’s. Die effek van veral die implementering van die Kinderwet (2005) en die beskikbaarheid van maatskaplikewerk-personeel, kan kapasiteitsbou van IGO’s deur diensorganisasies strem. Buiten die verwysing na die gevestigde maatskaplike diensorganisasies as “nasionale rade” (Departement van Maatskaplike Ontwikkeling 2006, 2012a), vind geen verdere groeperinge binne hierdie kategorie plaas nie. Binne die lidmaatskap van NACOSS (wat as verteenwoordigend van nasionale diensleweringsorganisasies beskou word) blyk dit uit die ondersoek dat die aard van dienslewering twee groepe in hierdie kategorie onderskei. Beide hierdie groeperings vorm deel van hierdie studie: • Die eerste groep verwys na gevestigde maatskaplike diensorganisasies wat uitgebreide generiese dienste aan kinders, gesinne en ouer persone insluit. • Die eerste groep verwys na gevestigde maatskaplike diensorganisasies wat uitgebreide generiese dienste aan kinders, gesinne en ouer persone insluit. • Die tweede groep verwys na gevestigde maatskaplike diensorganisasies wat spesialiteits­ dienste lewer en waarvan die omvang van werk dikwels meer beperk is as voorgaande groep juis vanweë die fokus op spesialiteitsvelde. Ten spyte van hul gevestigde aard lewer gevestigde maatskaplike diensorganisasies direk dienste in kwesbare gemeenskappe en is hulle oorwegend deur middel van strukture teenwoordig en toeganklik vir sodanige gemeenskappe. Hulle georganiseerdheid skep juis kanale waardeur ’n wye geografiese omvang van dienste gelewer kan word. Dit weerlê ’n moontlike persepsie dat gevestigde maatskaplike diensverskaffers nie direk self in gemeenskappe dienste lewer nie en dat IGO’s beter as gevestigde maatskaplike diensorganisasies geleë is om dienste aan kwesbare gemeenskappe te lewer. Daar bestaan nie tans statistiese bewys oor die hoeveelheid IGO’s waarvan die kapasiteit deur gevestigde diensorganisasies gebou word nie – iets wat onderhandelinge met die Staat en ander befondsers kan bemoeilik. BEVINDINGE Die bevindinge van hierdie studie word as ʼn sintese van die narratiewe van hoofuitvoerende beamptes van gevestigde organisasies, maatskaplike werkers betrokke by kapasiteitsbou van IGO’s en verteenwoordigers van IGO’s aangebied. Aangesien ʼn instrumentele gevallestudie, Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2 doi.10.17159/2224-7912/2016/v56n2-2a5 599 gebaseer op verskillende deelnemers in verskillende organisasies se belewenisse, as strategie in hierdie navorsing benut is, word die geïntegreerde aanbieding van bevindinge meer waardevol geag as die blote liniêre reflektering van deelnemers se narratiewe. Die verwerkte narratiewe van deelnemers word vervolgens in terme van vier hooftemas aangebied. Omskrywing van IGO’s Alhoewel IGO’s as maatskaplike diensverskaffers voldoen aan die oorhoofse onderskeidende kenmerke wat aan hierdie groep diensverskaffers toegeskryf word, verskil hul grootliks van die gevestigde diensorganisasies ten opsigte van die aard van hul vrywilligergrondslag, ongestruk­ tureerdheid en ongeorganiseerdheid in terme van wetlike vereistes en omvang van dienslewering (vergelyk Departement van Maatskaplike Ontwikkeling 2006, 2012a). Wat wel ooreenstem, is dat beide hierdie tipe diensverskaffers die nood van kwesbares aanspreek en dat IGO’s meewerk om die direkte nood (veral bestaansnood) van gemeenskapslede aan te spreek (vergelyk Ministerie van Welsyn en Bevolkingsontwikkeling 1997). Met inagneming van die wye omvang van IGO’s in die maatskaplike welsynsektor in Suid- Afrika in vergelyking met die beperkte hoeveelheid deelnemende IGO’s aan hierdie studie, is daar bevind dat daar twee groeperinge van IGO’s in hierdie studie na vore tree. Die oorgrote meerderheid van IGO’s het betrekking op informele groeperinge wat ’n beperkte diens in omvang aan kwesbare gemeenskappe lewer, wat waarskynlik in baie opsigte nie in omvang en grootte sal groei nie en sal voortgaan om informeel te funksioneer. ’n Klein groepering van IGO’s het sodanig gegroei en ontwikkel dat ’n meer gesofistikeerde diens as die deursnee-IGO aangebied word. Hier word spesifiek verwys na pleegsorghuise, waar IGO’s sodanig ontwikkel het dat hulle maatskaplike werkers in diens neem wat onder toesig van die gevestigde maatskaplike diensorganisasies funksioneer. As gevolg van die basiese vlak van organisasiestruktuur wat hierdie IGO’s bemeester en hul dienste uitgebrei het, beweeg hierdie IGO’s inderwaarheid uit die basiese informele sektor. Aangesien die oorgrote meerderheid van IGO’s waarvan die gevestigde maatskaplike diens­ verskaffers kapasiteitsboudienste lewer, op die basiese vlak van ontwikkeling lê, word daar ver­ volgens uitsluitlik verwys na die informele groepering van gemeenskapsgebaseerde organisasies, tensy dit spesifiek anders aangedui word. Tans word geen onderskeid getref in die Staat se beleidstukke tussen groeperinge van IGO’s nie en is die onderskeid tussen IGO’s en opkomende organisasies vaag (vergelyk Departement van Maatskaplike Ontwikkeling 2012a). Daar is dus geen onderskeid tussen IGO’s wat informeel funksioneer en dáárdie IGO’s wat effe meer gestruktureerd funksioneer nie. ’n Onderskeid tussen verskillende groeperinge van IGO’s en ’n beter omskrywing van wat die Staat “opkomende organisasies” noem, sou daartoe kon lei dat die Staat sou kon oorweeg om wél sekere tipe meer gestruktureerde funksionerende IGO’s toegang tot staatsfinansiering te verleen, aangesien sodanige tipe IGO’s tóg bewys sou kon lewer van ’n mate van verantwoordbare bestuur van sulke organi­ sasies aan die publiek en befondsers. Omskrywing van gevestigde maatskaplike diensorganisasies Daar is egter genoegsame bewys dat ’n beduidende hoeveelheid nasionale gevestigde diensorganisasies in ’n mindere of ’n meerdere mate betrokke is by kapasiteitsbou van IGO’s en ’n waardevolle rol speel in die versterking van IGO’s se kapasiteit. Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 600 Die beskikbare inligting kan dus as vertrekpunt dien en as bewyse aangevoer word in onderhandeling met die Staat. Die beskikbare inligting kan dus as vertrekpunt dien en as bewyse aangevoer word in onderhandeling met die Staat. Omskrywing van IGO’s IGO’s kom tot stand in kwesbare gemeenskappe om die nood van die gemeenskap en/of individue wat betrokke is aan te spreek (Departement van Maatskaplike Ontwikkeling 2012a; Ministerie van Welsyn en Bevolkingsontwikkeling 1997; Patel 1998). Die ligging van IGO’s is dus nie bepalend in die behoefte aan kapasiteitsbou nie. Selfs IGO’s in informele gemeenskappe aangrensend geleë tot groter stede, se kapasiteit is só beperk dat hulle sukkel om te bestaan, alhoewel die hulpbronne geografies veronderstel is om nader aan die IGO’s te wees as op die platteland. Die kapasiteitsbou van sommige IGO’s mag moontlik wél vanweë geografiese afgesonderdheid en groter ontoeganklikheid tot hulpbronne benadeel word. IGO’s is in kwesbare gemeenskappe geleë. Vrywilligers betrokke by IGO’s is ook afkomstig uit dieselfde kwesbare gemeenskappe (vergelyk Departement van Maatskaplike Ontwikkeling Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 601 2006; Patel 1998). Armoede en bestaansbeveiliging noodsaak vrywilligers om nie gratis vanuit ’n blote diensmotief dienste te lewer nie, maar om geleenthede te ontgin wat tot armoedeverligting en inkomstegenerering kan lei. Deelnemers se verwysing na spesifiek drie groepe uitdagings as deel van die omskrywing van IGO’s, te wete beperkte kapasiteit, wisseling van bestuurslede en die beduidende rol wat finansies speel, wys daarop dat hierdie genoemde aspekte as kenmerkend van IGO’s beskou kan word. Kerke speel ’n belangrike ondersteuningsrol in die identifisering van nood en die betrokke raak by gemeenskapslede in die aanpak daarvan – iets wat die volhoubaarheid van IGO’s verhoog. Hierdie tipe ondersteuning blyk meer deurlopend van aard te wees, omdat die kerke ook in die gemeenskappe geleë is waarin die IGO’s hulle bevind. Personeelbestuur verg bepaalde kennis en vaardighede. Aangesien weinige IGO’s oor personeel beskik of sodanig saamgestel is, sal die ontwikkeling van dergelike bestuursvaardighede tydens kapasiteitsbou van IGO’s ’n intensiewe uitdaging wees (vergelyk Departement van Maatskaplike Ontwikkeling 2006; Ministerie vir Welsyn en Bevolkingsontwikkeling 1997). Wanneer die besture van IGO’s as werkgewers begin optree en ’n duideliker rolverdeling tussen werkgewers en werknemers plaasvind, tree konflik dikwels in as gevolg van beperkte kapasiteit om die verskillende rolle te vervul en die vreemdheid van die rolverdeling vir individue wat vroeër almal op dieselfde vlak gewerk het. Die handhawing van gesagsverhoudinge tussen werkgewers en kernpersoneel blyk ’n noodsaaklike fokusarea in kapasiteitsbou te wees. Benewens personeelbestuur blyk IGO’s se beperkte finansiële vermoëns oor die algemeen ’n beduidende kenmerk van IGO’s te wees (vergelyk Departement van Maatskaplike Ontwikkeling 2012a). Omskrywing van IGO’s Die gebrek aan finansies word nie pertinent deur deelnemers in die omskrywing van IGO’s vermeld nie, maar wél beperkte finansiële vaardighede en stelsels. Dit gaan dus nie noodwendig bloot oor die beperkte hoeveelheid fondse wat IGO’s het nie, maar pertinent oor IGO’s se onvermoë om gesonde finansiële beginsels te handhaaf. Wanneer finansiering wel bekom word, tree konflik in oor die nakoming van finansiële vereistes, waar daar dan dikwels op die betrokke maatskaplike diensorganisasie gefokus word. Daar is nie by deelnemers ’n oortuiging dat alle IGO’s ’n georganiseerde struktuur wíl vorm nie. Baie IGO’s blyk onkundig te wees oor die implikasies en inhoud van die stigting van ’n organisasiestruktuur en die nakoming van staatsvereistes om aan staatsfinansiering te voldoen. Deelnemers vermeld ook hulle vertwyfeling of IGO’s oorwegend ’n behoefte het om gestruktureer te word. Te oordeel aan die belewenis van deelnemers van gevestigde diensorganisasies is IGO’s óf positief ingestel om meer gestruktureerd te werk, óf wil IGO’s in wese ongestruktureerd bly in terme van wetlike vereistes en voortgaan om informeel ’n diens aan die gemeenskap te lewer. Daar behoort dus nie sonder meer aanvaar te word dat alle IGO’s oor die begeerte of vermoëns beskik om aan staatsvereistes vir die verkryging van staatsfinansiering te voldoen nie. Suksesse behaal in kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensverskaffers Gevestigde diensorganisasies se sienings en ervarings van suksesse wat in kapasiteitsbou van IGO’s behaal is, gee ’n aanduiding van die ingesteldheid waarmee diensverskaffers kapasiteit van IGO’s bou en tot welke mate hierdie ingesteldheid die maatskaplike ontwikkelingsgerigte benadering weerspieël. Dit toon ook aan op welke wyse die suksesse aansluit by die geformuleerde doel van kapasiteitsbou van IGO’s soos gestel deur gevestigde maatskaplike diensorganisasies. Die beginsel van volhoubaarheid van IGO’s blyk relatief te wees. Dit word grotendeels deur die IGO’s se ingesteldheid bepaal en wanneer fondse vir oorlewing as genoegsaam geag word. Daaruit word afgelei dat IGO’s dikwels oorwegend in wese klein wil bly en slegs beperkte dienste wil lewer. Hulle wil wel genoegsame fondse hê om daardie dienste te kan lewer. Waarskynlik Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 602 veroorsaak eksterne finansiering spanning by IGO’s, omdat dit gepaardgaan met omvangryke vereistes van die befondsers of finansierders wat addisionele sisteme en vaardighede van IGO’s vereis as waaraan hulle gewoond is. g Eie aan ʼn maatskaplike ontwikkelingsgerigte benadering, fokus maatskaplike werkers betrokke by kapasiteitsbou van IGO’s op insigontwikkeling en die bereiking van individuele vaardighede3 en dui die deelnemers sodanige ontwikkeling as suksesse in kapasiteitsbou aan. Dit dien as verdere bewys dat maatskaplike werkers wél in die praktyk die maatskaplike ontwikkelingsgerigte benadering volg. Die meting van sukses aan die mate waartoe dienslewering aan kwesbare gemeenskappe verbeter, bevestig dat gevestigde maatskaplike diensorganisasies die kapasiteit van IGO’s as gemeenskapsbronne bou, eerder as om die belange van hul eie organisasies daardeur te wil uitbrei. Dit bevestig ook dat IGO’s ’n noodsaaklike rol in gemeenskappe speel om nood aan te durf, ongeag die beperkte omvang daarvan. g g p g Daar is ’n oënskynlike teenstrydigheid tussen enersyds die Staat se beskouing dat NPO- registrasie as suksesbereiking in kapasiteitsbou van IGO’s gereken word (vergelyk Departement van Maatskaplike Ontwikkeling 2012c), en andersyds gevestigde diensorganisasies se siening dat NPO-registrasie bloot één van die stappe en suksesse in organisasie-ontwikkeling is. Daaruit word afgelei dat NPO-registrasie wel ’n belangrike mylpaal in kapasiteitsbou is, maar dat dit nie noodwendig as die bereiking van selfstandige funksionering van IGO’s beskou kan word nie. Een van die groot redes hiervoor is dat NPO-registrasie oorhoofs bloot die samestelling van die bestuur en konstitusie weergee, maar nie die bestuursvaardighede van die organisasie evalueer nie. 3 Vergelyk Homfeldt & Reutlinger (2012); Lombard (2011); Lombard, Kemp, Viljoen-Toet & Booyzen (2012); Midgley (2014); Patel & Hochfeld (2008), (2012); Patel & Noyoo (2005). Suksesse behaal in kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensverskaffers Dit mag moontlik in ’n beperkte mate wel deur middel van die jaarverslag (wat jaarliks by die NPO- Direktoraat ingedien moet word) geëvalueer word. Dit blyk dat die indien van die jaarverslae nie noodwendig nagegaan word deur die NPO-Direktoraat om die aard en kategorie van vaardighede vas te stel en NPO’s of niewinsgerigte organisasies (veral IGO’s) verder deur kommentaar daaroor leiding te gee nie. Die afleiding oor die ontoereikendheid van NPO-registrasie as bewys van verantwoordbare bestuur van ’n niewinsgerigte organisasie stem ooreen met die Departement van Maatskaplike Ontwikkeling (2006) se eie kritiek op die Wet op Organisasies sonder Winsoogmerk (1997), te wete dat registrasie as NPOs nie noodwendig lei tot selfstandige funksionering van die meer informele organisasies nie, wat dié departement onder meer aan gebrekkige bestuurskapasiteit toeskryf. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s In teenstelling met bestuursteorie en organisasie-ontwikkeling in die algemeen, word gebrekkige organisasiestruktuur en menslike hulpbronne (veral vrywilligers as bestuurslede en personeel) van IGO’s uitgesonder as die grootste uitdaging in effektiewe kapasiteitsbou van IGO’s deur gevestigde diensorganisasies. Dit blyk dus dat die praktykervaring daarop dui dat daar ’n groepering van IGO’s is waar die vaardighede en beskikbaarheid van persone wat op die besture dien op só ’n ontwikkelingsvlak lê dat kapasiteitsbou meer op hulpverlening en ontwikkeling van individue neerkom, of op ’n meer vereenvoudigde vlak van organisasie-ontwikkeling of organisasiebou lê. Daaruit word afgelei dat IGO’s minder ontwikkeld is as wat bestuursteorie en organisasie- ontwikkeling (vergelyk Yachakaschi 1995) oor die algemeen as vertrekpunt neem. Die vereistes wat onder andere deur die Wet op Organisasies sonder Winsoogmerk (1997) en ander staatsbeleide Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 603 gestel word, hou ook waarskynlik nie tred met die uitdagings wat – weens die aard van die vrywilligers-besture en personeel as menslike hulpbronne, tesame met die vestiging van organisasiestrukture deur diensverskaffers – deur IGO’s ervaar word nie.i Deelnemers se response oor uitdagings verwys spesifiek na daardie uitdagings wat kapasiteits­ bou aan gevestigde maatskaplike diensorganisasies self stel, spesifiek verwysend na die finansiële impak van kapasiteitsbou van IGO’s op gevestigde diensorganisasies. Die rede daarvoor is diens­ organisasies se eie finansiële oorlewingstryd (vergelyk Davids 2012; NACOSS 2013; Swart 2012), wye omvang en intensiteit van hul eie fokusse van dienslewering op kwesbare gemeenskappe en groepe, asook die effek wat kapasiteitsbou op die tyd en omvang van werk van die maatskaplike werkers in hul diens het. Die tydsaamheid van IGO’s se kapasiteitsbou hou direk verband met die maatskaplike ontwikkelingsgerigte benadering wat gevolg word en wat as gegewe beskou behoort te word. ʼn Verdere uitdaging vermeld deur beide die gevestigde diensorganisasies en IGO’s is die finansiële beperkinge van laasgenoemde. Gevestigde organisasies beskou finansiële beperkinge egter veel wyer as IGO’s, wat suiwer fokus op bestaansnood en basiese finansiering van dienste en toerusting. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s Vir diensorganisasies sluit finansiële beperkinge die volgende aspekte in: gebrek aan finansies om dienste te lewer; gebrekkige infrastruktuur; geen of weinig insig en kennis ten opsigte van finansiële begrippe en stelsels; gebrekkige of baie beperkte finansiële bestuursvermoë; kompetisie tussen IGO’s om finansiering en befondsing; asook die wantroue wat IGO’s van donateurs en befondsers ervaar.i In aansluiting by die voorgaande uitdaging oor finansies blyk gevestigde maatskaplike diensorganisasies, wat reeds uit eie oortuiging en oorwegend uit eie fondse IGO’s se kapasiteit bou, in ’n onbenydenswaardige posisie geplaas te word wanneer IGO’s finansiering bekom. Konflik tussen informele en gevestigde diensorganisasies tree dikwels in wanneer IGO’s finansiering ontvang. Alhoewel baie tyd vooraf bestee word aan die insigontwikkeling van finansiële begrippe en die daarstel van finansiële stelsels, blyk die besteding van fondse wat nie voorheen deel van die IGO’s se ervaringswêreld was nie, in te druis teen die aard of wese van IGO’s se natuurlike ingesteldheid. Weens die verantwoordbaarheid vir terugvoer wat deur donateurs of finansierders van die gevestigde maatskaplike diensorganisasies verwag word, asook die gevestigde diensorganisasies se ingesteldheid op gestruktureerde finansiële bestuur, stel gevestigde diensorganisasies streng vereistes oor finansiering aan IGO’s − ’n benadering wat tot konflik lei. Aangesien IGO’s nie regstegnies deel vorm van die gevestigde organisasies se strukture nie, word die dilemma ten opsigte van afdwingbaarheid van vereistes verder verhoog. Hierdie dilemma word vererger deurdat die ooreenkoms of kontrak, as die enigste verbintenis tussen die diensorganisasie en die IGO, oorwegend informeel en mondelings van aard is juis om by die vlak van ontwikkeling van die IGO’s aan te pas.i Soms lei vorige kapasiteitsbou-aksies deur die Staat of spesialiteit-organisasies, firmas of individue tot IGO’s se wantroue teenoor gevestigde diensorganisasies. Dit word in verband gebring met die deurlopende behoefte van IGO’s aan praktykgerigte ondersteuning soos reeds aangetoon, wat waarskynlik nie deel vorm van tydbeperkende insette deur sommige kapasiteitsbouers nie. Alhoewel dit eerder dui op IGO’s se onrealistiese verwagting aan die Staat en ander diensver­ skaffers, ervaar IGO’s waarskynlik dat sodanige instansies hulle in die steek laat. Te oordeel aan die tydrowendheid van insette in die aanleer en oefening van vaardighede in kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensorganisasies, vorm praktykgerigte ondersteuning onlosmaaklik deel van kapasiteitsbou. Organisasiekultuur blyk by IGO’s ’n ander rol te speel as by meer gevorderde organisasies. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s By IGO’s blyk organisasiekultuur nie te verwys na hul ingesteldheid teenoor prestasie of die etos van die organisasie oor die manier hoe dinge in die organisasie gedoen word soos bevestig word Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 604 in die McKinsey-model (2001) nie. Dit blyk uit deelnemers se terugvoer dat die organisasiekultuur by IGO’s direk verband hou met die kultuur van die gemeenskap en manier van lewe, omdat IGO’s in dié opsig integraal deel van die gemeenskap vorm en nie ’n eiesoortige identiteit aanneem nie. Dit is waarskynlik daaraan toe te skryf dat IGO’s grotendeels eerder as individue in plaas van as organisasies funksioneer. IGO’s se ervaring van kultuur het dus ʼn bepalende effek op die model van kapasiteitsbou. p IGO’s se kapasiteitsbou oor kultuurgrense heen blyk nie een van die grootste uitdagings vir gevestigde diensorganisasies te wees nie, en word moontlik daaraan toegeskryf dat maatskaplike werkers gewoond daaraan is om oor taal- en kultuurgrense te werk en hulpsisteme te vestig om hierdie uitdaging die hoof te bied. Deelnemers beklemtoon egter die belangrikheid dat ’n sensitiwiteit teenoor kultuur deurentyd in dienslewering teenwoordig behoort te wees. y g g Die IGO’s se geïntegreerdheid met hulle gemeenskappe lei tot verdere uitdagings vir kapasiteitsbou. Die effek van die gemeenskappe se sienings, politieke beskouinge en groeperinge op IGO’s lei dikwels tot konflik tussen gemeenskapslede. Dit blyk ook dat wanneer onderskeidende vlakke in die organisasie gevorm word, soos met die aanstelling van personeel, of wanneer sekere vereistes gestel word oor die besteding van fondse, verwydering tussen die IGO’s en die betrokke gemeenskappe intree en konflik plaasvind. Vanweë IGO’s se ongestruktureerdheid en as gevolg van die integrering in die gemeenskap blyk IGO’s nie normaalweg oor maniere en meganismes te beskik om die konflik aan te spreek nie. Hierdie aspek vorm ’n spesifieke fokus in kapasiteitsbou van IGO’s. Die integrering met die gemeenskap lei direk daartoe dat die gemeenskap se behoeftes nét so belangrik geag word as die IGO’s se behoeftes. Die bepaling van die korrekte beginpunt in die kapasiteitsbouproses deur ’n behoeftebepaling bied ook ’n uitdaging weens die omvangrykheid en tydrowendheid van hierdie fase in kapasiteitsbou. Kapasiteitsbou het inherent verandering of transformasie ten doel, wat op sigself uitdagings bied. Verteenwoordigers van IGO’s het pertinent aangedui dat die veranderingsproses die grootste uitdaging in kapasiteitsbou bied, wat bevestig dat verandering binne normale organisasie- ontwikkeling ’n veeleisende proses is. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s By IGO’s is hierdie verandering lewensingrypend en dus uiters moeilik omdat beweeg word vanaf ’n informele groepering wat funksioneer soos en hoe die inisieerder van die diens besluit tot ’n georganiseerde entiteit wat aan wetsvereistes voldoen. AANBEVELINGS In onderhandeling met die Staat behoort gevestigde maatskaplike diensorganisasies die omvangryke implikasies van kapasiteitsbou vir gevestigde diensorganisasies te beklemtoon, sodat die Staat ’n beter begrip kan ontwikkel wanneer vereistes gestel word vir verpligte kapasiteitsbou van IGO’s deur gevestigde diensorganisasies. Die Staat behoort voorts erkenning te gee aan gevestigde maatskaplike diensorganisasies vir die feit dat hulle toeganklik is vir kwesbare gemeenskappe en behoort hul georganiseerdheid in terme van geografiese dienslewering as bate te benut. g g g gi g Gevestigde diensorganisasies behoort statistiek te bekom en by te hou oor die aantal IGO’s waarvan kapasiteit gebou word om die saak van gevestigde diensorganisasies in onderhandelinge met die Staat en ander befondsers te versterk. Desnieteenstaande behoort die Staat oorweging daaraan te verleen om die beste praktyke uit die bestaande voorbeelde van kapasiteitsbou deur gevestigde diensorganisasies te ondersoek en te identifiseer, sodat toepaslike beleide en vereistes gestel kan word en ook om ’n instaatstellende omgewing vir gevestigde maatskaplike diens­ organisasies te skep. g p Tans blyk IGO’s die Suid-Afrikaanse regering se fokus te wees in die herstrukturering van die maatskaplike diensleweringsisteem (vergelyk Departement van Maatskaplike Ontwikkeling 2012a; Ministerie van Welsyn en Bevolkingsontwikkeling 1997). In hierdie fokus behoort Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 605 kategorisering van IGO’s in onderskeie vlakke van gestruktureerdheid, bestuur en dienslewering mee te werk om prioriteite ten opsigte van strategiese doelwitte te bepaal en skaars hulpbronne meer effektief as tans te kan aanwend. Gevestigde maatskaplike diensorganisasies kan as een van die skaars hulpbronne in die proses van kapasiteitsbou van IGO’s in Suid-Afrika beskou word, maar sal beslis staatsfinansiering benodig om wel die kapasiteitsbou van IGO’s te kan implementeer. i g g p p Diensorganisasies behoort ernstig te oorweeg om ’n bondige geskrewe beleid neer te lê vir kapasiteitsbou van IGO’s vir besture en veral personeel in daardie spesifieke organisasies. Sodanige beleid sal rigting verskaf oor die tyd en aandag wat aan IGO’s se kapasiteitsbou bestee kan word. Vanweë die reeds bestaande uitdagings wat aan gevestigde maatskaplike diensorganisasies gestel word, word aanvaar dat hierdie beleid gereeld hersien sal word ten einde die interne kapasiteit van die diensorganisasies ten beste strategies en operasioneel te kan bestuur. AANBEVELINGS Indringende samesprekings tussen die gevestigde maatskaplike diensorganisasies en die Departement van Maatskaplike Ontwikkeling behoort plaas te vind, sodat die stel van verwagtinge aan hierdie groep diensverskaffers realisties kan wees, gedagtig daaraan dat kapasiteitsbou van IGO’s nie noodwendig deel van hulle kernfokus van dienslewering vorm nie. Die doel van die gesprekke in hierdie verband sou wees om insig by die Staat te ontwikkel oor die effek wat veral die implementering van die Kinderwet op kinderbeskermingsorganisasies blyk te hê, die effek van gebrekkige finansiering selfs op gevestigde diensorganisasies, en ten slotte wat die aard van kapasiteitsbou van IGO’s in die praktyk behels. ii Die groot uitdaging wat beperkte finansiële vaardighede op die hantering van finansies deur IGO’s het, moet onder die Departement van Maatskaplike Ontwikkeling se aandag gebring word, sodat beleidstukke spesifiek voorsiening kan maak vir die skep van kanale vir IGO’s vir toegang tot staatsfinansiering aan die een kant; maar aan die ander kant ook sodat ’n sekere vlak van finansiële vaardighede ontwikkel kan word alvorens staatsfinansiering toegeken word. Veral waar die Departement van Maatskaplike Ontwikkeling geld bewillig vir die opleiding van IGO’s in bestuur, moet aandag geskenk word aan die wyse waarop meer deurlopende ondersteuning aan sodanige IGO’s gebied kan word. Aangesien IGO’s se geïntegreerdheid met die gemeenskappe waarbinne hulle funksioneer een van hul wesenskenmerke is, moet hierdie aspek deurlopend op beleids- en diensleweringsvlak in ag geneem word in die kapasiteitsbou van IGO’s, ongeag die velerlei uitdagings wat die geïntegreerdheid tot gevolg het vir kapasiteitsbou. Veranderingsbestuur blyk inherent deel van organisatoriese kapasiteitsbou te wees en veral vanweë die impak daarvan op die kapasiteitsbouproses van IGO’s behoort maatskaplike werkers wat hierdie tipe maatskaplikewerk-intervensie doen, opleiding in veranderingsteorie te ontvang. SAMEVATTING Uit hierdie navorsing is dit duidelik dat gevestigde maatskaplike diensorganisasies reeds wel ʼn waardevolle bydrae tot die kapasiteitsbou van informele gemeenskapsgebaseerde organisasies lewer. In die stel van vereistes aan gevestigde diensorganisasies oor die kapasiteitsbou van IGO’s behoort daar allereers deur die Staat in ag geneem te word dat gevestigde organisasies rééds ’n finansiële oorlewingstryd en vele uitdagings in die aanspreek van nood in hul kerndienslewering ervaar. Die ernstige finansiële uitdaging aan gevestigde maatskaplike diensorganisasies word deur verskeie navorsers beklemtoon (Gray 2006; Lombard 2011; Patel & Hochfeld 2008; 2012). 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Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 doi.10.17159/2224-7912/2016/v56n2-2a5 Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016 Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 201 doi.10.17159/2224-7912/2016/v56n2-2a5 BIBLIOGRAFIE Qualitative research for social workers. Londen: Allyn & Bacon. organisations in the Eastern Cape. (MA tesis) Port Elizabeth: Nelson Mandela Metropolitan University. Tutty, L.M., Rothery, M.A. & Grinnell, R.M. 1996. Qualitative research for social workers. Londen: Allyn & Bacon. Tutty, L.M., Rothery, M.A. & Grinnell, R.M. 1996. Qualitative research for social workers. Londen: Allyn & Bacon. Whittemore, R., Chase, S.K. & Mandle, C.L. 2001. Validity in qualitative research. Qualitative Health Research, 11(4):522-537. Williams, M., Tutty, L.M. & Grinnell, R.M. 1995. Research in social work: an introduction. Ith Yachakaschi, S. 1995. 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Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma
Cancers
2,021
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UC Irvine Journal Cancers, 13(13) ISSN 2072-6694 Authors Lin, Min Kowolik, Claudia M Xie, Jun et al. Publication Date 2021 DOI 10.3390/cancers13133367 Peer reviewed UC Irvine UC Irvine Previously Published Works Title Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cel Lymphoma Permalink https://escholarship.org/uc/item/7t15q0p6 Journal Cancers, 13(13) ISSN 2072-6694 Authors Lin, Min Kowolik, Claudia M Xie, Jun et al. Publication Date 2021 DOI 10.3390/cancers13133367 Peer reviewed UC Irvine UC Irvine Previously Published Works Title Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cel Lymphoma Permalink https://escholarship.org/uc/item/7t15q0p6 Journal Cancers, 13(13) ISSN 2072-6694 Authors Lin, Min Kowolik, Claudia M Xie, Jun et al. Publication Date 2021 DOI 10.3390/cancers13133367 Peer reviewed   Abstract: Cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of debilitating, incurable malignancies. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes, accounting for ~65% of CTCL cases. Patients with advanced disease have a poor prognosis and low median survival rates of four years. CTCLs develop from malignant skin-homing CD4+ T cells that spread to lymph nodes, blood, bone marrow and viscera in advanced stages. Current treatments options for refractory or advanced CTCL, including chemotherapeutic and biological approaches, rarely lead to durable responses. The exact molecular mechanisms of CTCL pathology remain unclear despite numerous genomic and gene expression profile studies. However, apoptosis resistance is thought to play a major role in the accumulation of malignant T cells. Here we show that NT1721, a synthetic epidithiodiketopiperazine based on a natural product, reduced cell viability at nanomolar concentrations in CTCL cell lines, while largely sparing normal CD4+ cells. Treatment of CTCL cells with NT1721 reduced proliferation and potently induced apoptosis. NT1721 mediated the downregulation of GLI1 transcription factor, which was associated with decreased STAT3 activation and the reduced expression of downstream antiapoptotic proteins (BCL2 and BCL-xL). Importantly, NT1721, which is orally available, reduced tumor growth in two CTCL mouse models significantly better than two clinically used drugs (romidepsin, gemcitabine). Moreover, a combination of NT1721 with gemcitabine reduced the tumor growth significantly better than the single drugs. Taken together, these results suggest that NT1721 may be a promising new agent for the treatment of CTCLs. Citation: Lin, M.; Kowolik, C.M.; Xie, J.; Yadav, S.; Overman, L.E.; Horne, D.A. Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma. Cancers 2021, 13, 3367. https://doi.org/ 10.3390/cancers13133367 Received: 18 May 2021 Accepted: 1 July 2021 Published: 5 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Keywords: cutaneous T cell lymphoma (CTCL); epidithiodiketopiperazine; NT1721; GLI1; STAT3 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/ 4.0/). Article Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma a M. Kowolik 1,*,†, Jun Xie 1, Sushma Yadav 1,2 , Larry E. Overman 3 and David A. Horne 1,* Min Lin 1,†, Claudia M. Kowolik 1,*,†, Jun Xie 1, Sushma Yadav 1,2 , Larry E. Overman 3 and 1 Department of Molecular Medicine, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA; mlin@coh.org (M.L.); jxie@coh.org (J.X.); syadav@coh.org (S.Y.) 2 Department of Translational Research and Cellular Therapeutics, City of Hope National Medical Center 1500 E. Duarte Road, Duarte, CA 91010, USA 3 Department of Chemistry, University of California, Irvine, CA 92697-2025, USA; leoverma@uci.edu * Correspondence: ckowolik@coh.org (C.M.K.); dhorne@coh.org (D.A.H.) p y y * Correspondence: ckowolik@coh.org (C.M.K.); dhorne@coh.org (D.A.H.) p g † These authors contributed equally to this work. † These authors contributed equally to this work. † These authors contributed equally to this work. Simple Summary: Cutaneous T cell lymphomas (CTCLs) are a group of blood cancers that cannot be cured with current chemotherapeutical or biological drugs. Patients with advanced disease are severely immunocompromised due to the unchecked expansion of malignant T cells and have low survival rates of less than four years. Hence, new treatment options for CTCLs are urgently needed. In this study the anti-CTCL activity of a new compound, NT1721, was determined in vitro and in two CTCL mouse models. We found that NT1721 increased apoptosis (programmed cell death) in the malignant T cells and reduced tumor growth better than two drugs that are currently clinically used for CTCL treatment (i.e., gemcitabine, romidepsin). These results suggest that NT1721 may represent a potent new agent for the treatment of advanced CTCL.   Citation: Lin, M.; Kowolik, C.M.; Xie, J.; Yadav, S.; Overman, L.E.; Horne, D.A. Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma. Cancers 2021, 13, 3367. https://doi.org/ 10.3390/cancers13133367 Academic Editor: Daruka Mahadevan Received: 18 May 2021 Accepted: 1 July 2021 Published: 5 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 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/ 4.0/). Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org eScholarship.org cancers cancers cancers Citation: Lin, M.; Kowolik, C.M.; Xie, J.; Yadav, S.; Overman, L.E.; Horne, D.A. Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma. Cancers 2021, 13, 3367. https://doi.org/ 10.3390/cancers13133367 1. Introduction Cutaneous T cell lymphomas (CTCLs) are a biologically and clinically heterogeneous group of rare, incurable non-Hodgkin lymphomas that typically affect adults with a median age of 55 to 60 years [1–3]. The annual age-adjusted incidence rate in the United States is 6.4 per million persons [3]. CTCLs develop from malignant skin-homing CD4+ T cells that https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3367. https://doi.org/10.3390/cancers13133367 Cancers 2021, 13, 3367 2 of 15 spread to lymph nodes, blood, bone marrow and viscera in advanced stages [4]. The most common subtypes, mycosis fungoides (MF) and Sézary syndrome (SS), account for 65% of CTCL cases [5,6]: MF is an indolent disease with only 10% of patients progressing to advanced stages, while SS represents the aggressive leukemic variant of CTCL that can develop de novo or progress from MF [7]. Differences in molecular profiles and responses to therapies suggest that MF and SS are distinct diseases [8,9]. Patients with SS present with generalized erythroderma and lymphadenopathy and have a poor prognosis with low median survival rates of 2–4 years [10]. SS is characterized by the clonal expansion of circulating malignant T cells with cerebriform nuclei (Sézary cells), which leads to the loss of a normal T cell receptor repertoire and thus immunosuppression [10–14]. Hence, patients with advanced disease are highly susceptible for opportunistic infections, which represent the most common CTCL-related cause of death [5,14]. Current treatment options for CTCL depend on the tumor stage. Unlike early stage CTCL, which is treated with skin- directed therapies, refractory early stage and advanced stage CTCL requires systemic mono- or combination therapy such as photopheresis, interferon alpha, bexarotene, monoclonal antibodies (e.g., mogamulizumab), histone deacetylase inhibitors (e.g., romidepsin (FK228)) and chemotherapeutics (e.g., gemcitabine) [4,15]. Response rates for most treatments vary between 30 and 50% and are rarely durable (7.5 to 22.4 months) [15,16]. Relapses are common and treatment for the aggressive forms remains palliative, not curative despite recent advances with chemotherapeutic and biological approaches [4]. Moreover, current therapies often lead to progressive drug resistance [17]. Thus, new treatment options for advanced CTCL are urgently needed. Numerous genomic and gene expression profile studies have been performed over the past decade to discover genes that play a role in CTCL pathogenesis and to find potential new targets for therapy [11,14,18,19]. 2.1. Reagents NT1721 was synthesized as previously described [32]. Gemcitabine, romidepsin (FK228) and the ERK inhibitor SCH772984 were purchased from Selleckchem (Houston, TX, USA). 2.5. Cell Cycle Analysis Cells were treated with NT1721 as indicated and harvested after 48 h, stained with propidium iodide (PI) (ThermoFisher) as previously described and subjected to FACS analysis [27]. 2.4. Proliferation Assay Cells were stained with CFSE ((5(6)-carboxyfluorescein N-hydroxysuccinimidylester, CellTrace, ThermoFisher) according to the manufacturer’s instructions, and seeded at a concentration of 10,000 cells/well. The cells were treated with NT1721 or DMSO the next day, harvested after 48 h or 72 h, stained with 0.2 µg/mL DAPI and subjected to FACS analysis. Fluorescence data were collected on a CyAN flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (TreeStar, Ashland, OR, USA). 2.3. Determination of IC50 Values The CellTiter-GLO viability assay (Promega, Madison, WI, USA) was used according to the manufacturer’s instructions to determine cell viability: Briefly, 10,000 cells/well were seeded in 96-well plates, cultured overnight and then treated with various concentrations of NT1721 for 48 h or 72 h. Data from the assay were expressed as percent of viable cells compared to the vehicle control (0.1% DMSO). 2.2. Cell Culture HUT78 and HH cells were obtained from ATCC (Manassas, VA, USA), authenticated by STR-profiling at the source and passaged for less than 6 months after receipt or re- suscitation. Peripheral blood mononuclear cells (PBMCs) were obtained from ReachBio (Spokane, WA, USA). All cells were grown in RPMI supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO, USA). CD4+ cells were isolated from PBMCs using Dynabeads (ThermoFisher, Waltham, MA, USA). 1. Introduction However, the molecular mechanisms of CTCL pathogenesis remain obscure as there is little overlap among the study results, which highlights the genetic heterogeneity of CTCL [11,19,20]. Genes with the most frequently altered expression profiles in CTCL are associated with the constitutive production of cytokines characteristic for T helper type 2 cells, resistance to growth inhibition via TGFβ and apoptosis induction [11]. Importantly, apoptosis resistance rather than increased proliferation is thought to be the cause for the accumulation of malignant T cells in the early stages of CTCL [21,22]. Aberrantly activated STAT3 has been identified as one important factor in CTCL progression and apoptosis resistance [18,23–25]. Activated STAT3 has been associated with increased expression of antiapoptotic proteins (BCL2, BCL-xL) and decreased overall and progression-free survival in CTCL [23,26]. p g In this study, the anti-CTCL efficacy of an epidithiodiketopiperazine, NT1721, was investigated. Epidithiodiketopiperazines (ETPs) are a broad class of fungal metabolites with potent antitumor activity in multiple solid and non-solid tumors. We have shown in a previous study that NT1721, an ETP chosen from a library of ETPs because of its potent antitumor activity against various solid and non-solid tumors, attenuated hedge- hog/GLI signaling through downregulation of GLI transcription factors [27]. Aberrant hedgehog/GLI signaling promotes tumorigenesis, tumor progression, metastasis and drug resistance in various types of cancers [28,29]. Here we show that treatment with NT1721 mediated GLI1 downregulation in CTCL cells, which was associated with decreased STAT3 activation and reduced expression of downstream antiapoptotic proteins (BCL2 and BCL- xL). NT1721 led to potent apoptosis induction in vitro and significantly reduced tumor growth in two CTCL xenograft mouse models. Importantly, NT1721 impaired tumor growth significantly better than romidepsin or gemcitabine, two clinically used drugs for the treatment of refractory or relapsed CTCL [30,31]. Drug combinations of NT1721 with gemcitabine showed significantly improved antitumor effects in vitro and in vivo compared to the single agents. Our data suggest that NT1721 is, potentially, a promising new agent for the treatment of CTCL either by itself or in combination with gemcitabine. 3 of 15 Cancers 2021, 13, 3367 2.6. QPCR Total RNA was isolated using the Direct-zol kit (Zymo Research, Irvine, CA, USA) and reverse transcribed using the Tetro cDNA synthesis kit (Bioline, Taunton, MA, USA). The following qPCR primers were used: The following qPCR primers were used: BCL2: 5′AGTACCTGAACCGGCACCT/5′GCCGTACAGTTCCACAAAGG BCL-xL: 5′CTGAGTTACCGGCATCCC/5′TTCTGAAGGGAGAGAAAGAGATTC BMI1: 5′CCATTGAATTCTTTGACCAGAA/5′CTGCTGGGCATCGTAAGTATC CCNE1: 5′GGCCAAAATCGACAGGAC/5′GGGTCTGCACAGACTGCAT; GAPDH: 5′AGCCACATCGCTCAGACAC/5′GCCCAATACGACCAAATCC; GLI1: 5′ACCCGGGGTCTCAAACTG/5′GGCTGACAGTATAGGCAGAGC; Quantitative PCR was performed using a CFX96 Touch Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). Relative expression levels were calculated using the 2−∆∆Ct method and GAPDH as reference gene. g q p BCL2: 5′AGTACCTGAACCGGCACCT/5′GCCGTACAGTTCCACAAAGG BCL L 5′CTGAGTTACCGGCATCCC/5′TTCTGAAGGGAGAGAAAGAGATTC g q p BCL2: 5′AGTACCTGAACCGGCACCT/5′GCCGTACAGTTCCACAAAGG BCL-xL: 5′CTGAGTTACCGGCATCCC/5′TTCTGAAGGGAGAGAAAGAGATTC BMI1: 5′CCATTGAATTCTTTGACCAGAA/5′CTGCTGGGCATCGTAAGTATC CCNE1: 5′GGCCAAAATCGACAGGAC/5′GGGTCTGCACAGACTGCAT; GAPDH: 5′AGCCACATCGCTCAGACAC/5′GCCCAATACGACCAAATCC; GLI1: 5′ACCCGGGGTCTCAAACTG/5′GGCTGACAGTATAGGCAGAGC Quantitative PCR was performed using a CFX96 Touch Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). Relative expression levels were calculated using the 2−∆∆Ct method and GAPDH as reference gene. 2.7. Western Blots SDS-PAGE and Western blots were carried out as previously described [27]. Briefly, cells were lyzed using RIPA buffer (Cell signaling, Danvers, MA, USA) supplemented with Halt Protease inhibitor cocktail (ThermoFisher). The protein concentration was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). Equal amounts of protein were loaded on precast gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF Cancers 2021, 13, 3367 4 of 15 membranes (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h at RT in blocking buffer (5% w/v nonfat dry milk, 0.1% Tween-20 in TBS), then incubated with primary antibodies overnight at 4 ◦C. The NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher) was used for the preparation of cytoplasmic and nuclear extracts. Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): β-actin (#4970), BCL2 (#4223), BCL-xL (#2764), BMI1 (#6964), cleaved PARP (#5625), cleaved CASP3 (#9664), ERK (#4695), pERK1/2 (#4370), GAPDH (#5174), GLI1 (#3538), H2AX (#2595), γH2AX (#9718), H3 (#4499), p21 (#2947), p27 (#3686), STAT3 (#9139), pSTAT3 (Y705) (#9145), TBP (#44059); additionally, tubulin (05-829) was purchased from Sigma (St. Louis, MO, USA). The membranes were washed three times with TBS and then incubated 1 h at RT with the appropriate secondary antibodies. Bands were visualized using X-ray film or the ChemiDoc Imaging System (Bio-Rad). 2.8. In Vivo Studies Mouse care and experimental procedures were performed under pathogen-free condi- tions in accordance with approved protocols from the institutional animal care and use committee of City of Hope National Medical Center. For the xenograft mouse models, 5 million HuT78 or HH cells were injected subcutaneously into the flank of 6- to 8-week old NSG mice (Jackson Laboratory, Bar Harbor, ME, USA). The tumor volume was calculated using the equation: V = 1 2 × W2 × L with W and L representing the width and length. Treat- ment started when the tumor volume reached ~200 mm3. The mice were then distributed into groups bearing equal tumor burdens and treated with NT1721 by gavage, gemcitabine (i.p. injection), romidepsin (i.p. injection) or the vehicle control as indicated. PBS was used as vehicle control for treatments by i.p. injections; 30% solutol/3.3% DMSO in PBS or Ora-Blend (ThermoFisher)/10% saline were used as vehicle controls for gavage treatments. 2.9. Statistical Analysis The mean ± standard deviation (SD) was calculated for each treatment group. The two-tailed t-test (GraphPad Software Inc., La Jolla, CA, USA) was used to determine statistical significance between two treatment groups in the in vitro studies. The Mann– Whitney U test was used to determine statistical significance between treatment groups in mouse models. p values < 0.05 were considered significant. 3.1. NT1721 Led to Reduced CTCL Cell Viability, Proliferation and G2 Cell Cycle Arrest at Nanomolar Concentrations (B) Effect of NT1721 on normal cells. HuT78 cells, normal PBMCs and normal CD4+ cells were treated with 300 nM NT1721 or 0.3% DMSO. (C) Proliferation. CTCL cells were stained with CFSE, treated with NT1721 and subjected to FACS analysis after 48 or 72 h to determine the mean fluorescence intensity. The data were normalized to the controls. (D) Cell cycle analysis. HuT78 cells were treated with NT1721, stained with PI after 24 h and 48 h and analyzed by FACS. The graphs represent the mean ± SD from triplicate values. Figure 1. NT1721 decreased CTCL cell viability, proliferation and led to G2 cell cycle arrest. (A) Cell viability. HuT78 and HH cells were treated with increasing concentrations of NT1721. Cell viability and IC50 values were determined after 48 and 72 h. (B) Effect of NT1721 on normal cells. HuT78 cells, normal PBMCs and normal CD4+ cells were treated with 300 nM NT1721 or 0.3% DMSO. (C) Proliferation. CTCL cells were stained with CFSE, treated with NT1721 and subjected to FACS analysis after 48 or 72 h to determine the mean fluorescence intensity. The data were normalized to the controls. (D) Cell cycle analysis. HuT78 cells were treated with NT1721, stained with PI after 24 h and 48 h and analyzed by FACS. The graphs represent the mean ± SD from triplicate values. We then determined the effect of NT1721 on the cell cycle by staining NT1721-treated cells with PI. As shown in Figure 1D, NT1721 led to cell cycle arrest in CTCL cells. Treat- ment with NT1721 led to G2 cell cycle arrest as indicated by the dose- and time-dependent increase in the percentage of cells in the G2 phase and concomitant decrease in the percent- age of cells in the G1 phase. Treatment with 500 nM NT1721 decreased the number of cells in the G1 phase from ~70% to 30% and 22% after 24 h and 48 h, respectively; the number of cells in the G2 phase increased from ~12% to 35 and 38% in NT1721-treated cells after 24 h and 48 h, respectively. Taken together, the results indicate that NT1721-mediated preferen- tial reduction in CTCL cell viability (compared to normal CD4+ cells) was associated with decreased proliferation and G2 cell cycle arrest. 3.1. NT1721 Led to Reduced CTCL Cell Viability, Proliferation and G2 Cell Cycle Arrest at Nanomolar Concentrations To assess the potency of NT1721 against CTCL, we treated HuT78 and HH cells with increasing concentrations of NT1721 and determined viability after 48 and 72 h. The IC50 values were in the nanomolar range for both cell lines and time points: 125 nM and 6 nM in HuT78 cells after 48 and 72 h, respectively; 542 nM and 70 nM in HH cells after 48 and 72 h, respectively (Figure 1A). To assess the effect of NT1721 on normal cells we also treated Hut78 cells, normal peripheral blood mononuclear cells (PBMCs) and CD4+ cells from two healthy donors with NT1721. As shown in Figure 1B, 300 nM NT1721 reduced the viability of HuT78 by 83%, while only decreasing the viability of normal PBMCs and normal CD4+ by ~32%. The result suggests that NT1721 preferentially decreased the viability of CTCL cells. To investigate the effect of NT1721 on CTCL proliferation we stained HuT78 and HH cells with ((5(6)-Carboxyfluorescein N-hydroxysuccinimidylester (CFSE) and treated them with NT1721 (up to 300 nM) for 48 or 72 h. FACS analysis of live cells showed that NT1721 reduced proliferation faster and at lower concentrations in Hut78 compared to HH cells (Figure 1C). However, treatment with 100 nM NT1721 dramatically reduced proliferation in both cell lines after 72 h by 50–75%. 5 of 15 Cancers 2021, 13, 3367 Figure 1. NT1721 decreased CTCL cell viability, proliferation and led to G2 cell cycle arrest. (A) Cell viability. HuT78 and HH cells were treated with increasing concentrations of NT1721. Cell viability and IC50 values were determined after 48 and 72 h. (B) Effect of NT1721 on normal cells. HuT78 cells, normal PBMCs and normal CD4+ cells were treated with 300 nM NT1721 or 0.3% DMSO. (C) Proliferation. CTCL cells were stained with CFSE, treated with NT1721 and subjected to FACS analysis after 48 or 72 h to determine the mean fluorescence intensity. The data were normalized to the controls. (D) Cell cycle analysis. HuT78 cells were treated with NT1721, stained with PI after 24 h and 48 h and analyzed by FACS. The graphs represent the mean ± SD from triplicate values. Figure 1. NT1721 decreased CTCL cell viability, proliferation and led to G2 cell cycle arrest. (A) Cell viability. HuT78 and HH cells were treated with increasing concentrations of NT1721. Cell viability and IC50 values were determined after 48 and 72 h. 3.2. NT1721 Downregulated GLI1 Transcription Factor in CTCL Cells Since we previously found that NT1721 potently downregulated GLI transcription factors [27], we first determined the effect of NT1721 on GLI1 expression in CTCL cells. As shown in (Figure 2A,B), NT1721 dramatically reduced GLI1 expression on the mRNA and protein level in both HuT78 and HH cells after 24 h at concentrations of ≤100 nM. A comparison of GLI1 expression levels in the CTCL cell lines revealed ~3- fold higher GLI1 mRNA expression in Hut78 as well as higher GLI1 protein expression Cancers 2021, 13, 3367 6 of 15 compared to HH cells (Figure 2B,C). In contrast, no GLI1 protein expression was detected in two samples of normal CD4+ cells (Figure 2C), suggesting that GLI1 might be aberrantly expressed in the CTCL cell lines. Moreover, this result is in agreement with a previous report showing strong GLI1 expression in T cell lymphomas including CTCL while no GLI1 expression was observed in healthy peripheral blood T cells [33]. compared to HH cells (Figure 2B,C). In contrast, no GLI1 protein expression was detected in two samples of normal CD4+ cells (Figure 2C), suggesting that GLI1 might be aberrantly expressed in the CTCL cell lines. Moreover, this result is in agreement with a previous report showing strong GLI1 expression in T cell lymphomas including CTCL while no GLI1 expression was observed in healthy peripheral blood T cells [33]. Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05; ** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and NT1721-treated CTCL cells. Original Western Blots of Figure 2A,C available in Figure S4. Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05; ** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and NT1721-treated CTCL cells. 3.3. NT1721 Induced Apoptosis in CTCL In Vitro and In Vivo Since NT1721 mediated the downregulation of antiapoptotic BCL2 and BCL-xL, we decided to further investigate the effect of NT1721 on apoptosis. HuT78 cells were treated with NT1721 (up to 500 nM) and stained with annexin V after 24 h and 48 h. As shown in Figure 3A, low concentrations of NT1721 (10–30 nM) significantly increased the per- centage of apoptotic cells to 20–40% after 48 h. Treatment with a higher dose of NT1721 (500 nM) resulted in 35% and 62% apoptotic cells after 24 h and 48 h, respectively. To further investigate apoptosis induction by NT1721 on the molecular level, NT1721-treated HuT78 and HH samples were analyzed by Western blots. As shown in Figure 3B, NT1721 increased the expression of proteins related to apoptosis induction (i.e., cleaved CASP3 and cleaved PARP) in a concentration- and time-dependent manner. Moreover, this increase in apoptotic markers was accompanied by H2AX upregulation and increased levels of γ H2AX. Subcellular fractionation revealed that in NT1721-treated Hut78 cells the cytoplas- mic fractions contained the majority of γH2AX and total H2AX; only a small amount of these proteins was detected in the nuclear fractions (Figure S1). Also observed was a dra- matic increase in γH2AX and total H2AX levels in NT1721-treated HH cells; a significant portion of these proteins was also found in the cytoplasmic fractions. This H2AX upreg- ulation and cytoplasmic localization provided additional support that NT1721 induced apoptosis in CTCL. Increased H2AX levels have been identified as a cause of apoptosis in previous studies [35,36]. Since ERK activation has been linked to both apoptosis induction and G2 cell cycle arrest [37,38], the effect of NT1721 on ERK activation and p21 (CDKN1A) levels was investigated. As shown in Figure 3B, low concentrations of NT1721 (10–30 nM) led to concentration-dependent activation of ERK in Hut78 starting after 24 h. In HH cells, ERK activation was detected after 48 h of treatment with NT1721 and at higher NT1721 concentrations. Dramatic upregulation of p21 by NT1721 was detected in both cell lines (Figure 3B). To explore the importance of ERK phosphorylation on apoptosis induction, HuT78 cells were treated with an ERK inhibitor SCH772984, NT1721, or a combination of both agents. Western blot analyses showed that NT1721 alone increased phospho- ERK (pERK), p21, cleaved CASP3, cleaved PARP and γH2AX levels in a concentration- dependent manner (Figure S2A). In contrast, SCH772984 alone had no effect on these protein levels. 3.2. NT1721 Downregulated GLI1 Transcription Factor in CTCL Cells Original Western Blots of Figure 2A,C available in Figure S4. We next investigated the effect of GLI1 downregulation on the expression of GLI target Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05; ** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and NT1721-treated CTCL cells. Original Western Blots of Figure 2A,C available in Figure S4. Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05; ** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and NT1721-treated CTCL cells. Original Western Blots of Figure 2A,C available in Figure S4. We next investigated the effect of GLI1 downregulation on the expression of GLI target genes that are involved in apoptosis resistance and cell cycle progression (i.e., BCL2, BMI1, CCNE1) and that were also downregulated by NT1721 in our previous study [27]: NT1721 concentrations of 100 nM significantly reduced BMI1 and CCNE1 expression in HuT78 and HH cells (Figure 2A,B). NT1721 also significantly decreased BCL2 expression in HuT78 (Figure 2A,B). However, in line with previous reports [17,34], no BCL2 mRNA or protein expression could be detected in HH cells. We decided to determine the effect of NT1721 on STAT3 activation since several reports show that GLI1 inhibition or knockdown decreased phosho-STAT3 (pSTAT3) levels in T cell lymphomas and that pSTAT3 may be an important driver of CTCL, especially in advanced disease [26]. As shown in Figure 2C, strong pSTAT3 was only detected in the CTCL cells Cancers 2021, 13, 3367 7 of 15 and not in the normal CD4+ samples. Treatment of CTCL cells with NT1721 resulted in reduced pSTAT3 levels (Figure 2C). The reduced STAT3 activation was accompanied by a significant reduction in mRNA and protein levels of the STAT3 target gene BCL-xL in Hut78 (Figure 2B,C). 3.2. NT1721 Downregulated GLI1 Transcription Factor in CTCL Cells In contrast, very low BCL-xL expression was detected in HH and normal CD4+ cells. 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL Mouse Models 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL Mouse Models To assess the antitumor efficacy of NT1721 in vivo, NSG mice were injected subcu- taneously with HuT78 cells. When the tumor volumes reached ~200 mm3 the mice were treated with 20 mg/kg NT1721 by gavage three times per week on consecutive days. The control group received the vehicle control (30% solutol/3.3% DMSO in PBS). As shown in Figure 4A,B, NT1721 reduced the tumor volume and tumor weight significantly by 90%. To compare the efficacy of NT1721 to romidepsin, a clinically used drug for CTCL treatment [39], mice were treated with romidepsin (2 mg/kg) twice per week. Romidepsin did not significantly reduce the tumor weight (Figure 4A,B). Moreover, romidepsin-treated mice showed signs of toxicity, i.e., ascites and diarrhea, while no obvious signs of toxicity were observed in NT1721-treated mice. We next investigated the in vivo efficacy of NT1721 against CTCL in another mouse model. NT1721 also reduced the tumor volume and tumor weight of HH tumors by ~50% (Figure 4D–F). Treatment of HH tumors with romidepsin did not result in smaller tumor volumes (data not shown). These results indicate that NT1721 reduced CTCL tumor growth better than romidepsin and that NT1721 may be less toxic than romidepsin. To assess the antitumor efficacy of NT1721 in vivo, NSG mice were injected subcu- taneously with HuT78 cells. When the tumor volumes reached ~200 mm3 the mice were treated with 20 mg/kg NT1721 by gavage three times per week on consecutive days. The control group received the vehicle control (30% solutol/3.3% DMSO in PBS). As shown in Figure 4A,B, NT1721 reduced the tumor volume and tumor weight significantly by 90%. To compare the efficacy of NT1721 to romidepsin, a clinically used drug for CTCL treatment [39], mice were treated with romidepsin (2 mg/kg) twice per week. Romidepsin did not significantly reduce the tumor weight (Figure 4A,B). Moreover, romidepsin-treated mice showed signs of toxicity, i.e., ascites and diarrhea, while no obvious signs of toxicity were observed in NT1721-treated mice. We next investigated the in vivo efficacy of NT1721 against CTCL in another mouse model. NT1721 also reduced the tumor volume and tumor weight of HH tumors by ~50% (Figure 4D–F). Treatment of HH tumors with romidepsin did not result in smaller tumor volumes (data not shown). 3.3. NT1721 Induced Apoptosis in CTCL In Vitro and In Vivo Samples treated with combinations of NT1721 and SCH772984 displayed reduced ERK phosphorylation, cleaved PARP, cleaved CASP3, p21 and γH2AX induction compared to samples treated with NT1721 alone. To further investigate the effect of the drug combination on apoptosis induction we stained Hut78 cells that were treated with the drug combination with annexin V. As shown in Figure S2B, treatment with both NT1721 and SCH772984 reduced the percentage of apoptotic HuT78 cells significantly compared to cells treated with NT1721 alone. Taken together, these results suggest that increased ERK phosphorylation may be critical for efficient apoptosis induction by NT1721. To evaluate whether treatment with NT1721 could also induce apoptosis in vivo, NSG mice were injected with HuT78 cells subcutaneously. When the tumor volumes reached ~200 mm3 the mice were treated with a single oral dose of NT1721 or the vehicle control. The mice were euthanized 15, 24, 48 or 72 h later, and tumor tissue from individual mice was used for Western blot analysis. As shown in Figure 3C, NT1721 increased cleaved CASP3, cleaved PARP and p21 expression while decreasing GLI1 and BCL2 expression, thus recapitulating the changes in protein expression that were observed in vitro. These results suggest that NT1721 induces apoptosis in CTCL in vivo. 8 of 15 Cancers 2021, 13, 3367 Figure 3. NT1721 induced apoptosis in CTCL cells in vitro and in vivo. (A) HuT78 cells were treated with NT1721 for 24 h or 48 h, then stained with annexin V and subjected to FACS analysis. (B) HuT78 and HH cells were treated with NT1721 as indicated. Western blots were used to analyze the expression of genes related to apoptosis induction. (C) Apoptosis induction in vivo. NSG mice harboring HuT78 tumors (n = 4 per group) were treated by gavage with a single dose of NT1721 (20 mg/kg) or the vehicle (3.3% DMSO/30% solutol in PBS). The tumors were then harvested at the indicated time points and subjected to Western blot analysis. Original Western Blots of Figure 3B,C available in Figure S5. Figure 3. NT1721 induced apoptosis in CTCL cells in vitro and in vivo. (A) HuT78 cells were treated with NT1721 for 24 h or 48 h, then stained with annexin V and subjected to FACS analysis. (B) HuT78 and HH cells were treated with NT1721 as indicated. Western blots were used to analyze the expression of genes related to apoptosis induction. (C) Apoptosis induction in vivo. 3.3. NT1721 Induced Apoptosis in CTCL In Vitro and In Vivo NSG mice harboring HuT78 tumors (n = 4 per group) were treated by gavage with a single dose of NT1721 (20 mg/kg) or the vehicle (3.3% DMSO/30% solutol in PBS). The tumors were then harvested at the indicated time points and subjected to Western blot analysis. Original Western Blots of Figure 3B,C available in Figure S5. 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL Mouse Models 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL Mouse Models 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL Mouse Models These results indicate that NT1721 reduced CTCL tumor growth better than romidepsin and that NT1721 may be less toxic than romidepsin. 9 of 15 Cancers 2021, 13, 3367 Figure 4. NT1721 suppressed tumor growth significantly better than romidepsin in a CTCL mouse model. (A) NSG mice (n = 7/group) were injected s.c. with HuT78 cells and treated by gavage three times/week (on consecutive days) with the vehicle control (30% solutol/3.3% DMSO in PBS), 20 mg/kg NT1721 or with 2 mg/kg romidepsin by I.P. injection twice per week. Treatment with NT1721 reduced tumor volumes significantly better than romidepsin on Day 4, 7 and 11 (p values < 0.01). (B) Weight of HuT78 tumors from individual mice (p = 0.0003 for control vs. NT1721 and for romidepsin vs. NT1721; no significant difference for control vs. romidepsin: p = 0.317). (C) Picture of HuT78 tumors from the treated mice. (D) NSG mice were injected s.c. with HH cells and treated by gavage on three consecutive days per week with 20 mg/kg NT1721 or the vehicle control. The differences in tumor volume were significant from day 14 through day 25 (p values < 0.0012). (E) Weight of HH tumors from individual mice (p = 0.0003). (F) Picture of representative HH tumors from the treated mice. Figure 4. NT1721 suppressed tumor growth significantly better than romidepsin in a CTCL mouse model. (A) NSG mice (n = 7/group) were injected s.c. with HuT78 cells and treated by gavage three times/week (on consecutive days) with the vehicle control (30% solutol/3.3% DMSO in PBS), 20 mg/kg NT1721 or with 2 mg/kg romidepsin by I.P. injection twice per week. Treatment with NT1721 reduced tumor volumes significantly better than romidepsin on Day 4, 7 and 11 (p values < 0.01). (B) Weight of HuT78 tumors from individual mice (p = 0.0003 for control vs. NT1721 and for romidepsin vs. NT1721; no significant difference for control vs. romidepsin: p = 0.317). (C) Picture of HuT78 tumors from the treated mice. (D) NSG mice were injected s.c. with HH cells and treated by gavage on three consecutive days per week with 20 mg/kg NT1721 or the vehicle control. The differences in tumor volume were significant from day 14 through day 25 (p values < 0.0012). (E) Weight of HH tumors from individual mice (p = 0.0003). (F) Picture of representative HH tumors from the treated mice. 3.5. 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL Mouse Models DMSO-Free Formulation of NT1721 Potently Decreased CTCL Tumor Growth To assess the efficacy of NT1721 under more clinically relevant conditions we used a DMSO-free formulation of NT1721 for the treatment of mice bearing CTCL tumors: NT1721 was suspended in Ora-Blend containing 10% saline. Mice bearing HuT78 tumors were treated with the vehicle control, 30 mg/kg five times per week or 100 mg/kg NT1721 twice per week. As shown in Figure 5A–C, treatment with 30 mg/kg or 100 mg/kg NT1721 reduced the tumor volume and tumor weight significantly by 70% and 91%, respectively (p < 0.0001). No weight loss or other obvious signs of toxicity were observed with this formulation and drug schedule. In addition, mice bearing HH tumors were treated once or twice per week with 100 mg/kg NT1721 (in Ora-Blend). As shown in Figure 5D–F, the treatment led to a significant reduction in tumor growth by 35% and 55%, respectively. Taken together, the results show that treatment with NT1721 administered in a DMSO-free, more clinically relevant formulation potently suppressed CTCL tumor growth in mice. 10 of 15 Cancers 2021, 13, 3367 Figure 5. In vivo efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated with 30 mg/kg (five times per week), 100 mg/kg NT1721 (twice per week) or the vehicle control (Ora-Blend containing 10% saline). (A) Tumor volume. (B) Representative mice and tumors from the treatment and control groups. (C) Tumor weight in treated and control mice. The differences between control and treatment groups as well as between the treatment groups were statistically significant (p < 0.0001). (D–F) NSG mice bearing HH tumors were treated with 100 mg/kg NT1721 once a week, 100 mg/kg NT1721 twice per week or the vehicle control (Ora-Blend containing 10% saline). (D) Tumor volume. (E) Representative mice from the treatment and control groups. (F) Tumor weight in treated and control mice. The differences between control and treatment groups were statistically significant (control compared to 100 mg/kg 1×/week: p = 0.021 and control compared to 100 mg/kg 2×/week: p = 0.0002). o efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated Figure 5. In vivo efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated with 30 mg/kg (five times per week), 100 mg/kg NT1721 (twice per week) or the vehicle control (Ora-Blend containing 10% saline). 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL Mouse Models (A) Tumor volume. (B) Representative mice and tumors from the treatment and control groups. (C) Tumor weight in treated and control mice. The differences between control and treatment groups as well as between the treatment groups were statistically significant (p < 0.0001). (D–F) NSG mice bearing HH tumors were treated with 100 mg/kg NT1721 once a week, 100 mg/kg NT1721 twice per week or the vehicle control (Ora-Blend containing 10% saline). (D) Tumor volume. (E) Representative mice from the treatment and control groups. (F) Tumor weight in treated and control mice. The differences between control and treatment groups were statistically significant (control compared to 100 mg/kg 1×/week: p = 0.021 and control compared to 100 mg/kg 2×/week: p = 0.0002). Figure 5. In vivo efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated with 30 mg/kg (five times per week), 100 mg/kg NT1721 (twice per week) or the vehicle control (Ora-Blend containing 10% saline). (A) Tumor volume. (B) Representative mice and tumors from the treatment and control groups. (C) Tumor weight in treated and control mice. The differences between control and treatment groups as well as between the treatment groups were statistically significant (p < 0.0001). (D–F) NSG mice bearing HH tumors were treated with 100 mg/kg NT1721 once a week, 100 mg/kg NT1721 twice per week or the vehicle control (Ora-Blend containing 10% saline). (D) Tumor volume. (E) Representative mice from the treatment and control groups. (F) Tumor weight in treated and control mice. The differences between control and treatment groups were statistically significant (control compared to 100 mg/kg 1×/week: p = 0.021 and control compared to 100 mg/kg 2×/week: p = 0.0002). 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better Than the Single Agents 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better Than the Single Agents To determine if combinations of NT1721 with gemcitabine, a clinically used drug for CTCL treatment [39], could lead to improved anticancer responses, an in vitro viability assay was employed. As shown in Figure 6A, a combination of NT1721 with gemcitabine decreased the cell viability of HuT78 cells significantly better than the single drugs alone. To investigate what might cause this effect we assessed the expression of proteins related to apoptosis induction as well as GLI1 in HuT78 samples that were treated with NT1721, gemcitabine, or the drug combination. As shown in Figure 6B, the expression of cleaved PARP, pERK, and γH2AX was increased in samples treated with the drug combination compared to samples treated with the single agents. This result suggests that the drug combination may increase apoptosis induction and hence decrease cell viability. Treatment with the drug combination also seemed to slightly enhance GLI1 downregulation while treatment with gemcitabine alone did not affect GLI1 expression. To determine if combinations of NT1721 with gemcitabine, a clinically used drug for CTCL treatment [39], could lead to improved anticancer responses, an in vitro viability assay was employed. As shown in Figure 6A, a combination of NT1721 with gemcitabine decreased the cell viability of HuT78 cells significantly better than the single drugs alone. To investigate what might cause this effect we assessed the expression of proteins related to apoptosis induction as well as GLI1 in HuT78 samples that were treated with NT1721, gemcitabine, or the drug combination. As shown in Figure 6B, the expression of cleaved PARP, pERK, and γH2AX was increased in samples treated with the drug combination compared to samples treated with the single agents. This result suggests that the drug combination may increase apoptosis induction and hence decrease cell viability. Treatment with the drug combination also seemed to slightly enhance GLI1 downregulation while treatment with gemcitabine alone did not affect GLI1 expression. 11 of 15 Cancers 2021, 13, 3367 Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs. (B) Protein expression of GLI1 and proteins related to apoptosis iHut78 H. Induction (cleaved PARP, pERK, γH2A.x) in HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs for 48 h. 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better Than the Single Agents (C) Tumor weight in mice treated with NT1721, gemcitabine, a combination of the two drugs with the vehicle controls (Ora-Blend containing 10% saline or PBS, respectively) for 3 weeks. Representative HuT78 tumors treated with the single drugs or drug combinations are shown on the right side. (D) Expression of GLI1 and proteins related to apoptosis iHut78 H. Induction (BCL2, p21, pERK, γH2AX) in individual mice treated with 10 mg/kg NT1721, 20 mg/kg gemcitabine or the drug combination for 3 weeks. Original Western Blots of Figure 6B,D available in Figure S6. vo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721, Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells tre Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs. (B) Protein expression of GLI1 and proteins related to apoptosis iHut78 H. Induction (cleaved PARP, pERK, γH2A.x) in HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs for 48 h. (C) Tumor weight in mice treated with NT1721, gemcitabine, a combination of the two drugs with the vehicle controls (Ora-Blend containing 10% saline or PBS, respectively) for 3 weeks. Representative HuT78 tumors treated with the single drugs or drug combinations are shown on the right side. (D) Expression of GLI1 and proteins related to apoptosis iHut78 H. Induction (BCL2, p21, pERK, γH2AX) in individual mice treated with 10 mg/kg NT1721, 20 mg/kg gemcitabine or the drug combination for 3 weeks. Original Western Blots of Figure 6B,D available in Figure S6. Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs. (B) Protein expression of GLI1 and proteins related to apoptosis iHut78 H. Induction (cleaved PARP, pERK, γH2A.x) in HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs for 48 h. (C) Tumor weight in mice treated with NT1721, gemcitabine, a combination of the two drugs with the vehicle controls (Ora-Blend containing 10% saline or PBS, respectively) for 3 weeks. Representative HuT78 tumors treated with the single drugs or drug combinations are shown on the right side. (D) Expression of GLI1 and proteins related to apoptosis iHut78 H. 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better Than the Single Agents Induction (BCL2, p21, pERK, γH2AX) in individual mice treated with 10 mg/kg NT1721, 20 mg/kg gemcitabine or the drug combination for 3 weeks. Original Western Blots of Figure 6B,D available in Figure S6. To evaluate the efficacy of NT1721, gemcitabine, and the drug combination in vivo, mice bearing HuT78 tumors were treated with 10 mg/kg NT1721 (in Ora-Blend, twice per week), 10 mg/kg gemcitabine (twice per week), 20 mg/kg gemcitabine (twice per week) or the respective combinations. A comparison of the in vivo efficacy of NT1721 and gemcitabine showed that NT1721 significantly decreased tumor growth by 55%. Both 10 and 20 mg/kg gemcitabine only non-significantly decreased tumor growth by ~15% (Figure 6C). Better suppression of tumor growth by NT1721 was also observed in the other CTCL mouse model (HH tumors). As shown in Figure S3, NT1721 (100 mg/kg, 1×/week) decreased HH tumor growth by 52%, while gemcitabine (100 mg/kg, 2×/week) led to a 45% reduction. However, the difference between the two drugs did not reach statistical significance in this tumor model. Taken together, these results show that NT1721 led to a better suppression of CTCL tumor growth at a lower dose compared to gemcitabine. pp g p g Next, efficacies of the drug combinations (10 mg/kg NT1721 plus either 10 mg/kg or 20 mg/kg gemcitabine) against Hut78 tumors were compared. Mice treated with the drug combinations displayed 65% and 76% smaller tumors, respectively, while NT1721 alone only led to a 45% reduction (Figure 6C). The differences were significant: p = 0.0012 for NT1721 vs. NT1721 plus 10 mg/kg gemcitabine; p = 0.0003 for NT1721 vs. NT1721 plus Cancers 2021, 13, 3367 12 of 15 12 of 15 20 mg/kg gemcitabine. Thus, these results indicate that the drug combinations reduced tumor growth significantly better than the single drugs alone. 20 mg/kg gemcitabine. Thus, these results indicate that the drug combinations reduced tumor growth significantly better than the single drugs alone. g g y g g We then determined the expression of proteins related to the hedgehog pathway (GLI1, BCL2) and to apoptosis induction in individual mice treated with 10 mg/kg NT1721, 20 mg/kg gemcitabine or the drug combination. As shown in Figure 6D, NT1721 decreased the expression of GLI1 and antiapoptotic BCL2 and increased the expression of apoptosis induction-related proteins (p21, pERK, γH2AX), thus recapitulating what was observed in vitro (Figures 3B and 6B). 4. Discussion Apoptosis resistance has been identified as a major cause for the accumulation of malignant T cells in CTCL [22]. Patients with advanced disease have a poor prognosis with low survivals rates of less than 4 years [40]. Since current treatment options for advanced CTCL stages are rarely durable and remain largely palliative [4], new strategies for the treatment of advanced CTCL are urgently needed. g y Here we investigated the anti-CTCL activity of an ETP, NT1721. Treatment of CTCL cells derived from the blood of patients with either Sézary syndrome (Hut78) or aggres- sive, extensively pretreated CTCL (HH) (ATCC.org) with NT1721 downregulated GLI1 transcription factor, which was associated with decreased viability, reduced proliferation and increased apoptosis. This result aligns with previous reports showing that inhibition of aberrantly activated hedgehog/GLI in various types of T cell lymphomas by hedgehog inhibitors or GLI1 knockdown led to increased apoptosis and reduced proliferation and viability [33,41–43]. Decreased pSTAT3 levels as well as reduced expression of downstream targets were also observed in NT1721-treated cells. This agrees with a previous study show- ing that GLI1 knockdown or inhibition leads to a reduction in pSTAT3 levels in T cell lym- phomas including CTCL [33]. Aberrant STAT3 activation plays a major role in CTCL since it has been linked to apoptosis resistance, progression, and inferior clinical outcome [23–25]. Both GLI1 expression and STAT3 activation are absent from healthy peripheral T cells according to previous reports [24,33,43,44] and our results. This might partially explain why NT1721 preferentially reduces the viability of CTCL cells. The NT1721-mediated decreased GLI1 expression and STAT3 activation led to concentration-dependent downreg- ulation of downstream antiapoptotic proteins, i.e., BCL2 and BCL-xL, which contributed to apoptosis induction. Several recent studies have highlighted the importance of BCL2/BCL- xL inhibition for efficient apoptosis induction in CTCL. Adding BCL2 or BCL2/BCL-xL inhibitors to the treatment of CTCL cells with other agents (e.g., histone deacetylase or bromodomain inhibitors) synergistically increased apoptosis induction [7,17,34,45,46]. In contrast, no synergy was observed when BCL2-negative HH cells were treated with the drug combinations [17,34]. Moreover, it has been shown that antiapoptotic BCL2 proteins have a role in promoting hedgehog/GLI signaling by enhancing the turnover of a GLI antagonist, SUFU tumor suppressor. This inhibits GLI-SUFU interaction and thus increases the expression of GLI target genes such as BCL2 itself [47]. 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better Than the Single Agents BCL2 expression appeared to be decreased to a greater extend in mice treated with the drug combination compared to mice treated with the single agents. Upregulation of pERK, which is essential for NT1721-mediated increased expression of p21, yH2AX, cleaved PARP and cleaved CASP3 (Figure S2B), was elevated to a greater extent in mice treated with the drug combination compared to mice treated with NT1721 alone (Figure 6D). Taken together these results indicate that combining gemcitabine with NT1721 resulted in better antitumor effects compared to treatment with the single agents. This improved anticancer effect appears to be mediated by increased apoptosis. 4. Discussion Our in vivo results suggested that orally available, well-tolerated NT1721 inhibits CTCL tumor growth significantly better than two clinically used drugs, i.e., romidepsin or gemcitabine, in two CTCL mouse models. However, NT1721 reduced tumor growth in mice bearing Hut78 tumors significantly better than in mice bearing HH tumors (85% and 60% inhibition, respectively). Lower GLI1 and pSTAT3 levels as well as the lack of BCL2/BLC- xl expression in HH cells might at least partially explain the lower in vitro sensitivity Cancers 2021, 13, 3367 13 of 15 13 of 15 and relatively lower in vivo response to NT1721 compared to Hut78 cells. In summary, NT1721 may be a promising new agent for the treatment of CTCLs overexpressing GLI1 and activated STAT3 either as a single agent or in combination with gemcitabine. and relatively lower in vivo response to NT1721 compared to Hut78 cells. In summary, NT1721 may be a promising new agent for the treatment of CTCLs overexpressing GLI1 and activated STAT3 either as a single agent or in combination with gemcitabine. 5. Conclusions NT1721 potently induces apoptosis in CTCL in vitro and in vivo. Orally available, well-tolerated NT1721 reduces CTCL tumor growth better than two drugs that are currently clinically used for CTCL treatment (romidepsin and gemcitabine). Moreover, combinations of NT1721 with gemcitabine suppress CTCL growth better than the single agents. Thus, our results provide a rationale for testing the anti-CTCL efficacy of NT1721 in the clinic either as a single agent or in combination with gemcitabine. A patent related to this work was filed by City of Hope. A patent related to this work was filed by City of Hope. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13133367/s1. Figure S1: γH2AX expression. CTCL cells were treated with NT1721 as indicated for 24 h. γH2AX expression was detected in the cytoplasmic and nuclear extracts. Tubulin and TBP expression were used as loading controls; Figure S2: Combining NT1721 with an ERK inhibitor reduced apoptosis induction in HuT78 cells. (A) Protein expression in HuT78 cells treated with NT1721, the ERK inhibitor (SCH772984) or a combination. (B) Annexin V staining of HuT78 cells treated with NT1721, SCH772984 or combinations of both agents; Figure S3: In vivo efficacies of NT171 (in DMSO-free formulation) and gemcitabine against HH tumors. The mice were treated with NT1721, gemcitabine or the vehicle controls as indicated. (A) Tumor volume. (B) Tumor weight. (C) Picture of representative tumors from treatment and control groups; Figure S4: Original Western Blots of Figure 2A,C; Figure S5: Original Western Blots of Figure 3B,C; Figure S6: Original Western Blots of Figure 6B,D. Author Contributions: Conceptualization, C.M.K., L.E.O. and D.A.H.; methodology, formal analysis, investigation, M.L., C.M.K., S.Y.; resources: J.X.; writing—original draft preparation, C.M.K.; writing— review and editing, all authors; supervision, D.A.H.; funding acquisition, L.E.O. and D.A.H. All authors have read and agreed to the published version of the manuscript. Funding: The work was supported by the A. Gary Anderson Family Foundation (to D.A.H.), the Panda Charitable Foundation (to D.A.H.), and NIH grant R01GM098601 (to L.E.O.). Research reported in this publication included work performed in the Drug Discovery and Structural Bi- ology, Analytical Flow Cytometry, Pathology, and Light Microscopy Cores that are supported by the National Cancer Institute of the National Institutes of Health Cancer Center Support Grant (P30CA33572). Institutional Review Board Statement: Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with approved protocols from the institutional animal care and use committee of City of Hope National Medical Center. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article. Conflicts of Interest: The authors declare no conflict of interest. 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JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL. Blood Adv. 2020, 4, 2213–2226. [CrossRef] [PubMed] 47. Wu, X.; Zhang, L.S.; Toombs, J.; Kuo, Y.C.; Piazza, J.T.; Tuladhar, R.; Barrett, Q.; Fan, C.W.; Zhang, X.; Walensky, L.D.; et al. Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor. Nat. Cell Biol. 2017, 19, 1226–1236. [CrossRef]
https://openalex.org/W4392570306
https://bop.unibe.ch/linguistik-online/article/download/10789/13893
Italian
null
La fraseologia nei dizionari istrioto-italiano
Linguistik online
2,024
cc-by
10,506
Abstract In this paper, the treatment of phraseological elements in four bilingual dictionaries of Istriot varieties – Gallesanese, Vallese, and Rovignese – and Italian is analyzed. First, an overview of the sociolinguistic situation of Istriot in the complex linguistic reality of Croatian Istria is provided. This is followed by a concise outline of Istriot lexicography. In the empirical part, after describing the analyzed dictionaries, the research results of the treatment of phraseology in the latter are presented and discussed. Finally, some concluding remarks are made. 1 Per una discussione sul concetto di “esperto” in lessicografia, cf. Melchior (2024: 233–235), più in generale sul rapporto esperti/non esperti con riferimento al sapere e al discorso sulla lingua, cf. i contributi raccolti in Hoffmei- ster/Hundt/Naths (2021). 1 Introduzione Nel presente articolo si analizzerà il trattamento degli elementi fraseologici in quattro dizionari bilingui di varietà istriote – gallesanese, vallese e rovignese – e italiano. Innanzitutto, si illu- strerà a grandi linee la situazione sociolinguistica dell’istrioto nella complessa realtà linguistica dell’Istria croata. Si delineerà poi brevemente un profilo della lessicografia istriota, spesso opera di non esperti,1 per passare quindi all’analisi empirica. Dopo una descrizione dei voca- bolari in oggetto si procederà alla presentazione e discussione dei risultati della ricerca sul trattamento della fraseologia negli stessi e, infine, ad alcune considerazioni conclusive. Luca Melchior (Klagenfurt a. W./Graz) Luca Melchior (Klagenfurt a. W./Graz) 2 Per quanto assai ridotta, la diffusione territoriale dell’istrioto è dunque più ampia di quanto indicato ad es. da Banfi (2003: 629), che lo vedeva parlato solamente nelle località di Dignano e Rovigno. L’istrioto Parlando di istrioto ci si riferisce all’unico gruppo di idiomi autoctoni preveneti della penisola istriana, la cui estensione territoriale, un tempo verosimilmente estesa a un’area più ampia dell’Istria meridionale, al di sotto del Canale di Leme, si è ridotta ormai a sei – secondo alcune stime appena cinque – località dell’Istria meridionale, come visualizzato nella cartina rappre- sentata nella figura 1.2 Sia il termine istrioto, introdotto da Graziadio Isaia Ascoli (cf. Ascoli 1873: 436), cui si deve “il primo inquadramento scientifico, nel quale si colgono in nuce tutti gli elementi sviluppati nelle analisi successive” (Ursini 1989: 537), sia il più recente termine istroromanzo proposto da Petar Skok (1943) sono termini dotti, che non corrispondono alle denominazioni in uso in loco. Tradizionalmente i sei idiomi vengono infatti indicati ognuno con un suo nome, in genere 1 Per una discussione sul concetto di “esperto” in lessicografia, cf. Melchior (2024: 233–235), più in generale sul rapporto esperti/non esperti con riferimento al sapere e al discorso sulla lingua, cf. i contributi raccolti in Hoffmei- ster/Hundt/Naths (2021). 2 Per quanto assai ridotta, la diffusione territoriale dell’istrioto è dunque più ampia di quanto indicato ad es. da Banfi (2003: 629), che lo vedeva parlato solamente nelle località di Dignano e Rovigno. Linguistik online 125, 1/24 − http://dx.doi.org/10.13092/lo.125.10789 CC by 3.0 118 Linguistik online 125, 1/24 Figura 1: Località istriotofone3 Figura 1: Località istriotofone3 deonomastico, derivante dal nome della località in cui lo si parla: si ha dunque, procedendo da nord a sud, il ruvigniʃ ‘rovignese’ a Rovinj-Rovigno, il dignanìʃ ‘dignanese’ o boumbaro ‘bum- baro’ a Vodnjan-Dignano, il valeʃ ‘vallese’ a Bale-Valle, il galiʃaneʃ ‘gallesanese’ a Galižana- Gallesano, il faʃaneʃ ‘fasanese’ a Fažana-Fasana e il sisaneʃ ‘sissanese’ a Šišan-Sissano. Più in generale, i parlanti sembrano riferirsi alla propria varietà semplicemente con il termine dialetto. I sei idiomi sono sostanzialmente privi di codificazione sia a livello locale sia, e ancor di più, a livello di una norma di riferimento sovralocale. Considerato lingua “severly endangered” dall’UNESCO (cf. Moseley 32010: 40), l’istrioto nel suo complesso conta attualmente, secondo le stime di Cergna (2014: 317), “non più di circa 1070 individui” parlanti attivi residenti in Istria,4 a fronte di stime di circa 20.000 parlanti a inizio 1800 (cf. Buršić Giudici 2017: 59). 4 Per un totale di circa duemila potenziali parlanti, considerando anche gli istriotofoni emigrati fuori Istria e fuori Croazia (cf. Cergna 2014: 317). 3 L’immagine 1 è stata collocata sulla presente pagina per motivi di spazio e su richiesta delle curatrici E. Autelli e C. Konecny. 3 L’immagine 1 è stata collocata sulla presente pagina per motivi di spazio e su richiesta delle curatrici E. Autelli e C. Konecny. 4 Per un totale di circa duemila potenziali parlanti, considerando anche gli istriotofoni emigrati fuori Istria e fuori Croazia (cf. Cergna 2014: 317). L’istrioto In realtà, lo “stato di salute” nelle sei località in cui si parla(va)no idiomi istrioti è piuttosto di- verso: mentre per Valle e Gallesano la trasmissione intergenerazionale pare resistere (come deonomastico, derivante dal nome della località in cui lo si parla: si ha dunque, procedendo da nord a sud, il ruvigniʃ ‘rovignese’ a Rovinj-Rovigno, il dignanìʃ ‘dignanese’ o boumbaro ‘bum- baro’ a Vodnjan-Dignano, il valeʃ ‘vallese’ a Bale-Valle, il galiʃaneʃ ‘gallesanese’ a Galižana- Gallesano, il faʃaneʃ ‘fasanese’ a Fažana-Fasana e il sisaneʃ ‘sissanese’ a Šišan-Sissano. Più in generale, i parlanti sembrano riferirsi alla propria varietà semplicemente con il termine dialetto. I sei idiomi sono sostanzialmente privi di codificazione sia a livello locale sia, e ancor di più, a livello di una norma di riferimento sovralocale. Considerato lingua “severly endangered” dall’UNESCO (cf. Moseley 32010: 40), l’istrioto nel suo complesso conta attualmente, secondo le stime di Cergna (2014: 317), “non più di circa 1070 individui” parlanti attivi residenti in Istria,4 a fronte di stime di circa 20.000 parlanti a inizio 1800 (cf. Buršić Giudici 2017: 59). In realtà, lo “stato di salute” nelle sei località in cui si parla(va)no idiomi istrioti è piuttosto di- verso: mentre per Valle e Gallesano la trasmissione intergenerazionale pare resistere (come ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 119 mostrano dati di una ricerca inedita effettuata dallo scrivente con Isabella Matticchio nel 2021), a Dignano5 la lingua sembra fortemente in pericolo, andando ad assumere funzioni postverna- colari (cf. Shandler 2004) più che comunicative. Per l’ultima località, Fasana, Cergna (2014: 317) la indica addirittura come completamente scomparsa ed effettivamente, nel corso del pro- getto DERSII – Documentazione e rivitalizzazione dei sei idiomi istrioti, attivo dal 2013, non sono stati individuati parlanti di fasanese. Una ricerca più recente riguarda Sissano: qui si regi- stra un deciso calo sia di uso sia di trasmissione intergenerazionale del sissanese a favore di istroveneto, ciacavo e croato standard (cf. Giudici 2018: 103–105), ma al contempo offre dati meno pessimistici di precedenti ricerche per quanto riguarda il numero di parlanti. Sia dal punto di vista linguistico sia da quello sociolinguistico i sei idiomi istrioti si trovano in una situazione assai complessa. Dal primo, una classificazione di tali idiomi come parte del continuum italoromanzo settentrionale o come gruppo autonomo è tuttora dibattuta6 – e non scevra di implicazioni politiche –, come si vedrà anche più avanti. 5 In questa località si registra però un certo fiorire di produzioni letterarie in istrioto. 7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto, di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita- lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres- sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi di parlare in un italiano ricercato’. L’istrioto Lo spiccato plurilinguismo che caratterizza l’Istria rende anche il quadro sociolinguistico non meno intricato: oltre al croato standard, nelle località in cui si parla ancora istrioto, l’italiano ha co-ufficialità; queste due lin- gue vanno a coprire il polo alto della situazione poliglossica. Fanno infatti parte del repertorio sociale – e spesso di quello individuale dei parlanti di istrioto – anche varietà ciacave, ben distanti dallo standard croato, nonché dialetti istroveneti locali, che sono di regola la prima lingua dei parlanti appartenenti alla Comunità Nazionale Italiana (CNI)7 e lingua della quoti- dianità comunicativa “romanza” e infine un istroveneto koineizzante, sorta di “lingua franca” per gli “italofoni” della penisola istriana. Alla CNI appartengono di regola anche i parlanti istrioto, che non di rado hanno a lungo considerato e talora ancora “considerano l’istrioto una delle varianti dell’istroveneto” (Filipi 2009), subordinandolo sociolinguisticamente a questo (sul senso di appartenenza, anche identitaria, cf. anche Filipi 2005).8 Come accennato, l’uso dei termini istrioto e istroromanzo può prestarsi – e si è purtroppo prestato – a possibili interpreta- zioni ideologico-politiche: mentre al primo, infatti, è stata attribuita talora la funzione di postu- lare una continuità geolinguistica tra l’istrioto e i dialetti italoromanzi, al termine istroromanzo è stata contestata una presunta affermazione dell’autonomia di questo gruppo (cf. al riguardo Fanciullo 2011: 116s., ripreso anche in Covino 2019: 84s.).9 A prescindere da tali questioni, g p ( ) 7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto, di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita- lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres- sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi di parlare in un italiano ricercato’. ISSN 1615-3014 5 In questa località si registra però un certo fiorire di produzioni letterarie in istrioto. 6 Una discussione al riguardo in Filipi (2002). 7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto, di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita- lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres- sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi di parlare in un italiano ricercato’. 8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018). Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei parlanti, cf. anche Giudici (2018: 107–109). 9 Il che non significa che la scelta dell’uno o dell’altro termine implichi necessariamente una componente ideolo- gico-politica; così Giudici (2022: 13s.) nega valore politico all’uso del termine istroromanzo da parte di Pavao Tekavčić. In questo contributo si preferisce istrioto, che pare il termine maggiormente utilizzato nella ricerca. sione parlar in cicara, che si ritrova in forme simili anche nell Italia settentrionale con il significato di sforzarsi di parlare in un italiano ricercato’. 8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018). Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei parlanti, cf. anche Giudici (2018: 107–109). 6 Una discussione al riguardo in Filipi (2002). 10 I quattro vocabolari, così come quello di Dalla Zonca (1978) e quelli, di varietà istrovenete, di Manzini/Rocchi (1995), di Dussich (2008, 2019) e di Orbanić/Giudici (2009), sono liberamente scaricabili in formato PDF. Al riguardo si vedano le rassegne bibliografiche a cura di Muljačić (1969, 1981, 1988, 1998, 2009) e di Buršić Giudici (1996). Lessicografia istriota 3 Non solo esiste una notevole mole di lavori scientifici dedicati a diversi aspetti delle parlate istriote,10 ma vi è anche una certa produzione lessicografica, in particolare a partire dalla fine del secolo scorso, sebbene le parole di Tekavčić (1986: 5) non abbiano perso la loro attualità: “non abbiamo ancora nemmeno un vocabolario moderno dell’insieme dei dialetti istroro- manzi”, bensì esistono dizionari bilingui delle singole parlate (in coppia con l’italiano).11 Così si hanno per la parlata di Valle il dizionario di Cernecca (1986) e quello di Cergna (2015), che del primo “rappresenta la naturale continuazione” (Cergna 2015: 9); per il rovignese esiste il ponderoso vocabolario in due volumi a cura di Pellizzer/Pellizzer (1992), mentre alla parlata di Gallesano è dedicato il vocabolario di Balbi/Moscarda Budić (2003). Tutti i vocabolari pre- sentano la direzione di consultazione varietà istriota > italiano, di cui costituiscono delle sorte di repertori lessicali dal carattere più documentario che d’uso, come esplicitamente dichiarato nella prefazione di Pellizzer/Pellizzer (1992: 15): Tuttavia sarebbe ingiusto non riconoscere la motivazione affettiva nella quale è da ricercarsi il primo vigoroso impulso impresso alla volontà, se non di “fermare”, ma almeno di documentare nel tempo, un aspetto della vita, quella specificatamente “ruvigniʃa”, nella dimensione che era ed è, congiuntamente con i suoi usi e costumi e con il suo patrimonio culturale tout court, attraverso la sua più autentica realtà storica e sociale: quella della comunicazione, in presenza di una diaspora sofferta e mutilante su entrambi i versanti. 12 A quelli citati va aggiunto il Vocabolario giuliano (Rosamani 1958), che contiene anche elementi afferenti i diversi idiomi istrioti (al riguardo, cf. Tekavčić 1986; Holtus 1987). 11 Crevatin (2015: 159) scrive al proposito della lessicografia istriota: “[l]’Istria meridionale, il cui dialetto è defi- nito in genere ‘istrioto’, è coperta in maniera ragionevole anche se non completa”. tuttavia, si è visto che, percettivamente, i parlanti tendono a riconoscere la pertinenza delle proprie varietà al sistema italoromanzo, senza peraltro disconoscerne le peculiarità. tuttavia, si è visto che, percettivamente, i parlanti tendono a riconoscere la pertinenza delle proprie varietà al sistema italoromanzo, senza peraltro disconoscerne le peculiarità. L’istrioto g p ( ) 7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto, di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita- lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres- sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi di parlare in un italiano ricercato’. 8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018). Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei parlanti, cf. anche Giudici (2018: 107–109). 8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018). Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei parlanti, cf. anche Giudici (2018: 107–109). 9 Il che non significa che la scelta dell’uno o dell’altro termine implichi necessariamente una componente ideolo- gico-politica; così Giudici (2022: 13s.) nega valore politico all’uso del termine istroromanzo da parte di Pavao Tekavčić. In questo contributo si preferisce istrioto, che pare il termine maggiormente utilizzato nella ricerca. ISSN 1615-3014 Linguistik online 125, 1/24 120 3.1 Le somiglianze tra i vocabolari analizzati sono numerose. In tutti e quattro, la lingua utilizzata nei testi esterni, nella descrizione microstrutturale e nei rinvii nella mediostruttura è l’italiano, che è anche la lingua obiettivo dei dizionari, che, come detto, sono bilingui, ma monodirezio- nali. Tutti i dizionari hanno una serie di testi esterni, che comprende una presentazione o intro- duzione dell’autore/degli autori, una lista delle abbreviazioni e una bibliografia delle opere con- sultate, sostituita in Cergna (2015) da una lista delle persone intervistate. A Pellizzer/Pellizzer (1992) e a Cergna (2015) è premessa una presentazione per opera del linguista Franco Crevatin. In Balbi/Moscarda Budić (2003), premessa alla lista delle abbreviazioni (e dunque al lemmario) vi è una sezione con cenni storici sulla varietà in oggetto; posposte al lemmario invece sono alcune note grammaticali. Cernecca (1996) contiene un’appendice posposta al lemmario, nella quale sono presentati i paradigmi di verbi regolari e irregolari, alcuni testi in vallese e un breve riassunto dell’opera in croato (Sažetak). Sia Cernecca (1986) per Valle sia Balbi/Moscarda Budić (2003) per Gallesano hanno raccolto il materiale linguistico sulla base di fonti esclusivamente orali – informanti, di regola anziani – , poiché affermano che il rispettivo idioma non conosce usi scritti. Pellizzer/Pellizzer (1992) per Rovigno e Cergna (2015) per Valle si basano invece sia su fonti orali che scritte, ma mentre i primi fanno uno spoglio il più completo possibile della scritturalità, di regola letteraria, rovi- gnese, il secondo si basa sostanzialmente su “dieci Quaderni manoscritti di Giovanni Obrovaz – ‘Zaneto’, compilati dallo stesso tra gli anni Sessanta e Settanta del Novecento ed oggi accu- ratamente custoditi nell’archivio del Centro di ricerche storiche di Rovigno” (Cergna 2015: 10, corsivo nell’originale), concepiti a loro volta come sorta di repertorio lessicale. I quattro dizio- nari hanno una mole – e di conseguenza un lemmario – dalle dimensioni assai diverse tra loro: 140 pagine in totale Cernecca (1986), di cui 106 dedicate al lemmario, 293 pagine (di cui 266 di lemmario, con diverse illustrazioni) Balbi/Moscarda Budić (2003), 421 pagine Cergna (2015), di cui 400 dedicate al lemmario (con alcune illustrazioni), e ben 1140 pagine Pelliz- zer/Pellizzer (1992), di cui 1096 di lemmario (518 nel primo, 578 nel secondo volume). Diversa non è solo l’estensione del lemmario, ma anche, come visto, la ricchezza di testi esterni, il numero di abbreviazioni registrate – su cui si tornerà tra poco – e l’esaustività della microstrut- tura. 13 Su tale vocabolario si veda Tekavčić (1986; con particolare riguardo alla trattazione di elementi fraseologici in senso lato, ibd.: 14s.). (ibd: 15, corsivo nell’originale) Ai vocabolari citati si aggiungono inoltre l’edizione, nel 1978, del Vocabolario dignanese-ita- liano, compilato dall’erudito istriano Giovanni Andrea Dalla Zonca a metà Ottocento, e infine il Vocabolario italiano-rovignese di Benussi (2013) che, pur contenendo un’appendice a Pelliz- zer/Pellizzer (1992) rovignese-italiano, è altrimenti concepito nella direzione opposta. Manca- no repertori lessicografici delle varietà di Fasana e di Sissano. Per quest’ultima è in preparazio- ne un repertorio etimologico e fraseologico (cf. Buršić Giudici 2018, 2021; Giudici/ Buršić Giudici 2022).12 Con l’eccezione di Benussi (2013), tutti i vocabolari sono usciti per il Centro di Ricerche Storiche di Rovigno, emanazione dell’Unione Italiana, che rappresenta i membri della Comunità Nazionale Italiana di Croazia e Slovenia. Per lo stesso Centro di Ricerche sono apparsi anche dizionari dei dialetti istroveneti di Capodistria (Manzini/Rocchi 1995), di Buie d’Istria (Dussich 2008, 2019) e di Pola (Orbanić/Giudici 2009). Sebbene questi non si occupino di varietà istriote, sono accomunabili ai primi non solo perché ne condividono la casa editrice, ma perché gli autori degli uni e degli altri pertengono allo stesso ambiente culturale. I diversi 10 I quattro vocabolari, così come quello di Dalla Zonca (1978) e quelli, di varietà istrovenete, di Manzini/Rocchi (1995), di Dussich (2008, 2019) e di Orbanić/Giudici (2009), sono liberamente scaricabili in formato PDF. Al riguardo si vedano le rassegne bibliografiche a cura di Muljačić (1969, 1981, 1988, 1998, 2009) e di Buršić Giudici (1996). 11 Crevatin (2015: 159) scrive al proposito della lessicografia istriota: “[l]’Istria meridionale, il cui dialetto è defi- nito in genere ‘istrioto’, è coperta in maniera ragionevole anche se non completa”. ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 121 dizionari mostrano chiaramente il debito reciproco che hanno tra loro. Nel presente contributo si illustreranno i risultati dell’analisi dei quattro vocabolari con direzione istrioto > italiano (Balbi/Moscarda Budić 2003 per il gallesanese, Cergna 2015; Cernecca 1986, entrambi per il vallese; e Pellizzer/Pellizzer 1992 per il rovignese); in fase di ricerca non sono mancati tuttavia numerosi confronti con i restanti. Si è scelto di escludere Dalla Zonca (1978)13 in quanto esso non solo riporta uno stato della lingua decisamente anteriore agli altri, ma anche nella sua strut- turazione risponde a criteri ottocenteschi; Benussi (2013) è stato invece escluso perché la dire- zione di lemmatizzazione e ricerca è opposta a quella dei precedenti, non è dunque con questi facilmente comparabile. 3.1 Così, mentre in Cernecca (1986), nella microstruttura, oltre all’indicazione della parte del discorso o del genere (per i nomi) si trova pressoché solamente il traducente italiano, Sia Cernecca (1986) per Valle sia Balbi/Moscarda Budić (2003) per Gallesano hanno raccolto il materiale linguistico sulla base di fonti esclusivamente orali – informanti, di regola anziani – , poiché affermano che il rispettivo idioma non conosce usi scritti. Pellizzer/Pellizzer (1992) per Rovigno e Cergna (2015) per Valle si basano invece sia su fonti orali che scritte, ma mentre i primi fanno uno spoglio il più completo possibile della scritturalità, di regola letteraria, rovi- gnese, il secondo si basa sostanzialmente su “dieci Quaderni manoscritti di Giovanni Obrovaz – ‘Zaneto’, compilati dallo stesso tra gli anni Sessanta e Settanta del Novecento ed oggi accu- ratamente custoditi nell’archivio del Centro di ricerche storiche di Rovigno” (Cergna 2015: 10, corsivo nell’originale), concepiti a loro volta come sorta di repertorio lessicale. I quattro dizio- nari hanno una mole – e di conseguenza un lemmario – dalle dimensioni assai diverse tra loro: 140 pagine in totale Cernecca (1986), di cui 106 dedicate al lemmario, 293 pagine (di cui 266 di lemmario, con diverse illustrazioni) Balbi/Moscarda Budić (2003), 421 pagine Cergna (2015), di cui 400 dedicate al lemmario (con alcune illustrazioni), e ben 1140 pagine Pelliz- zer/Pellizzer (1992), di cui 1096 di lemmario (518 nel primo, 578 nel secondo volume). Diversa non è solo l’estensione del lemmario, ma anche, come visto, la ricchezza di testi esterni, il numero di abbreviazioni registrate – su cui si tornerà tra poco – e l’esaustività della microstrut- tura. Così, mentre in Cernecca (1986), nella microstruttura, oltre all’indicazione della parte del discorso o del genere (per i nomi) si trova pressoché solamente il traducente italiano, ISSN 1615-3014 122 Linguistik online 125, 1/24 accompagnato, sporadicamente, da esempi, compresi tra le virgolette «» e non tradotti, in Pel- lizzer/Pellizzer (1992) la microstruttura prevede esempi e attestazioni, con indicazione della fonte e traduzione, confronti con altri idiomi (istrioti e non solo), talora l’etimologia o informa- zioni di stampo enciclopedico. Tra questi due estremi si pongono Balbi/Moscarda Budić (2003) e Cergna (2015), che presentano un buon apparato di esempi e attestazioni (con traduzione). Il trattamento della fraseologia Punto di partenza per l’analisi è stato il più corposo vocabolario di Pellizzer/Pellizzer (1992), per poi allargare le osservazioni agli altri tre. L’analisi è stata effettuata in due modi: innanzi- tutto, si è osservato come vengano utilizzate, nella microstruttura, le abbreviazioni che si rife- riscono alla fraseologia, cioè quali elementi vengano da queste introdotti e quale sistematica vi stia alla base; dall’altra parte è stato analizzato il trattamento dei fraseologismi menzionati sotto determinati lemmi (càn, bàrca, lògo), se e quali marcature essi presentano e se vi sono eventuali rimandi mediostrutturali. 3.2 Riferimenti espliciti alla fraseologia Solamente Pellizzer/Pellizzer (1992: 17) fanno esplicito, seppur indiretto, riferimento alla fra- seologia nell’introduzione al loro lavoro. Essi scrivono infatti: “Certi lemmi vengono presentati in più varianti, appunto perché si è voluto dare un quadro completo (alle volte anche contrad- ditorio rispetto alla normativa linguistica) della situazione odierna del dialetto, alla quale si è voluto subordinare anche la fraseologia.” A loro volta, Balbi/Moscarda Budić (2003: 10) si limitano ad affermare di aver raccolto anche elementi fraseologici: “Non abbiamo preteso entrare nel campo dell’etimologia, poiché non di nostra competenza, ma ci siamo limitate alla raccolta documentata di voci, ma anche frasi tipi- che, detti, proverbi, modi di dire, usanze che riflettono momenti e aspetti della vita quotidiana, contadina, familiare della nostra gente.” Con l’eccezione di Cernecca (1986), nei restanti vocabolari sono registrate, nella rispettiva lista, abbreviazioni utilizzate nella microstruttura per indicare elementi fraseologici. Queste sono “loc.” e “loc. avv.” in Cergna (2015: 14), “locuz.”, “locuz. avv.” e “loc. inf.” (glossata come “locuzione infantile”) in Balbi/Moscarda Budić (2003: 15). In Pellizzer/Pellizzer (1992) vi è una maggiore copia di abbreviazioni relative agli elementi fraseologici. Si trovano infatti “espress.” per espressione ed espressivo; “loc., locuz.” per locuzione, “locuz. avv.” e “locuz. verb.” per locuzione avverbiale e locuzione verbale, oltre che “fras.” per fraseologia, fraseolo- gico (ibd.: 21s.). L’abbondanza di soluzioni sembra creare più confusione che portare chiarezza, anche se in realtà l’abbreviazione “espress.” viene usata solamente in casi in cui essa non va ad assumere, nella microstruttura, la funzione dell’indicazione sintattico-semantica, ma si trova nel commento semantico o introduce esempio d’uso, come nei casi di danàro e danèri (cf. figure 2–3). ISSN 1615-3014 Figura 2: Voce danàro (Pellizzer/Pellizzer 1992: 298) Figura 3: Voce danèri (Pellizzer/Pellizzer 1992: 298) Figura 2: Voce danàro (Pellizzer/Pellizzer 1992: 298) Figura 3: Voce danèri (Pellizzer/Pellizzer 1992: 298) ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 123 In realtà, l’analisi delle voci mostra che in Balbi/Moscarda Budić (2003), nella microstruttura, sia presente due volte anche “loc. avv.” (mentre l’abbreviazione “loc.” normalmente significa ‘località’); in Pellizzer/Pellizzer (1992), inoltre, modi di dire e proverbi sono introdotti dalla nota “Detti e prov. rov.”. 14 L’allineamento spostato a destra nelle riproduzioni degli articoli lessicografici sta a indicare che l’articolo stesso, nel dizionario, continua su una seconda colonna o una seconda pagina. 4.1 È stato possibile indagare l’uso delle abbreviazioni tramite una ricerca automatica delle stesse nei file PDF. Dell’abbreviazione “espress.” e del suo uso in Pellizzer/Pellizzer (1992) si è detto sopra. Per quanto riguarda l’abbreviazione “fras.”, essa si trova solamente tre volte, alle voci Càiser, ecùte e ièsi (cf. figure 4–6). Interessante che nel primo caso tale abbreviazione introduca un fraseologismo in dialetto bisiaco, portato a confronto con l’istrioto rovignese, e non per in- trodurre quello in quest’ultimo idioma, che viene presentato solo come esempio. Per quanto riguarda i fraseologismi alla voce ièsi, stupisce il fatto che essi siano presentati solamente sotto il lemma della copula e non sotto quello degli altri elementi che li compongono. Figura 4: Voce Càiser (Pellizzer/Pellizzer 1992: 166)14 Figura 5: Voce ecùte (Pellizzer/Pellizzer 1992: 345s.) Figura 5: Voce ecùte (Pellizzer/Pellizzer 1992: 345s.) 14 L’allineamento spostato a destra nelle riproduzioni degli articoli lessicografici sta a indicare che l’articolo stesso, nel dizionario, continua su una seconda colonna o una seconda pagina. ISSN 1615-3014 124 Linguistik online 125, 1/24 Figura 6: Voce ièsi (Pellizzer/Pellizzer 1992: 449s.) Figura 6: Voce ièsi (Pellizzer/Pellizzer 1992: 449s.) Nel caso di ecùte, la classificazione come fraseologismo non pare comprensibile: si tratta infatti di un’interiezione monolessicale, formata da un avverbio e da un pronome oggetto, la cui fun- zione può sicuramente essere formulaica, ma che non ricade sotto una definizione assai ampia di fraseologismo. La caratterizzazione come “fras. avv.”, interpretabile come ‘fraseologismo avverbiale’, pare inoltre essere un ἄπαξ nella lessicografia e fraseografia: L’abbreviazione “locuz. avv.” viene utilizzata in Pellizzer/Pellizzer (1992) per diversi tipi di espressioni, in realtà solo in parte con valore avverbiale, come nei casi di a baloûchi, a bruʃaca- meîʃa e in canpanièla, come si vede nelle figure 7–9. Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88) Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88) ISSN 1615-3014 Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88) Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88) Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) 15 Fermo restando che biʃièfe è componente unicale, come giustamente nota un recensore del presente articolo, sebbene tale caratteristica non sia rilevante per l’analisi presentata in questo punto. Sui componenti unicali cf. tra gli altri Veland (2005, 2006) e Konecny (2018). L’indicazione dell’unicalità per gli elementi da questa caratteriz- zati non è presente nei dizionari in esame, come si vede anche nella figura 24. 16 Solamente la maggiore trasparenza di dabànda rispetto a cusiveîa pare forse poter giustificare la decisione per un trattamento diverso da parte degli autori del dizionario. ISSN 1615-3014 125 Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 9: Voce canpanièla (Pellizzer/Pellizzer 1992: 181) Figura 9: Voce canpanièla (Pellizzer/Pellizzer 1992: 181) Per quanto riguarda la lemmatizzazione, in questi tre casi il fraseologismo è riportato sotto il termine con maggiore valore lessicale, mentre la preposizione, parte integrale della locuzione, viene indicata tra parentesi in coda al lemma, prima dell’indicazione sintattica. In altri casi, strutturalmente non diversi da quelli appena discussi, la strutturazione dell’articolo lessicogra- fico è diversa. Si veda il caso di a biʃièfe (figura 10): anche qui il fraseologismo è registrato sotto l’elemento con valore lessicale, il quale viene già indicato come “locuz.”, salvo poi spe- cificare “Usato nella locuz. avv.: a biʃièfe”. La strutturazione dell’articolo risulta dunque assai diversa dalle precedenti, pur essendo gli elementi descritti, per struttura morfosintattica, pres- soché identici.15 Figura 10: Voce biʃièfe (Pellizzer/Pellizzer 1992: 122) Figura 10: Voce biʃièfe (Pellizzer/Pellizzer 1992: 122) In altri casi ancora, l’indicazione “locuz. avv.” caratterizza elementi univerbali con valore av- verbiale. Si osservi l’esempio in figura 11: Figura 11: Voce cusuveîa (Pellizzer/Pellizzer 1992: 289) Figura 11: Voce cusuveîa (Pellizzer/Pellizzer 1992: 289) Interessante è che, per elementi simili, si trovino anche soluzioni diverse, come nel caso di dabànda (figura 12), senza che sia comprensibile quali ragioni stiano alla base della disparità di trattamento:16 15 Fermo restando che biʃièfe è componente unicale, come giustamente nota un recensore del presente articolo, sebbene tale caratteristica non sia rilevante per l’analisi presentata in questo punto. Sui componenti unicali cf. tra gli altri Veland (2005, 2006) e Konecny (2018). L’indicazione dell’unicalità per gli elementi da questa caratteriz- zati non è presente nei dizionari in esame, come si vede anche nella figura 24. 16 Solamente la maggiore trasparenza di dabànda rispetto a cusiveîa pare forse poter giustificare la decisione per un trattamento diverso da parte degli autori del dizionario. 17 Seppure il rovignese pare sia storicamente, tra gli idiomi istrioti, l’unico a presentare una serie quasi completa di pronomi enclitici (cf. Giudici 2022: 195); tuttavia tale tematica non viene trattata nella grammatica di Benussi (2015), il che fa presumere “che questo tratto sia ormai uscito dall’uso” (Giudici 2022: 196). 18 Da notare però che la forma con pronome atono femminile ʃìla è trattata ben diversamente. Anch’essa è lem- matizzata come tale, seguita da “(ùla)”, ma la microstruttura è la seguente: “f. verb. - Forma verbale contratta corrispondente a: dove è lei. Ùla ʃìla, dove (lei) è?; i nu la trùvo, dùve ʃìla? non la trovo, dove è (lei)” (Pelliz- zer/Pellizzer 1992: 911). ISSN 1615-3014 ISSN 1615-3014 126 Linguistik online 125, 1/24 Figura 12: Voce dabànda (Pellizzer/Pellizzer 1992: 293) Infine, l’indicazione “locuz. avv.” viene utilizzata anche per elementi che non hanno valore avverbiale, come nel caso seguente (figura 13): Fi 12 V d bà d (P lli /P lli 1992 293) Figura 12: Voce dabànda (Pellizzer/Pellizzer 1992: 293) Infine, l’indicazione “locuz. avv.” viene utilizzata anche per elementi che non hanno valore avverbiale, come nel caso seguente (figura 13): Figura 13: Voce bài (Pellizzer/Pellizzer 1992: 84) Figura 13: Voce bài (Pellizzer/Pellizzer 1992: 84) L’abbreviazione “locuz. verb.” è presente un’unica volta; essa introduce, in realtà, una forma interrogativa con inversione di verbo alla terza persona singolare (da ièsi ‘essere’) e pronome soggetto enclitico maschile, che fa parte del verbo alla forma interrogativa, presentata nel con- testo di una domanda e lemmatizzata come tale. La scelta di caratterizzare questo elemento come locuzione verbale risulta incomprensibile; si può ipotizzare che l’inversione interrogativa, cristallizzata e non più in uso,17 sia ritenuta poco riconoscibile ai parlanti.18 Figura 14: Voce ʃìlo (Pellizzer/Pellizzer 1992: 911) Figura 14: Voce ʃìlo (Pellizzer/Pellizzer 1992: 911) Sia “locuz.” che “locuz. avv.” vengono utilizzate però non solo immediatamente dopo il lemma, ma anche all’interno della descrizione microstrutturale per introdurre elementi fraseologici composti con il lemma in questione, come nell’esempio in figura 15, al lemma cuòrda. ISSN 1615-3014 127 Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 15: Voce cuòrda (Pellizzer/Pellizzer 1992: 277) Figura 15: Voce cuòrda (Pellizzer/Pellizzer 1992: 277) Analoghe incoerenze si trovano anche in Balbi/Moscarda Budić (2003), dove peraltro il numero di voci introdotte da una delle indicazioni elencate nell’indice delle abbreviazioni è assai limi- tato: appena 31, di cui 28 da “locuz. avv.” e tre da “locuz.”. Si confronti il diverso trattamento di a cavaloto, a cao e n batùda nelle figure 16–18. Figura 16: Voce cavaloto (Balbi/Moscarda Budić 2003: 56) Figura 16: Voce cavaloto (Balbi/Moscarda Budić 2003: 56) Figura 17: Voce cao (Balbi/Moscarda Budić 2003: 56) Figura 17: Voce cao (Balbi/Moscarda Budić 2003: 56) ISSN 1615-3014 128 Linguistik online 125, 1/24 Non vi sono lemmi marcati con l’abbreviazione “locuz. inf.”, presente tuttavia nell’indice delle abbreviazioni. In alcuni casi l’uso dell’abbreviazione “locuz.” risulta difficilmente comprensi- bile. Si osservi per esempio il caso in figura 19: qui “locuz.” caratterizza un secondo significato del verbo. Fraseologico non è però l’utilizzo del verbo istrioto gallesanese, bensì il suo tradu- cente italiano; a sua volta, però, l’esempio proposto viene tradotto con un altro fraseologismo italiano: Figura 19: Voce ʃbafà (Balbi/Moscarda Budić 2003: 217) Figura 19: Voce ʃbafà (Balbi/Moscarda Budić 2003: 217) Più coerenza nella caraterrizzazione degli elementi introdotti dalle abbreviazioni “loc.” e “loc. avv.” mostra Cergna (2015), che però lemmatizza le espressioni fraseologiche talora nell’arti- colo di uno degli elementi che le compongono, talaltra come lemmi autonomi, talaltra sotto l’elemento lessicale, seguito dalla preposizione (o in alcuni casi da un verbo) tra parentesi. Si confrontino le figure 20–23: ISSN 1615-3014 Figura 20: Voce ala una el can lavòra (Cergna 2015: 22) Figura 21: Voce babalòca (Cergna 2015: 34) Figura 22: Voce badinè (Cergna 2015: 35) Figura 23: Voce basilà (Cergna 2015: 40) Figura 20: Voce ala una el can lavòra (Cergna 2015: 22) Figura 20: Voce ala una el can lavòra (Cergna 2015: 22) 19 Da non confondere con l’omonimo can ‘murice’. Figura 22: Voce badinè (Cergna 2015: 35) Luca Melchior: La fraseologia nei dizionari istrioto-italiano 129 Come si è accennato più sopra, Cernecca (1986) non contiene un indice delle abbreviazioni. Una scansione del testo nel documento PDF con possibili abbreviazioni relative a fraseologismi permette di individuare un caso di utilizzo dell’abbreviazione “locuz.”, come nella figura 24: Come si è accennato più sopra, Cernecca (1986) non contiene un indice delle abbreviazioni. Una scansione del testo nel documento PDF con possibili abbreviazioni relative a fraseologismi permette di individuare un caso di utilizzo dell’abbreviazione “locuz.”, come nella figura 24: 22 Nei lemmi pubblicati in anteprima del futuro dizionario di sassanese vi è il lemma can (cf. Buršic Giudici/Giu- dici 2022: 129). Nella microstruttura dell’articolo vi è un elemento che pare avere carattere proverbiale (Tùti i càni bàja) con rispettiva traduzione. Tale elemento non è introdotto da alcuna indicazione, ma è in corsivo come tutti gli esempi presentati in questi stralci di dizionario. 21 Il vocabolario lemmatizza anche fiòl d’oùn cein, caratterizzato come “locuz.” e spiegato come “Forma attenuata di ‘fiòl d’oûn can’” (Pellizzer/Pellizzer 1992: 374). 20 Che peraltro non sono tutti lemmatizzati: nel vocabolario non si trova infatti il lemma bòia. 4.2 Il trattamento dei fraseologismi all’interno degli articoli Oltre all’uso delle abbreviazioni, la ricerca ha previsto anche un’analisi esemplare del tratta- mento dei fraseologismi all’interno degli articoli lessicografici. Si è scelto di indagare le voci riguardanti tre sostantivi (barca, can ‘cane’,19 logo ‘luogo’), tre verbi (cantà ‘cantare’, magnà ‘mangiare’, ʃeî (o equivalente ʃì) ‘andare’) e tre aggettivi (alto ‘alto’, bon ‘buono’, viècio (o equivalente vècio) ‘vecchio’). Punto di partenza è stato anche in questo caso il vocabolario rovignese di Pellizzer/Pellizzer (1992), per poi verificare la presenza delle unità eventualmente identificate negli altri dizionari. Come si può vedere in figura 25, al lemma can sono presenti diversi elementi fraseologici in senso lato: Figura 25: Voce can (Pellizzer/Pellizzer 1992: 176) Figura 25: Voce can (Pellizzer/Pellizzer 1992: 176) La presentazione di tali elementi non avviene in modo sistematico e coerente: a una serie di unità fraseologiche (di tipo diverso) priva di qualunque indicazione sulla loro natura segue una sezioni più breve introdotta da “Modi di dire, detti e prov. rov.”. In questa, oltre a due proverbi si trova tuttavia anche una voce polirematica (can da vàche). La stessa voce è lemmatizzata anche come voce a sé stante (Pellizzer/Pellizzer 1992: 179), ma non è registrata alla voce vàca. Solamente can barbòn e can da firma sono, infatti, presenti anche ai lemmi corrispondenti agli ISSN 1615-3014 130 Linguistik online 125, 1/24 altri elementi che li formano:20 il primo al lemma barbòn, ma sotto forma di esempio, il secondo alla voce firma, a sua volta in un esempio che ha però la funzione di spiegazione semantica di questo significato del lemma. Come can da vàche, anche fiòl d’oûn can è presentato inoltre come lemma autonomo e caratterizzato come “locuz. scherz.” (ibd: 374).21 All’interno dell’articolo relativo si trova un rimando mediostrutturale al lemma fiòl (non però a can). Alla voce fiòl il fraseologismo è presente solo in un commento di stampo comparatistico, senza che se ne spieghi il significato (cf. figura 26): Figura 26: Voce fiòl (Pellizzer 1992: 373s.) Figura 26: Voce fiòl (Pellizzer 1992: 373s.) Figura 26: Voce fiòl (Pellizzer 1992: 373s.) Cernecca (1986) presenta, alla voce can, il solo fraseologismo vita de can (cf. figura 27). Questo è introdotto da virgolette, assieme all’espressione can del s’ciopo, dove can ha però significato di ‘grilletto’.22 Più ricco è Balbi/Moscarda Budić (2003), che al lemma can presenta voci polirematiche, non introdotte come categoria a sé stante, mentre Xe lavor de cani viene indicato come modo di dire e Fiol de un can! come imprecazione. Segue una sezione di proverbi, alcuni dei quali corri- spondono a quelli contenuti anche in Pellizzer/Pellizzer (1992). ISSN 1615-3014 131 Figura 29: Voce bàrca (Pellizzer/Pellizzer 1992: 95s.) Luca Melchior: La fraseologia nei dizionari istrioto-italiano Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 28: Voce can (Balbi/Moscarda Budić 2003: 4 Figura 28: Voce can (Balbi/Moscarda Budić 2003: 45) Per quanto riguarda gli altri elementi che formano tali espressioni, le voci polirematiche non sono riportate ai rispettivi lemmi; lavor, bando e fiol non sono registrati nemmeno come lemmi. Lemma a sé stante forma invece fiolduncan, univerbato, e indicato come aggettivo con il signi- ficato di ‘bricconcello’; non vi sono rinvii alla voce can (cf. Balbi/Moscarda Budić 2003: 91). Cergna (2015: 69) non presenta elementi fraseologici alla voce can. Anche alla voce bàrca, troviamo diverse forme fraseologiche, incluse collocazioni e proverbi – questi ultimi introdotti dall’indicazione “Modi di dire e prov. rov.”. Di tutte le unità fraseolo- giche, solo Bàrca rùta cònti fàti e bàrca nìta nu vadàgna sono menzionati anche altrove, rispet- tivamente alla voce rùto (Pellizzer/Pellizzer 1992: 793) e alla voce vadagnà (ibd.: 1099). Stupisce dunque il rinvio mediostrutturale a paròn, sotto il quale l’elemento fraseologico non è presente (cf. ibd.: 658). Figura 29: Voce bàrca (Pellizzer/Pellizzer 1992: 95s.) Figura 29: Voce bàrca (Pellizzer/Pellizzer 1992: 95s.) ISSN 1615-3014 Linguistik online 125, 1/24 132 Cernecca (1986: 20) e Cergna (2015: 38) non hanno elementi fraseologici alla voce barca, che non è lemmatizzata in Balbi/Moscarda Budić (2003: 28), dove si trova solo barcon.23 Al lemma lògo Pellizzer/Pellizzer (1992) riporta il fraseologismo ni fògo ni lògo e la colloca- zione avì lògo, non introdotti da alcuna indicazione. Entrambi sono registrati soltanto al lemma lògo. Anche in questo caso, le due espressioni sono presenti solo qui. Figura 30: Voce lògo (Pellizzer/Pellizzer 1992: 534) Figura 30: Voce lògo (Pellizzer/Pellizzer 1992: 534) Negli altri tre dizionari il fraseologismo è attestato, al rispettivo lemma (lógo in Cernecca 1986: 60; lògo in Balbi/Moscarda Budić 2003: 124; lógo in Cergna 2015: 186). In Cernecca (1986) tale unità è presentata come “no vé né fógo né l.”, il cui traducente è indicato come “essere povero in canna”. Si è già visto più sopra che in Cernecca (1986) le virgolette hanno molteplici funzioni, introducendo esempi, fraseologia, ecc.; analogamente polifunzionale è in Cergna (2015) e Balbi/Moscarda Budić (2003) il corsivo, con cui è indicata l’unità fraseologica in oggetto. 23 Il lemma barca è uno di quelli che andranno a formare il futuro dizionario di sissanese presentati in anteprima in Buršic Giudici (2021: 554); alla rispettiva voce non vi sono però fraseologismi. 24 Il lemma cantà dal futuro dizionario di sissanese è presentato in anteprima in Buršic Giudici/Giudici (2022: 131); nell’articolo corrispondente non vi sono elementi fraseologici. Luca Melchior: La fraseologia nei dizionari istrioto-italiano In Balbi/Moscarda Budić (2003) essa è presente solo in un esempio, tradotto prima letteralmente, poi nel senso figurato: Figura 31: Voce lògo (Balbi/Moscarda Budić 2003: 124) Figura 31: Voce lògo (Balbi/Moscarda Budić 2003: 124) Al lemma cantà si trovano in Pellizzer/Pellizzer (1992: 182) un fraseologismo comparativo, sotto forma di esempio (el cànta cùme oûn deîo), e due proverbi, questi ultimi introdotti con l’indicazione, già menzionata sopra, “Modi di dire, detti e prov. rov.”. Tutti e tre i fraseologismi sono registrati solo al lemma cantà. ISSN 1615-3014 133 25 Nella bibliografia di Pellizzer/Pellizzer (1992: 1134) è indicata la ristampa della seconda edizione del dizionario del Boerio, datata 1861, che non è a mia disposizione. Il brano citato in Pellizzer/Pellizzer (ibd.: 165) si trova nella seconda edizione (Boerio 1856: 114). Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 35: Voce ʃeî (Pellizzer/Pellizzer 1992: 880) Figura 35: Voce ʃeî (Pellizzer/Pellizzer 1992: 880) ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 135 Di questa copia di fraseologismi, solo sei sono registrati anche ai lemmi corrispondenti di altri elementi che li compongono – tra questi tre sono verbi sintagmatici. Alla voce uòrsa si trova il fraseologismo in un esempio: “Si ti ʃbàndi màsa, va a l’uòrsa, se sbandi troppo, va all’orza; uòrsa” (Pellizzer/Pellizzer 1992: 1087, corsivo nell’originale). Lo stesso sintagma è riportato tuttavia nella forma base e in un altro esempio anche con un significato diverso: “ʃeî a l’uòrsa, prendere una storta: i son ʃeî a l’uòrsa cul peîe e dièso el ma sa ʃgiònfa, mi sono preso una storta al piede e adesso mi si gonfia”. Alla voce ʃboûʃa si trova “ʃeî ʃboûʃa, andare buca, andare male, non aver successo” (ibd.: 832, corsivo nell’originale), seguito da due esempi con relativa traduzione. Alla voce foûmo, nella sezione “Motti, detti e prov. rov.” si trova, dopo altre due unità fraseologiche, “ʃeî in foûmo, svanire, sparire, nel nulla” (ibd.: 381), corsivo nell’originale). Per quanto riguarda i verbi sintagmatici, alla voce vànti (ibd.: 1105) vi sono alcuni esempi per i quali si propongono da una parte il traducente letterale ‘andare avanti’, in altri casi però anche quello di ‘continuare, proseguire’; alla voce dreîo è presente ʃeî dreîo (ibd.: 335, corsivo nell’originale), ma con due significati (e relativi traducenti) diversi da quello proposto alla voce ʃeî, ovverosia ‘dare ascolto, credere, seguire, prestar fede, badare’ e ‘corteggiare, far la corte’. Infine, ʃeî ʃù è presente, in un esempio, alla voce ʃù: “i ʃèmo ʃù, (o in ʃù), andiamo giù e anche usciamo” (ibd.: 991, corsivo nell’originale). Di questa copia di fraseologismi, solo sei sono registrati anche ai lemmi corrispondenti di altri elementi che li compongono – tra questi tre sono verbi sintagmatici. Alla voce uòrsa si trova il fraseologismo in un esempio: “Si ti ʃbàndi màsa, va a l’uòrsa, se sbandi troppo, va all’orza; uòrsa” (Pellizzer/Pellizzer 1992: 1087, corsivo nell’originale). Lo stesso sintagma è riportato tuttavia nella forma base e in un altro esempio anche con un significato diverso: “ʃeî a l’uòrsa, prendere una storta: i son ʃeî a l’uòrsa cul peîe e dièso el ma sa ʃgiònfa, mi sono preso una storta al piede e adesso mi si gonfia”. Luca Melchior: La fraseologia nei dizionari istrioto-italiano Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 32: Voce cantà (Pellizzer/Pellizzer 1992: 182) Figura 32: Voce cantà (Pellizzer/Pellizzer 1992: 182) In Cergna (2015: 70) alla voce cantà non vi sono fraseologismi, come nemmeno in Bal- bi/Moscarda Budić (2003: 47). In Cernecca (1986: 28) si trova “c. da galo” (non riportato alla voce galo, cf. Cernecca 1986: 51), presentato senza traducenti, che non è conosciuto ai parlanti vallesi interpellati come fraseologismo e che pare dunque una combinazione libera.24 Anche al lemma magnà (Pellizzer/Pellizzer 1992: 558) si trovano alcune espressioni fraseolo- giche. Tra queste, la collocazione magnà fòra ‘mangiare al ristorante’ e la locuzione idiomatica magnà la fòia ‘mangiare la foglia, scoprire l’inganno’ sono presentati senza particolari indica- zioni, il primo in un esempio, il secondo alla forma base. Seguono tre proverbi, appositamente introdotti. Tra questi, magnà puòco e duòrmi mòndo è presente anche al lemma duòrmi (ibd.: 341) e ti iè vulìsto, màgna da quìsto al lemma vulì; gli altri non sono presenti altrove. Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) ISSN 1615-3014 134 Linguistik online 125, 1/24 Cernecca (1986: 61), alla voce magnà riporta “‘m. la fùia’ accorgersi dell’inganno”, non regi- strato alla voce fùia (Cernecca 1986: 49). Cergna (2015: 194) riporta il detto o magna l oso o salta l foso, il cui carattere fraseologico è indicato non tramite un’abbreviazione o mezzi tipo- grafici, ma con un’esplicita spiegazione nel commento semantico (cf. figura 34). Il detto è men- zionato solo a questa voce, non a quelle degli altri elementi che lo compongono. Figura 34: Voce magnà (Cergna 2015: 194) Figura 34: Voce magnà (Cergna 2015: 194) In Balbi/Moscarda Budić (2003: 128, corsivo nell’originale), alla voce magnà troviamo “Prov. gall.: Chi magna pian, lavora pian; chi magna ʃvelto, lavora ʃvelto. Chi mangia piano, lavora piano; chi mangia veloce, lavora veloce”. Il proverbio è registrato solo a questa voce (il lemma ʃvelto manca nel dizionario). Alla voce ʃei in Pellizzer/Pellizzer (1992: 880) vengono presentati, in maniera indifferenziata, numerose unità fraseologiche di tipo diverso. 26 Il lemma álto dal futuro dizionario di sissanese è presentato in anteprima in Buršic Giudici (2018: 211); nell’ar- ticolo relativo non vi sono elementi fraseologici. 27 Il pronome cheî non è lemmatizzato, viene introdotto al lemma chi come allomorfo interrogativo dello stesso (Pellizzer/Pellizzer 1992: 215). Benussi (2015: 75) indica che chei si riferisce a persone, chi a cose o animali. 26 Il lemma álto dal futuro dizionario di sissanese è presentato in anteprima in Buršic Giudici (2018: 211); nell’ar- ticolo relativo non vi sono elementi fraseologici. 27 Il pronome cheî non è lemmatizzato viene introdotto al lemma chi come allomorfo interrogativo dello stesso ticolo relativo non vi sono elementi fraseologici. 27 Il pronome cheî non è lemmatizzato, viene introdotto al lemma chi come allomorfo interrogativo dello stesso (Pellizzer/Pellizzer 1992: 215). Benussi (2015: 75) indica che chei si riferisce a persone, chi a cose o animali. Luca Melchior: La fraseologia nei dizionari istrioto-italiano Alla voce ʃboûʃa si trova “ʃeî ʃboûʃa, andare buca, andare male, non aver successo” (ibd.: 832, corsivo nell’originale), seguito da due esempi con relativa traduzione. Alla voce foûmo, nella sezione “Motti, detti e prov. rov.” si trova, dopo altre due unità fraseologiche, “ʃeî in foûmo, svanire, sparire, nel nulla” (ibd.: 381), corsivo nell’originale). Per quanto riguarda i verbi sintagmatici, alla voce vànti (ibd.: 1105) vi sono alcuni esempi per i quali si propongono da una parte il traducente letterale ‘andare avanti’, in altri casi però anche quello di ‘continuare, proseguire’; alla voce dreîo è presente ʃeî dreîo (ibd.: 335, corsivo nell’originale), ma con due significati (e relativi traducenti) diversi da quello proposto alla voce ʃeî, ovverosia ‘dare ascolto, credere, seguire, prestar fede, badare’ e ‘corteggiare, far la corte’. Infine, ʃeî ʃù è presente, in un esempio, alla voce ʃù: “i ʃèmo ʃù, (o in ʃù), andiamo giù e anche usciamo” (ibd.: 991, corsivo nell’originale). Cernecca (1986: 102) alla voce ʃeî presenta solo un esempio e parte del paradigma; in Bal- bi/Moscarda Budić (2003: 233), nell’articolo di ʃì si trovano solo il traducente e alcuni esempi con relativi traducenti. Cergna (2015: 346, corsivo nell’originale) riporta come secondo signi- ficato del verbo ʃì ‘(Figura) ʃì per le longhe, prolungare una questione, un impegno’, seguito da un’attestazione. Delle unità fraseologiche proposte alla voce àlto (Pellizzer/Pellizzer 1992: 44, cf. figura 36), delle quali alcune sono introdotte dall’indicazione “Come loc. avv. e fig.”, solo una è presente anche al lemma corrispondente a un altro elemento costituente, ovverosia “Àlta de cagadùr”, per la quale vi è anche un rinvio mediostrutturale al lemma cagadùr. A questa voce viene però presentato solamente un brano dal dizionario veneziano di Boerio (31867), che registra la stessa unità fraseologica in veneziano, spiegandone il significato.25 ISSN 1615-3014 136 Linguistik online 125, 1/24 Figura 36: Voce àlto (Pellizzer/Pellizzer 1992: 44) 27 Il pronome cheî non è lemmatizzato, viene introdotto al lemma chi come allomorfo interrogativo del (Pellizzer/Pellizzer 1992: 215). Benussi (2015: 75) indica che chei si riferisce a persone, chi a cose o ani Figura 36: Voce àlto (Pellizzer/Pellizzer 1992: 44) Gli altri dizionari lemmatizzano l’aggettivo nella forma alto (Cernecca 1986; Balbi/Moscarda Budić 2003: 20; Cergna 2015: 24), alle rispettive voci non sono però riportati elementi fraseo- logici.26 Gli altri dizionari lemmatizzano l’aggettivo nella forma alto (Cernecca 1986; Balbi/Moscarda Budić 2003: 20; Cergna 2015: 24), alle rispettive voci non sono però riportati elementi fraseo- logici.26 Nell’ampio articolo dedicato al lemma bon (Pellizzer/Pellizzer 1992: 126s.) vi sono diversi elementi fraseologici. Indicati come tali (“nelle locuz. avv. e agg ibd.: 127) sono quelli in cui l’aggettivo viene in realtà nominalizzato alla forma femminile e quattro “modi di dire e pro- verbi”, non però le collocazioni tignìse bon, da bon grado e stà bon. Tra tutti gli elementi fra- seologici, solo il proverbio Biègna stà in bòna cui treîsti ca cui bòni nu mànca mài si trova registrato anche altrove, alla voce treîsto (ibd.: 1062).27 È inoltre classificato come “loc. avv.” bon bon, in realtà segnale discorsivo, per cui si propongono i traducenti “sì, sì, d’accordo”. ISSN 1615-3014 137 Luca Melchior: La fraseologia nei dizionari istrioto-italiano Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 37: Voce bon (Pellizzer/Pellizzer 1992: 126s.) Cernecca (1986: 23) riporta il fraseologismo comparativo “b. como l pan” e le due collocazioni “ʃì cun le bone”, per il quale propone il traducente ‘persuadere con le buone maniere’, e ‘tégnise n bonÄ, per cui propone il traducente ‘vantarsi’. Tutte e tre le unità fraseologiche sono registrate ISSN 1615-3014 138 Linguistik online 125, 1/24 solo al lemma bon. Cergna (2015: 51, corsivo nell’originale) ha il fraseologismo comparativo “Bon como il pan” e la collocazione “tégnise n bona”. Entrambi sono presenti solo al lemma bon e non sono classificati come elementi fraseologici. Balbi/Moscarda Budić (2003: 34, corsivo nell’originale) caratterizza il lemma bon come “agg. e locuz. avv.” e presenta, come esempi, i fraseologismi “Al xe bon como ’l pan” e “I xe n bona”, con i relativi traducenti (per il secondo “Hanno dei buoni rapporti”). Anche in questo dizionario si registra, in un esempio, bon bon, non ulteriormente caratterizzato, per cui si propone il traducente “Va bene”. Infine, per viècio, che conosce un’unica lemmatizzazione come aggettivo e sostantivo (i due elementi non vengono differenziati nemmeno nell’articolo), in Pellizzer/Pellizzer (1992: 1119) si trovano quattro proverbi (più altri due in cui l’elemento è sostantivo), introdotti da “Detti e prov. rov.”. Di tali proverbi, tre su quattro sono presenti anche sotto lemmi corrispondenti ad altri elementi che li compongono: galeîna viècia fa bon brù (ibd.: 138, corsivo nell’originale) si trova anche al lemma brù, can viècio nu bàia a la loûna (ibd.: 84, corsivo nell’originale) ai lemmi baià e loûna (ibd.: 536), e i moûri vièci fà pànsa al lemma pànsa (ibd.: 649s., corsivo nell’originale; i proverbi con viècio sostantivo sono registrati solo a questa voce). In Balbi/Moscarda Budić (2003: 276) non vi sono unità fraseologiche alla voce vecio. Cernecca (1986: 116) ha solo “pan v.” e “l me vècio” ‘mio padre’, che non sono elementi fraseologici; gli stessi sono riportati da Cergna (2015: 410, corsivo nell’originale), che ha anche la collocazione “vècio cudinaro, vecchissimo”. L’articolo del lemma cudinaro (Cergna 2015: 99) consiste solo di un rinvio mediostrutturale a vècio. Figura 38: Voce viècio (Pellizzer/Pellizzer 1992: 1119) Figura 38: Voce viècio (Pellizzer/Pellizzer 1992: 1119) 5 Conclusioni 5 L’analisi dei quattro dizionari ha rivelato diverse incoerenze, sia nell’utilizzo delle abbrevia- zioni per indicare elementi fraseologici, sia nel trattamento degli stessi. In alcuni casi le abbre- viazioni – poco differenziate per le diverse categorie di unità fraseologiche – introducono ele- ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 139 menti che non sono fraseologici, spesso invece mancano completamente; all’interno dell’arti- colo lessicografico vengono utilizzate in modo e posizione diversi. La maggior parte delle unità fraseologiche è presentata solo sotto un lemma; in pochi casi anche ai lemmi corrispondenti ad altri elementi che la formano. Anche in tal caso però si notano numerose incongruenze: talora sotto un lemma non si trovano tutte le possibili unità fraseologiche registrate invece all’altro o non sono presenti tutti i traducenti possibili. Altre volte i traducenti proposti per la stessa unità fraseologica, attestata sotto diversi lemmi, differiscono tra loro. Infine, alcune unità fraseologi- che vanno a costituire lemmi a sé stanti (eventualmente in forma grafica diversa), senza che sia comprensibile il criterio che sta alla base della scelta di quali siano autonomamente lemmatiz- zati e quali no. Pressoché assenti sono i rinvii mediostrutturali; se presenti, talora paiono fuor- vianti. Dei quattro dizionari, Pellizzer/Pellizzer (1992) presenta una fraseologia relativamente ricca, trattata in maniera differenziata, anche se in questo caso spesso non coerente. Balbi/Mo- scarda Budić (2003: 276) e Cergna (2015) hanno una discreta presenza di fraseologismi, date le dimensioni più ridotte di questi dizionari, però sicuramente inferiore a quella del precedente. Pur costituendo indubbiamente fonti documentarie preziose per le varietà di istrioto in esse trattate, i dizionari mostrano un certo carattere di “amatorialità” lessicografica, che conduce appunto alle incoerenze strutturali qui evidenziate. Auspicabile è dunque che gli interessanti materiali qui e altrove raccolti (cf. p. e. Moscarda 1998; Trgovčić 1998; Moscarda Mirković 2001, 2002, 2013; Cergna 2013; Tamaro 2021), integrati con ulteriori materiali da raccogliere sul campo, vengano ulteriormente sistematizzati in future opere lessicografiche e fraseogra- fiche. Bibliografia Ascoli, Graziadio Isaia (1873): “Saggi ladini”. Archivio Glottologico Italiano 1: 1–556. Ascoli, Graziadio Isaia (1873): “Saggi ladini”. Archivio Glottologico Italiano 1: 1–556 Banfi, Emanuele (2003): “Evoluzione delle frontiere delle lingue romanze: Romania del Sud- Est”. In: Ernst, Gerhard et al. (eds.): Romanische Sprachgeschichte./Histoire linguistique de la Romania. Vol. 1. Berlin/New York, de Gruyter: 622–631. (= Handbücher zur Sprach- und Kommunikationswissenschaft 23.1). Benussi, Libero (2015): Grammatica del dialetto di Rovigno d’Istria. Rovigno: Comunità degli italiani “Pino Budicin” – Rovigno. Blagoni, Robert (2012): “L’italiano in Croazia e in Istria tra opportunità giurilinguistiche, at- teggiamenti sociolinguistici e squilibri linguistico-politici”. In: Telmon, Tullio/Raimondi, Gianmario/Revelli, Luisa (eds.): Coesistenze linguistiche nell’Italia pre- e postunitaria. Atti del XLV Congresso internazionale della Società di Linguistica Italiana (Aosta/Bard/Torino, 26–28 settembre 2011). Roma, Bulzoni: 449–458. (= Pubblicazioni della Società di Lingui- stica Italiana 57). Blagoni, Robert/Poropat Jeletić, Nada/Blecich, Kristina (2018): “Istroveneto e italofonia in Istria: prospettive e visioni di un’insularità etnolinguistica”. In: Šimičić, Lucija/Škevin, Ivana/Vuletić, Nikola (eds.): Le isole linguistiche dell’Adriatico. Canterano, Aracne: 69– 992. (= Neuropaideia. Didattica, lingue e culture 19). doi: 10.4399/97888255162345. p g Buršić Guidici, Barbara (1996): “Bibliografia dell’istrioto”. Annales 8: 261–270. Buršić Giudici, Barbara (2017): “Il dialetto sissanese quasi cent’anni dopo: per uno studio com- parativo in diacronia tra l’ALI e la parlata di oggi”. In: Gudurić, Snežana/Radić-Bojanić, Buršić Giudici, Barbara (2017): “Il dialetto sissanese quasi cent’anni dopo: per uno studio com- parativo in diacronia tra l’ALI e la parlata di oggi”. In: Gudurić, Snežana/Radić-Bojanić, ISSN 1615-3014 Linguistik online 125, 1/24 140 Biljana (eds.): Jezici i culture u vremenu i prostoru 6. Novi Sad, Sveučilište u Novom Sadu: 59–68. Biljana (eds.): Jezici i culture u vremenu i prostoru 6. Novi Sad, Sveučilište u Novom Sadu: 59–68. Buršić Giudici, Barbara (2018): “Approccio al Vocabolario del dialetto di Sissano – lettera A”. In: Cossutta, Rada (ed.): Tromeje dialektov brez meja. Koper, Znanstveno-raziskovalno sre- dišče, Založba Annales ZRS: 205–220. Buršić Giudici, Barbara (2021): “La lettera ‘B’ del futuro vocabolario del dialetto sissanese”. In: Gudurić, Snežana/Radić-Bojanić, Biljana (eds.): Jezici i culture u vremenu i prostoru 7. Novi Sad, Sveučilište u Novom Sadu: 549–560. Cergna, Sandro (2013): “La tradizione paremiologica di Valle d’Istria”. Atti del Centro di Ri- cerche Storiche di Rovigno XLIII: 723–775. Cergna, Sandro (2014): “L’istrioto: cenni storici”. Atti del Centro di Ricerche Storiche di Ro- vigno XLIV: 317–332. Covino, Sandra (2019): Linguistica e nazionalismo tra le due guerre mondiali. Scienza e ideo- logia negli epigoni ascoliani. Bibliografia Bologna: Il Mulino. Crevatin, Franco (2015): “Lessicografia istriana ed etimologia: un bilancio”. Rivista Italiana di Dialettologia XXXIX: 157–167. Fanciullo, Franco (2011): “Lingua e lingue nell’Europa e nel Mediterraneo”. In: Barbero, Ales- sandro (ed.): Storia d’Europa e del Mediterraneo. II. Dal Medioevo all’età della globaliz- zazione, Sezione V. L’età moderna (secoli XVI–XVIII). Vol. XI: Culture, religioni, saperi, a cura di Roberto Bizzocchi. Roma, Salerno: 85–121. Filipi, Goran (2002): “ Istriotisch”. In: Okuka, Miloš (ed.): Lexikon der Sprachen des europäi- schen Ostens. Klagenfurt-Celovec/Wien/Ljubljana, Wieser: 87–89. (= Wieser Enzyklopädie des Europäischen Ostens (WEEO) 10). Filipi, Goran (2005): “istriotski ili istroromanski jezik”. In: Bertoša, Miroslav/Matijašić, Robert (eds.): Istarska enciklopedija. Zagreb, Leksikografski zavod Miroslav Krleža: 343. Filipi, Goran (2009): “Istrioto (lingua istroromanza)”. Istrapedia. Istarska internetska enciklo- pedija. istrapedia.hr/it/natuknice/1312/istrioto-lingua-istroromanza [26.01.2023]. Giudici, Alberto (2018): “Un’isola linguistica in una penisola linguistica. Il caso del sissanese”. In: Šimičić, Lucija/Škevin, Ivana/Vuletić, Nikola (eds.): Le isole linguistiche dell’Adriatico. Canterano, Aracne: 93–115. (= Neuropaideia. Didattica, lingue e culture 19). doi: 10.4399/ 97888255162346. Giudici, Alberto (2022): Grammatica storica del dialetto di Sissano d’Istria. Tesi di dottorato, Universität Zürich. Giudici, Alberto/Buršić Giudici, Barbara (2022): “Alcuni lemmi della lettera C del futuro Di- zionario etimologico e fraseologico del dialetto di Sissano d’Istria”. In: Todorović, Su- zana/Baloh, Barbara (eds.): Kontaktna dijalektologija na območju med Alpami in Jadranom. V spomin akademiku Goranu Filipiju. Capodistria/Koper, Libris: 119–144. Hoffmeister, Toke/Hundt, Markus/Naths, Saskia (2021): Laien, Wissen, Sprache: Theoretische, methodische und domänenspezifische Perspektiven. Berlin/Boston: de Gruyter. doi: 10.1515/9783110731958. Holtus, Günter (1987): “Zur Lexikographie des Istroromanischen: der ‘Vocabolario giuliano’ von Enrico Rosamani”. In: Holtus, Günter/Kramer, Johannes (eds.): Romania et Slavia Adriatica. Festschrift für Žarko Muljačić. Hamburg, Buske: 525–535. ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 141 Konecny, Christine (2018): “La monocollocabilità: un fenomeno di interfaccia tra sincronia e diacronia”. PHRASIS. Rivista di studi fraseologici e paremiologici 2: 60–76. Melchior, Luca (2024): “9 Lexicographie”. In: Becker, Lidia/Herling, Sandra/Wochele, Holger (eds.): Manuel de linguistique populaire. Berlin/Boston, de Gruyter: 231–254. (= Manuals of Romance Linguistics 34). Moscarda, Eliana (1998): “I proverbi dei mesi nell’agricoltura a Gallesano d’Istria”. In: Milani, Nelida (ed.): Civiltà istriana. Ricerche e proposte. Trieste/Rovigno, Unione Italiana – Fiume/Università popolare di Trieste/Società “Pietas Iulia” – Pola: 91–118. (= Etnia. Extra serie 1). Moscarda Mirković, Eliana (2001): “La tradizione paremiologica a Gallesano (Parte I)”. Atti (Centro di Ricerche Storiche di Rovigno) XXXI: 371–468. Moscarda Mirković, Eliana (2002): “La tradizione paremiologica a Gallesano (Parte II)”. Bibliografia Atti (Centro di Ricerche Storiche di Rovigno) XXXII: 515–626. Moscarda Mirković, Eliana (2013): “La fede, la religione, la provvidenza nella saggezza popo- lare gallesanese”. El portego 11: 16–17. Moseley, Christopher (ed.) (32010): Atlas of the World’s Languages in Danger. Paris: UNESCO. unesdoc.unesco.org/ark:/48223/pf0000187026 [26.01.2023]. Muljačić, Žarko (1969): “Bibliographie de linguistique romane. Domaine dalmate et istriote avec les zones limitrophes (1906–1966)”. Revue de linguistique romane 33/129–130: 144– 167 : 356–391. Muljačić, Žarko (1981): “Bibliographie de linguistique romane : domaine dalmate et istriote avec les zones limitrophes (1966–1976)”. Revue de linguistique romane 45/177–178: 158– 214. Muljačić, Žarko (1988): “Bibliographie de linguistique romane : domaine dalmate et istriote avec les zones limitrophes (1976–1987)”. Revue de linguistique romane 52/205–206: 183– 239. Muljačić, Žarko (1998): “Bibliographie de linguistique romane domaine dalmate et istriote avec les zones limitrophes (1987–1996)”. Revue de linguistique romane 62/245–246: 183–223. Muljačić, Žarko (2009): “Bibliographie dalmate et istriote. Bibliographie de linguistique ro- mane: domaine dalmate et istriote avec les zones limitrophes (1996–2006)”. Revue de lin- guistique romane 73/291–292: 485–527. Shandler, Jeffrey (2004): “Postvernacular Yiddish. Language as a Performance Art”. The Drama Review 48/1: 19–43. Skok, Petar (1943): “Considérations générales sur le plus ancien istro-roman”. Sache, Ort und Wort. Jakob Jud zum sechzigsten Geburtstag, 12. Januar 1942. Genève/Zürich-Erlenbach, Droz/Rentsch: 472–485. (= Romanica Helvetica 20). Tamaro, Sandra (2021): “Processi di metaforizzazione nella fraseologia istriota e istroveneta”. Studia Romanica et Anglica Zagrabiensia LXVI: 305–312. doi: 10.17234/SRAZ.66.35. Tekavčić, Pavao (1986): “Lessicografia istroromanza ieri, oggi, domani”. Revue de linguistique romane 50/197–198: 5–35. Trgovčić, Patricija (1998): “Analisi contrastiva dei proverbi in istroveneto, istrioto e ciacavo: convergenze e divergenze”. In: Milani, Nelida (ed.): Civiltà istriana. Ricerche e proposte. Trieste/Rovigno, Unione Italiana – Fiume/Università popolare di Trieste/Società “Pietas Iu- lia” – Pola: 119–136. (= Etnia. Extra serie 1). ISSN 1615-3014 142 Linguistik online 125, 1/24 Ursini, Flavia (1989): “Istroromanzo. Storia linguistica interna”. In: Holtus, Günter/Metzeltin, Michael/Schmitt, Christian (eds.): Lexikon der Romanistischen Linguistik (LRL). Vol. III: Die einzelnen romanischen Sprachen und Sprachgebiete von der Renaissance bis zur Ge- genwart. Rumänisch, Dalmatisch/Istroromanisch, Friaulisch, Ladinisch, Bündnerromani- sch. Tübingen, Niemeyer: 537–548. Veland, Reidar (2005): “I vocaboli a collocazione unica nell’italiano di oggi”. In: Garavelli Enrico/Suomela-Härmä, Elina (eds.): Atti del VII Congresso degli Italianisti Scandinavi: Helsinki, 3–6 giugno 2004. Helsinki, Societé Néophilologique: 331–339. (= Mémoires de la Société néo-philologique à Helsingfors 68). Veland, Reidar (2006): “Il concetto di collocazione unica e il valore di predizione della dicitura «solo nella loc.» in uso nella pratica lessicografica”. Bibliografia Zeitschrift für Romanische Philologie 122/2: 260–280. Dizionari Balbi, Maria/Moscarda Budić, Maria (2003): Vocabolario del dialetto di Gallesano d’Istria. Rovigno/Trieste: Unione Italiana – Fiume/Università Popolare – Trieste. (= Collana degli Atti Centro di Ricerche Storiche – Rovigno 20). Benussi, Libero (2013): Vocabolario italiano-rovignese e appendice del vocabolario del dia- letto di Rovigno d’Istria 1992–2013. Rovigno: Comunità degli italiani “Pino Budicin”. Boerio, Giuseppe (21856 [ristampa 1861]; 31867): Dizionario del dialetto veneziano. Venezia: Cecchini. Cergna, Sandro (2015): Vocabolario del dialetto di Valle d’Istria. Rovigno: Unione Italiana – Fiume/Università popolare – Trieste/Comunità degli italiani – Valle. (= Centro di Ricerche Storiche – Rovigno. Collana degli Atti 41). Cernecca, Domenico (1986): Dizionario del dialetto di Valle d’Istria. Trieste: Lint. (= Collana degli Atti Centro di Ricerche Storiche – Rovigno 8). Dalla Zonca, Giovanni Andrea (1978): Vocabolario dignanese-italiano. A cura di Miho Debe- ljuh. Trieste: Lint. (= Collana degli Atti del Centro di Ricerche Storiche – Rovigno 2). DERSII – Documentazione e rivitalizzazione dei sei idiomi istrioti (2013). youtube.com/ @istriotoDERSII/about [26.01.2023]. Dussich, Marino (2008): Vocabolario della parlata di Buie d’Istria. Rovigno/Trieste: Unione Italiana – Fiume//Università Popolare di Trieste. (= Collana degli Atti Centro di Ricerche Storiche – Rovigno 29). Dussich, Marino (2019): Dizionario italiano-buiese. Rovigno/Trieste: Unione Italiana – Fiume//Università Popolare di Trieste. (= Centro di Ricerche Storiche – Rovigno Collana degli Atti 46). Manzini, Giulio/Rocchi, Luciano (1995): Dizionario storico fraseologico etimologico del dia- letto di Capodistria. Trieste/Rovigno: Unione Italiana – Fiume//Università Popolare di Trie- ste/Istituto Regionale per la Cultura Istriana – Trieste/Regione del Veneto – Venezia. (= Col- lana degli Atti Centro di Ricerche Storiche – Rovigno 12). Orbanić, Barbara/Giudici, Giuseppe (2009): Dizionario del dialetto di Pola. Rovigno: Unione Italiana – Fiume//Università Popolare di Trieste/Società di Studi e Ricerche “Mediteran” – Pola. (= Centro di Ricerche Storiche – Rovigno Collana degli Atti 31). ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 143 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 143 Pellizzer, Antonio/Pellizzer, Giovanni (1992): Vocabolario del dialetto di Rovigno d’Istria. 2 voll. Trieste/Rovigno: La mongolfiera. (= Collana degli Atti Centro di Ricerche Storiche – Rovigno 10). Rosamani, Enrico (1958): Vocabolario giuliano dei dialetti parlati nella regione giuliano-dàl- mata quale essa era stata costituita di comune accordo tra i due stati interessati nel conve- gno di Rapallo del 12-XII-1920. Bologna: Cappelli. Pellizzer, Antonio/Pellizzer, Giovanni (1992): Vocabolario del dialetto di Rovigno d’Istria. 2 voll. Trieste/Rovigno: La mongolfiera. (= Collana degli Atti Centro di Ricerche Storiche – Rovigno 10). Rosamani, Enrico (1958): Vocabolario giuliano dei dialetti parlati nella regione giuliano-dàl- mata quale essa era stata costituita di comune accordo tra i due stati interessati nel conve- gno di Rapallo del 12-XII-1920. Bologna: Cappelli.
https://openalex.org/W4233042127
https://inria.hal.science/hal-01640274/document
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Productive Gaming
Lecture notes in computer science
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To cite this version: Ulrich Brandstätter, Christa Sommerer. Productive Gaming. 15th International Conference on Enter- tainment Computing (ICEC), Sep 2016, Wien, Austria. pp.260-265, ￿10.1007/978-3-319-46100-7_27￿. ￿hal-01640274￿ Distributed under a Creative Commons Attribution 4.0 International License 1 “[. . . ] a game’s value proposition is in how it might make its player think and feel and fun is the ultimate emotional state that they expect to experience as a consequence of playing.” [2] Ulrich Brandstätter and Christa Sommerer Interface Culture, University of Art and Design Linz, Austria {Ulrich.Brandstaetter, Christa.Sommerer}@ufg.at Abstract. Video games can be appropriated for productive purposes. Commercial games and game engines are often used for video productions, and game development companies provide development kits and modding environments to gaming communities and independent developers. With gamification, game principles are deployed in non-game contexts for benefits beyond pure entertainment. Most approaches are more focused on using games and their design elements rather than the process of playing. We propose a video game category wherein productivity is achieved by playing video games, and present a forthcoming productive game as an example. Keywords: Serious games; media production; video games; play HAL Id: hal-01640274 https://inria.hal.science/hal-01640274v1 Submitted on 20 Nov 2017 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 Productive gaming Ulrich Brandstätter and Christa Sommerer 1 Introduction We propose a new video game category for video games that yield productive results by being played: productive gaming transforms playing activities into creative processes, facilitating creations with validity beyond game virtualities. In the language of McLuhan and Nevitt, who conceived the concept for electronic technology in 1972, we allow gamers to become Prosumers [1], i.e., consumers turned into producers. The proposed video game category increases the value propositions1 of video games. Implicitly, “all games express and embody human values” [3]. However, with the exception of serious games [4], the explicit gain of playing video games beyond pure recreation is disputable: as Malaby points out, play dissociates from everyday life by being consequence free and pleasurable and, especially, dissociates from work by staying within a magic circle [5]. Caillois divides play and productivity, and implies that game play must not become productive or be conducted in pursuit of profit, otherwise it becomes corrupted [6]. Consequently, the conjunction of play and productivity makes the introduction of specific frame- work conditions necessary. These are described as a set of attributes that apply to video games that enable productive gaming. However, we refrain from giving an ultimate definition to describe productive gaming. The reasoning behind this approach is given by Wittgenstein, who argues that “elements of games, such Ulrich Brandstätter and Christa Sommerer 2 as play, rules, and competition, all fail to adequately define what games are” [7], and by Sutton-Smith, who considers the description and definition of play and games in non-paradoxical terms almost impracticable [8]. Games facilitating productive gaming are serious games with additional charac- teristics: – productivity is a subject matter of the game, not to be enforced as game goals, but enabled as goals of the meta game.l – Consistently, productivity is not reflected in victory conditions, creational objectives are set and determined before or during play sessions by the players themselves, who retain unrestrained artistic freedom.l Creation processes are aligned towards (and do not break) the game flow. – Gameplay results are perceivable without the game and, ideally, even without knowledge of the game. These nominal characteristics describe a non-exclusive video game category with emphasis on the game-play. Consistently, productive gaming requires games that provide mechanisms to start and realize creative processes, but also constructive- minded players. In this paper, we describe related work with a concise disambiguation in section 2. 1 Introduction In section 3, we provide a more detailed introduction to productive gaming. We then present Forever loops (section 4), an ongoing project that facilitates productive gaming for the creation of audio-visual compositions. In section 5, we discuss our findings and conclusions and present suggestions for future work. 2 Related work We differentiate from similar concepts using the previously introduced productive gaming characteristics. A related concept can be found in the industrial use of construction games, e.g. the utilization of Lego [9] for architectural prototypes, or using Minecraft [10] (in creative mode) as a 3D modeling environment. In both cases, productivity is a subject matter. The main distinction originates from the objectives. The industrial use of construction games typically involves predefined ambitions given by constituents, whereas in productive gaming the players themselves determine creational objectives before or during play. Another approach can be found in human-based computation games, or games with a purpose, where “people playing computer games could, without consciously doing so, simultaneously solve large-scale problems” [11]. Here, human game interaction is used to acquire labeling data for specific meta game goals, such as the analysis of gene sequences in Play to Cure: Genes in Space [12]. By being played, results with validity beyond the game virtuality are achieved. However, the creational goals usually are fixed by the developers and cannot be influenced by the players. A different, yet very popular concept is the utilization of game engines and modifications for media productions, e.g. Machinima communities using suitable computer games for video productions. Here, results typically are perceivable 3 Productive gaming without the game and yield audio-visual makings. Nevertheless, productivity is achieved by using games rather than playing games. Finally, we dissociate productive gaming from gamification (i.e., the use of game design elements in non-game contexts [13]) because the latter usually aims at goal-oriented play: “Finally, academic as well as industry critiques of gamified applications have repeatedly emphasized that these focus almost exclusively on design elements for rule-bound, goal-oriented play (i.e., ludus), with little space for open, exploratory, free-form play (i.e., paidia)” [14] 3 Productive gaming A game example facilitating productive gaming can be found in Sim Tunes [15]. It resembles a drawing game in which players populate a canvas with up to four virtual bugs of different color. These AI-controlled bugs traverse the displayed virtual space and trigger music playback. Specific pixel colors represent different musical notes that are played by the instrument according to what is assigned to the bug. In terms of our proposed characteristics, Sim Tunes does not employ victory conditions, i.e., players set their own objectives and produce audio-visual creations by playing the game. In general, productive gaming is closer to free form (paidia) than to more structured (ludus) playing and takes advantage of the games also being simulations: “[. . . ] Espen Aarseth has argued that [. . . ] simulation is what drives most”serious games" [. . . ]. In simulations, and I quote, “knowledge and experience is created by the player’s actions and strategies”. Aarseth calls for recognition of simulation as “a major new hermeneutic discourse mode, coinciding with the rise of computer technology, and with roots in games and playing.” [16] The proximity to simulations makes a clear distinction from productivity software necessary. Barr describes the main differences as follows: “First, the motivations for playing video games differ from productivity application use. Specifically, players play games for their own sake, while they generally use productivity applications to achieve some other task. Second, video game interfaces are not neutral, presenting carefully designed narratives and complex graphics to the player. Third, video games frequently dictate goals to players, while productivity applications generally facilitate user goals. Finally, video game designs purpose- fully involve conflict and constraints on the player, while productivity applications are designed to minimize them.” [17] Juul and Norton consider game obstacles and challenges as the distinguishing features of games as opposed to usability for productivity software [18]. For our purposes, the capability of seducing users into a lusory attitude [19] is the distinctive feature, i.e., play requires a proper mindset and engagement, which is established upon and complements game rules [20]. Concerning both the level of engagement and the level of game structure, productive gaming involves substantial oscillation effects on the paidia- ludus continuum [21] and play structure flexibility. During play sessions, users experiment and play with the simulations without pre-defined goals, and then apply their discoveries and new techniques to their performances. 3 Productive gaming Ulrich Brandstätter and Christa Sommerer 4 In this context, the notion of serious storytelling must be contemplated, as well as its relation between serious games and productive games. Serious storytelling refers to “storytelling outside the entertainment context, where the narration as artefact is impressive in quality and relates to a matter of importance and seriousness.” [22] A narrative, i.e. the “[. . . ] chain of events related by cause and effect occurring in time and space and involving some agency” [23], is important for both serious storytelling and productive gaming. Serious storytelling constitutes a broader concept with several application scenarios, including serious games, but also eLearning, qualitative journalism, virtual training environments, or forensics. Productive gaming on the other hand builds upon serious games and at the same time concerns only a subset. In this context, the notion of serious storytelling must be contemplated, as well as its relation between serious games and productive games. Serious storytelling refers to “storytelling outside the entertainment context, where the narration as artefact is impressive in quality and relates to a matter of importance and seriousness.” [22] A narrative, i.e. the “[. . . ] chain of events related by cause and effect occurring in time and space and involving some agency” [23], is important for both serious storytelling and productive gaming. Serious storytelling constitutes a broader concept with several application scenarios, including serious games, but also eLearning, qualitative journalism, virtual training environments, or forensics. Productive gaming on the other hand builds upon serious games and at the same time concerns only a subset. 5 Conclusion To some extent, video games are already used for productive purposes, e.g., supporting the modification of existing games, asset export and import capability, or video capture support, which are popular game features that facilitate usage scenarios beyond pure entertainment. Specific video game genres, such as active games, educational games, and art games, attempt to offer additional benefits. The proposed new video game category specifically describes video games that facilitate productivity by being played. Productive games as described in this paper are biased towards free-form play and can be considered as a specialization of serious games. Another approach towards productive gaming is the segregation of victory conditions and productivity, i.e., results correlate with and are affected by game progression, as they can actively be influenced by the player. Creative freedom is more limited, whereas the underlying game genre can be chosen more arbitrarily. 4 Forever loops Forever loops by Marlene and Ulrich Brandstätter is a productive gaming project that produces audio-visual compositions. The underlying simulation involves an interface that primarily comprises graphical gears, as shown in Figure 1. Fig. 1. Forever loops screen-shot. A gear train comprising multiple interconnected gears is playfully arranged and programmed by a user. The canvas in the upper left corner gives a visual representation of the audio-visual composition. The window to the right is required for media selection. Fig. 1. Forever loops screen-shot. A gear train comprising multiple interconnected gears is playfully arranged and programmed by a user. The canvas in the upper left corner gives a visual representation of the audio-visual composition. The window to the right is required for media selection. 5 Productive gaming Relevant mechanisms are based on real-world gears and their machinery, which is well known and understood by most people. Users begin with a fundamental understanding of the system mechanics, which quickly evolves into impressive skills regarding (musical) composition and performance. Concerning productivity, the virtual gears can be interfaced with audio-visual material, including sounds, images, and videos. Playback of the media goes hand in hand with playfully arranging gears and gear trains with their respective programming. Game play results include musical compositions, video remixes, slide shows, VJ acts, and even performances. In contrast to other productivity games, including the aforementioned Sim Tunes, players can import and are encouraged to use personal media material. On top of this, satisfactory results can be exported as a video file at user request, which can than be viewed without the game and even without knowledge of the game. Users are encouraged to experiment with the underlying physics of the gear simulation, and to use their findings to produce new compositions at their own pace. A central aspect of our approach is the rejection of the notions of winning or losing. From a design perspective, it is common for video games to blur the borders between interface and game play [18]. Here the gear wheels and their mechanics become the central interface, and are tightly interwoven with the game-play. 3. Mary Flanagan and Helen Niessenbaum. Values at Play in Digital Games. The MIT Press, Cambridge, Massachusetts, London, England, 2014. 1. M. McLuhan and B. Nevitt. Take Today: The Executive As Dropout. Harcourt, Brace, Jovanovich, Incorporated, 1972. ISBN 9780151878307. 1. M. McLuhan and B. Nevitt. Take Today: The Executive As Dropout. Harcourt, Brace, Jovanovich, Incorporated, 1972. ISBN 9780151878307. 2. Laura Ermi and Frans Mäyrä. Fundamental components of the gameplay experience: Analysing immersion. In In DIGRA. DIGRA. 2005. 3. Mary Flanagan and Helen Niessenbaum. Values at Play in Digital Games. The MIT Press, Cambridge, Massachusetts, London, England, 2014. 2. Laura Ermi and Frans Mäyrä. Fundamental components of the gameplay experience: Analysing immersion. In In DIGRA. DIGRA. 2005. References Ulrich Brandstätter and Christa Sommerer 6 4. Fedwa Laamarti, Mohamad Eid, and Abdulmotaleb El Saddik. An overview of serious games. International Journal of Computer Games Technology, 2014. 5. Thomas M. Malaby. Beyond play: A new approach to games. Games And Culture, 2:95–113, 2007. 6. R. Caillois and M. Barash. Man, Play, and Games. University of Illinois Press, 1961. ISBN 9780252070334. 7. Ludwig Wittgenstein. Philosophical Investigations. Blackwell Publishers, 1953. 8. Bo Kampmann Walther. Playing and gaming reflections and classifications. inter- national journal of computer game research, 3, 2003. j f g 9. Ole Kirk Christiansen and TheLegoGroup. Lego, 1949.f 10. Markus Persson, Mojang, Jens Bergensten, Kristoffer Zetterstrand, Markus Toivo- nen, and Daniel Rosenfeld. Minecraft, 2009. URL https://minecraft.net/. 11. Luis von Ahn. Games with a purpose. Computer, 39(6):92–94, June 2006. ISSN 0018-9162. doi: 10.1109/MC.2006.196. 12. CancerResearchUK. Play to cure: Genes in space, 2014. 13. Sebastian Deterding, Rilla Khaled, Lennart Nacke, and Dan Dixon. Gamification: Toward a definition. In CHI 2011 Gamification Workshop Proceedings, Vancouver, BC, Canada, 2011. 14. Sebastian Deterding, Dan Dixon, Rilla Khaled, and Lennart Nacke. From game design elements to gamefulness: Defining "gamification". In Proceedings of the 15th International Academic MindTrek Conference: Envisioning Future Media En- vironments, MindTrek ’11, pages 9–15, New York, NY, USA, 2011. ACM. ISBN 978-1-4503-0816-8. 15. Toshio Iwai, Heather Mace, Benimaru Itoh, Uruma Delvi, Jerry Martin, and Maxis. Simtunes, 1996.f 16. Geoffrey M. Rockwell and Kevin Kee. The leisure of serious games: A dialogue. Game studies: the international journal of computer game research, 2011. 17. Pippin Barr. Video Game Values Play as Human-Computer Interaction. PhD thesis, Victoria University of Wellington, 2007.fi 18. Jesper Juul and Marleigh Norton. Easy to use and incredibly difficult: On the mythical border between interface and gameplay. In Proceedings of the 4th Inter- national Conference on Foundations of Digital Games, FDG ’09, pages 107–112, New York, NY, USA, 2009. ACM. ISBN 978-1-60558-437-9. 19. Christa Sommerer, Ulrich Brandstätter, and Laurent Mignonneau. Aesthetics of interactive art: Art-game interfaces. Handbook of Digital Games and Entertainment Technologies, 2015. 20. K. Salen and E. Zimmerman. Rules of Play: Game Design Fundamentals. MIT Press, 2004. ISBN 9780262240451. 21. Graham H. Jensen. Making sense of play in video games: Ludus, paidia, and possibility spaces. Eludamos. Journal for Computer Game Culture., 7:69–80, 2013. 22. Jarkko Suhonen, Artur Lugmayr, and Erkki Sutinen. Serious storytelling – serious digital story telling. All links were last followed on July 17, 2016. References Proceedings of the 7th International Workshop on Semantic Ambient Media Experiences (SAME 2014) – Ambient Media Usability, Interaction, and Smart Media Technologies, 2014. 23. Todd Wilkens, Anthony Hughes, Barbara M. Wildemuth, and Gary Marchionini. The role of narrative in understanding digital video: An exploratory analysis. Proceedings of the American Society for Information Science and Technology, 40 (1):323–329, 2003. ISSN 1550-8390. doi: 10.1002/meet.1450400140. All links were last followed on July 17, 2016.
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Changes of Spatial Characteristics: Socio-Cultural Sustainability in Historical Neighborhood in Beijing, China
Sustainability
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sustainability sustainability sustainability sustainability   Keywords: socio-cultural sustainability; typological approach; urban morphology; neighborhood; spatial characteristics; urban transformation Academic Editor: Manuel Duarte Pinheiro Received: 23 April 2021 Accepted: 26 May 2021 Published: 31 May 2021 Article Changes of Spatial Characteristics: Socio-Cultural Sustainability in Historical Neighborhood in Beijing, China Huiming Liu and Bin Li * Laboratory for Future City, School of Urban Planning and Design, Shenzhen Graduate School, Peking University, Shenzhen 518055, China; liuhuiming@pkusz.edu.cn * Correspondence: libin@pkusz.edu.cn; Tel.: +86-186-2870-6470 Laboratory for Future City, School of Urban Planning and Design, Shenzhen Graduate School, Peking University, Shenzhen 518055, China; liuhuiming@pkusz.edu.cn * Correspondence: libin@pkusz.edu.cn; Tel.: +86-186-2870-6470 Abstract: This paper uses a typological approach as a tool to establish an analytical framework from a physical perspective to understand ‘place‘ and to identify key spatial characteristics that could adapt to local needs to deliver socio-cultural sustainability. Six representative housing types with their spaces and uses that were introduced in a historic neighborhood in Beijing, China are selected as case studies. Their morphological characteristics at the building, open space and neighborhood scales are examined, and typological transformations among the cases in terms of the degree of spatial continuity are identified. The paper proposes an analytical framework consisting of fifteen indicators to assess socio-cultural sustainability at the different morphological scales (building, open space and block/neighborhood) of the residents of the six cases. The score of changes from its original design is brought into calculations of continuities of spatial characteristics, which present the transitions and transformations of morphological characteristics in relation to adaptation of local needs and uses. The analysis results show that the spatial characteristics were changed when political-socioeconomic ideologies changed, and local needs and uses were transformed to follow these mutations, and finally, the methods of use in different morphological scales mostly differed from historical norms. Although the continuities of spatial characteristics were significantly changed, they are positively and continually accommodating the transformations and transitions of local needs and uses. On the other hand, the invariant spatial characteristics are important, which last despite transformation of the city development and changing of political-social-economic ideologies, and could be maintained for future development to enhance sociocultural sustainability. Citation: Liu, H.; Li, B. Changes of Spatial Characteristics: Socio-Cultural Sustainability in Historical Neighborhood in Beijing, China. Sustainability 2021, 13, 6212. https://doi.org/10.3390/su13116212 1. Introduction In the past few decades, the international architecture has been accused of incom- patibility with the local cultures [1], and is commonly believed to be responsible for placelessness, particularly in China [2–5]. Many Chinese cities are suffering from a typolog- ical crisis and a loss of adaptation to local needs. Especially in Beijing, due to dramatically increased population, traditional courtyard housing types were largely transformed into court-no-yard houses, and turned from one family owned to multi-family occupied [6]. Meanwhile, since the Modernist movement, mass production of standardized houses was free planned into historical neighborhoods to fulfill emerging housing demands. As a result, they devised a built environment, and transformed the spatial network organically to balance the changing needs and transitions of the physical setting [7,8]. This leads to two questions which need to be asked: Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 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/ 4.0/). (1) Is current built environment (both historic and early modern) responsive to socio- cultural needs in use? (2) What spatial characteristics should be continued, which could adapt local socio- cultural needs to guide future development? https://www.mdpi.com/journal/sustainability Sustainability 2021, 13, 6212. https://doi.org/10.3390/su13116212 2 of 28 Sustainability 2021, 13, 6212 In order to achieve local socio-cultural sustainability in historical neighborhood devel- opment, the theory of place helps to understand what people’s needs should be considered. Butina-Watson and Bentley [9] (p. 6) define place as a ‘set of meanings associated with any particular cultural landscape which any particular person or group of people draws on in the construction of their own personal or social identities.’ Castello [10] (p. 62) goes further, reinforcing the idea of time and suggesting the importance of heritage by defining place as ‘a collection of images that can transmit, at a glance, a suggestive reminder of the historical urban forms the city has acquired all along its evolution’ [11]. Although the concept of place is rooted in history, it is continuously changing and is neither static nor uniform [9,12]. 1. Introduction Morphologists explain these changes of urban form as typological studies that is cumulative and continuous [13], and consider that continuities of urban form and typological transformation could enhance socio-cultural sustainability and benefit people’s satisfaction with living [14–16]. Nurse [17] (p. 33) supports this argument, and states that socio-culture is more than just the manifestations of culture, it is the ‘whole social order’, the ‘whole way of life’ and a dynamic process driving sustainable development with the forms of engagement [18]. Therefore, typological approach can be seen as a great tool for defining spatial characteristics and distinctiveness of old and new physical forms based on spatial forms, urban culture and social practice through transformation of the built form that delivers socio-cultural sustainability [19–22]. Recent typological studies in China have not brought much socio-cultural sustain- ability to light. Gu et al. [23] studied the transformation process of traditional houses in Guangzhou, but focused on the building level. Feng [24] studied the continuities of spatial changes on shikumen (linong) houses with their relationships to layout, plot and street. Chen and Thwaites [14] employed Nanjing as a case in which urban forms were comprehensively studied and described its transformation in relation to a design process for identifying key factors to improve local policy making. Whitehand et al. [7] compared the transformation process of terrace house types and lilong houses between England and Shanghai, and aims to identify whether the latter had been influenced by the former around the late nineteenth and early twentieth centuries. However, although they have contributed to either large-scale city developments or regeneration of specific urban heritage building typology, none of studies look at what spatial characteristics could adapt to local needs and continue to be applied to future devel- opment to enhance socio-cultural sustainability, especially in the context of Beijing, China. In addition, it has always been challenging to identify types through a transformation pro- cess, firstly due to the degree of judgement in relation to determining the types of changes to be continued, which is largely subject to the individual researcher’s viewpoints [7]. Secondly, it is unclear which types of spatial characteristics and the level of specificity in the investigation that should be considered. Thirdly, there are limitations in empirical studies of spatial types at articulated scales, although many studies focus on individual scales with limited levels of specificity (such as [25]). 1. Introduction Based on discussions above, this paper develops an analytical framework from a physical perspective to define changes of spatial characteristics, which could adapt to the changing of needs to deliver socio-cultural sustainability in historical neighborhoods (vernacular neighborhoods) in Beijing. Six housing types with their spaces and uses were investigated. The research findings contribute to a conceptual and analytical framework for assessing local needs from a physical perspective in order to sustain socio-cultural sustain- ability; the discontinuity of adaptation of local needs requires more attention to design in order to improve the physical setting to improve socio-cultural sustainability. In addition, the developed analytical framework improves efficiency of analysis in identifications of which spatial characteristics adapt to local needs through physical transformations. This paper proposes three strategies for analysis: 2. Research Strategies This paper proposes three strategies for analysis: Sustainability 2021, 13, 6212 3 of 28 1. Based on the understanding of place, define key spatial characteristics by employing a typological approach 1. Based on the understanding of place, define key spatial characteristics by employing a typological approach 2. To select cases through identified morphological periods in the neighborhood for analysis 2. To select cases through identified morphological periods in the neighborhood for analysis 3. To apply the analytical framework to cases that identify continuity of spatial charac- teristics that could adapt local needs in order to achieve socio-cultural sustainability in historical neighborhoods in Beijing In the first step, an analytical framework is developed for delivering socio-cultural sustainability in historical development in Beijing by employing a typological approach to define continuity of spatial characteristics. It reviews literature on place [9,10,12,26–28] and seeks to understand links to typological transformation [22,29–32] from a physical perspective to look at how built forms could adapt to local uses and needs. In the second step, case selections should be representative of their specific historical and geographic characteristics in each morphological period. The identification of residen- tial building types based on different morphological periods is defined by the analysis of the historical transformation period of Beijing from imperial times (before 1911) through the period of Soviet influence (1949–1978) and post Reform period (1978–1990) to the contemporary period (after 1990) [33]. Starting with the building types with their spaces and uses that correspond to imperial traditions and continuing contemporary lifestyles, the case study neighborhood has thus accommodated a greater variety of building types than other areas in Beijing (details refer to analysis in later sections). The third step implements the analytical framework developed in the first step, to cases identified in the second step. It first studies the neighborhood/ block layout to understand the context, then it analyzes the transformation of cases from its original design to contemporary configurations by employing a typological approach, which identifies the adaptation of changes of local cultural values, social patterns and uses experiences and perceptions following a chronological order. It then compares the degree of changes in the spatial transformation. Based on each spatial characteristic in three morphological layers, this research considers continue, partly continue, and change as evaluation measurements, and a formula is developed in order to calculate the precise value to manage the continuities of these spatial characteristics between cases. 2. Research Strategies Finally, the changes between typological studies as to which spatial characteristics could adapt to local needs and sustain socio- cultural sustainability are presented. 3. Development of Analytical Framework Key Theories to Understand Place Key Authors Sense of belonging [2,37] Potpourri of memories, interpretations and feelings in relation to the physical setting [38] Character, uniqueness, distinctiveness and sense of place are evolving [9,26,27] Metal registration of physical space ‘mind map’ [39] Set of meanings associated with cultural landscape and morphological characteristics [9] Act of embedding meanings in a place, rootedness, memories and feelings within layers of the physical setting [2,9,27,39,40] Physical environment and the meanings feelings associates with it [2] Physical representation of place [10,15] Unique places with strong images [15] Result of a continuous evolutionary process over time, that includes socio-economic and political relationships and dynamics [12,28,40–44] , p g ation to the physical setting [38] iqueness, distinctiveness and sense of place are evolving [9,26,27] ration of physical space ‘mind map’ [39] associated with cultural landscape and orphological characteristics [9] ding meanings in a place, rootedness, d feelings within layers of the physical setting [2,9,27,39,40] onment and the meanings feelings asso- ciates with it [2] sical representation of place [10,15] ue places with strong images [15] inuous evolutionary process over time, cio-economic and political relationships and dynamics [12,28,40–44] al setting needs to adapt to a sense of place rootedness of feelings and Key Theories to Understand Place g iveness and sense of The physical setting needs to adapt to a sense of place, rootedness of feelings and memories and perceptions through urban transformation [2,9,10,15,37,38], while in order to carry out these experiences of use, physical form needs to be developed that corresponds to spatial characteristics through different morphological scales (block, open spaces and buildings) [9,30]. Thereby, the place can be simply seen as the result of the interactions between people and the physical setting. The changes to the physical setting need to adapt to people’s needs, and to sustain people’s needs, the regeneration of physical environment then is as an opportunity to enhance social and economic satisfaction [45,46]. These needs are sense of place, sense of belonging, set of meanings and memories and feelings (Figure 1). erceptions through urban transformation [2,9,10,15,37,38], while in order se experiences of use, physical form needs to be developed that corre- al characteristics through different morphological scales (block, open dings) [9,30]. Thereby, the place can be simply seen as the result of the ween people and the physical setting. 3. Development of Analytical Framework In order to achieve the first research goal, developing an analytical framework, it is first important to understand people’s needs in place (Table 1), and then to clarify whether these needs are adapted to by physical form through time—this is the key to achieving socio-cultural sustainability. Christian Norberg-schulz [34] suggests that the connection between people and places involves a sort of guardian-spirit that accompanies them from birth to death, a genius loci, that determines their characters, which is identity for a place. Therefore, the relationship between an individual and a place is not just between oneself and one’s surroundings, but it has to do with a much deeper process to identify where people create a friendly connection with a particular environment [2,9,15]. The process of shaping place is then formed by interactions between people and the physical setting; human actions influence physical settings, and, in return, physical settings stimulate human actions [35,36]. Sustainability 2021, 13, 6212 Table 1. Un 4 of 28 Table 1. Understanding of needs in place. emories, interpretations and feelings in ti t th h i l tti Table 1. Understanding of needs in place. 3. Development of Analytical Framework The changes to the physical setting people’s needs, and to sustain people’s needs, the regeneration of physi- t then is as an opportunity to enhance social and economic satisfaction eds are sense of place, sense of belonging, set of meanings and memories gure 1). nging of people’s needs in a local place Figure 1. The changing of people’s needs in a local place. nging of people’s needs in a local place Figure 1. The changing of people’s needs in a local place. nging of people’s needs in a local place Figure 1. The changing of people’s needs in a local place. whether a place is considered as socio-culturally sustainable depends on ort or sustainability of the local needs: sets of meanings, sense of belong- ce, and memories from and feelings about a physical setting. The core of However, whether a place is considered as socio-culturally sustainable depends on the level of support or sustainability of the local needs: sets of meanings, sense of belonging, sense of place, and memories from and feelings about a physical setting. The core of socio-cultural sustainability [47] then involves two aspects: futurity and equity. The most often-quoted definition is “(sustainable development) is development that meets the needs of the present without compromising the ability of future generations to meet their own needs”[48] (p. 28). However, this definition lacks consideration of the distinction between equity within a generation, and equity between generations [49]. The needs of local people should be emphasized for the least advantaged in society and a fair treatment of future generations should be ensured [50]. Lim [51] states that culture can be considered a complex matrix of collective experiences and knowledge that formulates a meaning of life, so the values that people hold, the rules and norms they obey and the material objects they use are keys to shaping the built environment into a sustainable place [52,53]. Zetter and Butina Watson [54] go further to say that how people shape their own places and spaces, how Sustainability 2021, 13, 6212 5 of 28 they adapt local technologies and traditions, and how they deploy innate capacities to adapt cultural precepts to the modern idiom are crucial for sustainability implementations. 3. Development of Analytical Framework Therefore, the sets of meanings, sense of place, sense of belonging and memories and feelings that people attach in shaping a place becomes the essence of the local needs, while continually fulfilling these needs for contemporary and future development is the key of the theory of socio-cultural sustainability [17,55]. On the other hand, when we go to study in detail how the transformations of physical settings could adapt local needs to achieve socio-cultural sustainability, the difficulties include how to precisely define which spatial characteristics could continually contribute to uses in public open spaces, and what spatial characteristics of urban forms should be involved, and which can fulfill the local socio-cultural needs. Therefore, the typological approach is a tool because it goes beyond the analysis of physical forms of urban trans- formation and studies how physical transformation adapts to the changing of needs to understand human actions [22,56]. Moudon [57] states that urban forms can be understood by investigating layout and configuration of forms and spaces in order to clarify the pro- cesses of local change—how forms and spaces are locally built and why. Zetter and Butina Watson [54] argue that physical forms are the products of social consensus and productions of urban forms are the process by which socio-spatial construction take place. The typological approach aims to provide an understanding of the built environment by examining the historical process of its formation and its hierarchical structure, in order to identify the adaptation to changing needs. It interprets the history of the civilization and its needs for transformation through architecture [58] by analyzing building materials, the structure of buildings and the arrangement of rooms [32]. Then, it uses buildings as elements in order to understand forms and their memories and values, and the meanings beyond. On the other hand, Goodey [59] claims that physical forms should create a sense of belonging and a sense of place that adapts to people’s feelings as though individuals belong to the people, to the buildings and to the life there (Figure 2). 6 of 31 Figure 2 The interrelationship between spatial characteristics and changing of needs Figure 2. The interrelationship between spatial characteristics and changing of needs. re 2 The interrelationship between spatial characteristics and changing of needs Figure 2. The interrelationship between spatial characteristics and changing of needs. p p g g ng to Kropf [30] (p. 3. Development of Analytical Framework The diachronic type is relevant to the typological study, where the types evolve and adapt to the transformation of socio-cultural needs through different historical periods, with the essential spatial characteristics which remain becoming a foundation type [19]. while others are more robust and can survive over consecutive time periods in the same place [32]. The diachronic type is relevant to the typological study, where the types evolve and adapt to the transformation of socio-cultural needs through different historical periods, with the essential spatial characteristics which remain becoming a foundation type [19]. Based on the discussion above, the understanding of an interactional relationship between people and physical setting is clear—in order to design a socio-culturally sustainable place, one must fully understand how people’s changing needs associate with continuities of spatial characteristics that deliver socio-cultural sustainability to the local place (Figure 3). In other words, in order to identify what spatial characteristics can be continued in the transformation process, it must comprehensively understand what spatial characteristics continually associate with local uses. Then, the key components of the analytical framework are identified and associated with each morphological scale (building, open space and neighborhood block), and will be used for analyzing cases, identified throughout the morphological period in this research. 7 of 31 Figure 3 Achievement of socio cultural sustainability with continuities of spatial characteris Figure 3. Achievement of socio-cultural sustainability with continuities of spatial characteristics. Figure 3 Achie ement of socio cultural sustainability with continuities of spatial characteris Figure 3. Achievement of socio-cultural sustainability with continuities of spatial characteristics. y p o understand people’s changing needs, and the adaptation of these needs characteristic through physical transformation, the study of physical form ee morphological scales: building structures, open spaces, and neighbor- out. The spatial characteristics related to each morphological scale are ex- heories (Tables 2 and 3). Researching the neighborhood/block layout in- ntification of the relationship of the buildings to the block, size and shape rhood, land coverage, density and spatial flow and street configurations. open space concentrates on building/plot arrangements along public open ng height and width, active front coverage and spatial access patterns and ween building-building and building-open space. The building structure y involves the building layout, functions, number of floors, spatial sequence to private property and efficiency of access. 3. Development of Analytical Framework 112), ‘forms found at different levels are identified as are conceived as cultural entities rooted in, and specific to the local process velopment’ [60]. In Caniggia and Maffei’s [29] work, they divided the types gories: synchronic and diachronic. In synchronic types, production decreases and remains strictly confined to the period in which it was introduced, while c type undergoes a series of progressive transformations throughout an im- period [32]. Some of types can be observed in different places within a par- According to Kropf [30] (p. 112), ‘forms found at different levels are identified as types, which are conceived as cultural entities rooted in, and specific to the local process of cultural development’ [60]. In Caniggia and Maffei’s [29] work, they divided the types into two categories: synchronic and diachronic. In synchronic types, production decreases through time and remains strictly confined to the period in which it was introduced, while the diachronic type undergoes a series of progressive transformations throughout an important time period [32]. Some of types can be observed in different places within a particular time, p p g g ing to Kropf [30] (p. 112), ‘forms found at different levels are identified as h are conceived as cultural entities rooted in, and specific to the local process evelopment’ [60]. In Caniggia and Maffei’s [29] work, they divided the types egories: synchronic and diachronic. In synchronic types, production decreases e and remains strictly confined to the period in which it was introduced, while nic type undergoes a series of progressive transformations throughout an im- period [32]. Some of types can be observed in different places within a par- According to Kropf [30] (p. 112), ‘forms found at different levels are identified as types, which are conceived as cultural entities rooted in, and specific to the local process of cultural development’ [60]. In Caniggia and Maffei’s [29] work, they divided the types into two categories: synchronic and diachronic. In synchronic types, production decreases through time and remains strictly confined to the period in which it was introduced, while the diachronic type undergoes a series of progressive transformations throughout an important time period [32]. Some of types can be observed in different places within a particular time, Sustainability 2021, 13, 6212 6 of 28 while others are more robust and can survive over consecutive time periods in the same place [32]. 3. Development of Analytical Framework Figure 4 presents these spatial In order to understand people’s changing needs, and the adaptation of these needs to each spatial characteristic through physical transformation, the study of physical form focuses on three morphological scales: building structures, open spaces, and neighbor- hood/block layout. The spatial characteristics related to each morphological scale are extracted from theories (Tables 2 and 3). Researching the neighborhood/block layout involves the identification of the relationship of the buildings to the block, size and shape of the neighborhood, land coverage, density and spatial flow and street configurations. The analysis of open space concentrates on building/plot arrangements along public open spaces, building height and width, active front coverage and spatial access patterns and hierarchy between building-building and building-open space. The building structure analysis mainly involves the building layout, functions, number of floors, spatial sequence from entrance to private property and efficiency of access. Figure 4 presents these spatial characteristics and their relationship with each morphological scale that constitutes the framework of adaptation of changing of use in place. s and their relationship with each morphological scale that constitutes the adaptation of changing of use in place. l characteristics of typological study. aracteristics Key Authors g layout e.g., [23,25,31,61–63] g façade e.g., [23,31,62,63] r of floors e.g., [61] Table 2. Spatial characteristics of typological study. Spatial Characteristics Key Authors Building layout e.g., [23,25,31,61–63] Building façade e.g., [23,31,62,63] Number of floors e.g., [61] Building materials e.g., [58,62] and their relationship with each morphologica adaptation of changing of use in place. Table 2. Spatial characteristics of typological study. 7 of 28 Sustainability 2021, 13, 6212 Table 2. Cont. Spatial Characteristics Key Authors Spatial hierarchy e.g., [24,31,64] Access efficiency e.g., [35,58] Open space structures e.g., [65,66] Plot and block layout, size and shapes e.g., [25,30,62,65,67,68] Density and land coverage e.g., [64,65] Spatial relationship between building, plot, open space and block e.g., [24,31,62,69] Buildings with its relations to open spaces e.g., [31,35,64,69] Site configurations e.g., [67] Table 3. The components of Analytical framework. Layout Building/Plot Arrangement Site/Block Arrangement Public/private zones are strictly separated, partly separated or not separated? Buildings’ spatial relationships (Back-to-back/Side-to-side); Any set back, front garden, back garden? Diversity and continuity of buildings Identical or irregular plots in shape and size? Open plan or gated? Any defined site entrance or boundary? 4. Identification of Cases in Transformation of the Neighborhood 4. Identification of Cases in Transformation of the Neighborhoo In the second research step, six cases of building typologies are identified in a trans- formation study of the neighborhood. Qian Yuan’en Si neighborhood (Figure 5) is one of the historical living areas where mainly vernacular but also a variety of other building typologies were free planned after the 1980s [6,69]. During the urban transformation, the main driver of changes in socio-spatial patterns was the shift in formation of the economy from a politically-controlled to a market-driven mechanism [70–72]. Although horizon- tally the original fabric can still be recognized in the current urban tissue, vertically, the building facades and high-rise plots show dramatic changes (Figure 6). The neighborhood contributes to the overall urban analysis because its built form combined a variety of building types in a vernacular context. Table 4 illustrates four morphological periods in the neighborhood that present transformation of political administration, economic formation, as well as socio-cultural transitions. g In the second research step, six cases of building typologies are identified in a trans- formation study of the neighborhood. Qian Yuan’en Si neighborhood (Figure 5) is one of the historical living areas where mainly vernacular but also a variety of other building typologies were free planned after the 1980s [6,69]. During the urban transformation, the main driver of changes in socio-spatial patterns was the shift in formation of the economy from a politically-controlled to a market-driven mechanism [70–72]. Although horizon- tally the original fabric can still be recognized in the current urban tissue, vertically, the building facades and high-rise plots show dramatic changes (Figure 6). The neighborhood contributes to the overall urban analysis because its built form combined a variety of building types in a vernacular context. Table 4 illustrates four morphological periods in the neighborhood that present transformation of political administration, economic for- mation as well as socio-cultural transitions (a) (b) Figure 5. The location of Qian Yuan’en Si neighborhood. The location of the neighborhood in the city (a), the area of the neighborhood (b). Figure 5. The location of Qian Yuan’en Si neighborhood. The location of the neighborhood in the city (a), the area of the neighborhood (b). (b) (b) (a) (a) (b) Figure 5. The location of Qian Yuan’en Si neighborhood. The location of the neighborhood in the city (a), the area of the neighborhood (b). Figure 5. The location of Qian Yuan’en Si neighborhood. 3. Development of Analytical Framework Spatial Hierarchy Functions Building height/street width Size and shape Functions are defined/partly defined/ not defined W = H/2; W = H/3; W = 2H; W = H Size defined by footprint in terms of accessibility Numbers of floors Active frontage Land coverage Original designed/ professional retrofit/ self-regenerated Active front coverage (%)? Land construction coverage (%) (20%—low/50%—medium/over 70%—high) Numbers of residents Spatial Sequence Spatial hierarchy Density and Spatial Flow From the public entrances to private property; Spatial relationship from public to private Location of the case in density areas; Accessibility efficiency and privacy; Access efficiency to open space Spatial Movement Street configurations Total number of steps from private property to public open spaces; Spatial transition of access; Steps from buildings to private property Total number of steps from private property to out of neighborhood Types of roads/streets/paths in site; Are they mix of pedestrian and vehicles or separate? Table 2. Cont. 8 of 28 vehi- Sustainability 2021, 13, 6212 Spatial transition of acce Steps from buildings to p Figure 4. The analytical framework of changing of needs. R REVIEW 9 Figure 4. The analytical framework of changing of needs. Figure 4. The analytical framework of changing of needs. Figure 4. The analytical framework of changing of needs. 4. Identification of Cases in Transformation of the Neighborhood 4. Identification of Cases in Transformation of the Neighborhood 4. Identification of Cases in Transformation of the Neighborhood 4. Identification of Cases in Transformation of the Neighborhoo The location of the neighborhood in the city (a), the area of the neighborhood (b). 9 of 28 he Sustainability 2021, 13, 6212 Figure 5. The loca Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6 The section of Qian Yuan’en Si neighborhood Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6 The section of Qian Yuan’en Si neighborhood Figure 6. The section of Qian Yuan’en Si neighborhood. g g g yp Morphological Periods and Transforming of Buildings Period 1 Imperial Period—before 1949 Morphological Periods and Transforming of Buildings Period 1 Imperial Period—before 1949 Traditional housing types, open space structure and block layout, which address imperial power and social hierarchy in spatial structure. Period 2 Soviet Influence Period—1949–1978 Soviet socialist idea is spread into China, the housing types changed to early modern style, to emphasize socialism and collectivism in lifestyle. Period 3 Post-reform and Contemporary—1978-current period Modern building types with more private property layout design were imported into China, and the lifestyles were transformed into market lead The neighborhood came into existence during the Ming dynasty (1368–1644). It was mainly developed during the Qing dynasty (1636–1912) in terms of its morphological tissues. The neighborhood was developed for government institutions and government owned workshops. Thus, the road network within the neighborhood was characterized by one-way roads (cul-de-sacs) designed to have one entrance accessible to the hutong (internal road within the historic block) from the main streets, but all hutongs ended inside in order to maintain a high level of privacy [73,74]. The significant change of the neighborhoods started during the Communist period, when there was a movement to remove the ‘Four Olds’ and the population dramatically increased, and therefore, the function of the neighborhoods completely changed from institutional and residential use to mostly residential use by adapting to the changes in the political and social environment in order to respond to political needs [8,73,75]. Thus, the housing types were changed from private to shared houses, and hutong networks were transformed from private design to open access. 4. Identification of Cases in Transformation of the Neighborhood 4. Identification of Cases in Transformation of the Neighborhoo Before the Post-Reform (1970s), due to population growth, the work-unit based social structure and resources allocation had a serious effect on property allocation, so a movement to reform the political, economic and social structure was formed [75]. However, before the City Planning Law of People’s Republic of China (1989) was enacted, due to the dramatically increased population, the residential houses grew organically with occupation of public space and leveled up horizontally in order to meet the enormous demand for living spaces [8,75]. Meanwhile, during the transformation process, economic influences started to play an important role because daily commercial needs increased due to population density growth, the changing needs in public space based on uses of automobile, and the lifestyle transit because of cultural effects from globalization, which all meant the area started to engage with urban growth in a more complex way [71] (Figure 7). 10 of 28 31 Sustainability 2021, 13, 6212 nability 2021, 13, x FOR PEER R Figure 7. Land use transformation of the neighborhoods. Figure 7. Land use transformation of the neighborhoods. d f the neighborhoods. Figure 7. Land use transformation of the neighborhoods. Through morphological layers for identifying continuities of spatial characteristics deliver socio-cultural sustainability, Table 5 presents the analysis of a contemporary hborhood/block structure, which provides current uses in the neighborhood as ground and context. In the following sub-sections, each case is identified based on hological period, and is introduced and analyzed at building scale and open space in order to find out changes of spatial characteristics in relation to uses. Through morphological layers for identifying continuities of spatial characteristics that deliver socio-cultural sustainability, Table 5 presents the analysis of a contemporary neigh- borhood/block structure, which provides current uses in the neighborhood as background and context. In the following sub-sections, each case is identified based on morphological period, and is introduced and analyzed at building scale and open space scale in order to find out changes of spatial characteristics in relation to uses. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis Site/Block Arrangement Open side (organically and internally grown) Externally no strict defined boundaries Size and shape Max. 7 turns (approx. 180 m) from primary streets to private prop- erty. Regular and historical continued layout shape with walkable The entire neighborhood was developed based on imperial context; traditional court- yard houses and open space networks are commonly discovered in the neighborhoods. However, since 1949, due to changing socio-political ideology, dramatically increased populations and other factors, the traditional courtyard houses were transformed into chaotic-no-yard houses, and one family-owned houses become multi-family occupied residential plots [6,69]. Therefore, its original spatial design is largely changed to fulfill the transitions of access, uses of facilities and ways of living (Figures 8 and 9). Case 1 and 2 are all originally part of traditional courtyard houses, but during urban transformation and socio-political transition, the original inner private yard is built with more rooms to fulfill increased demands for housing, and existing rooms start opening up access directly to the public street (Tables 6 and 7). The differentiations between these cases are: case 1 has occupied public space and built an extension garden as transitional space; and case 2 has converted from residential to commercial uses with transparent doors that visually increase spatial connections between indoor and outdoor. These types of cases represent the largest number of residential houses within the neighborhood. 11 of 28 en space en space Sustainability 2021, 13, 6212 Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis scale in order to find out changes of spatial characteristics in relation to uses. Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis Site/Block Arrangement Open side (organically and internally grown) Externally no strict defined boundaries Size and shape Max. 7 turns (approx. 180 m) from primary streets to private property. Regular and historical continued layout shape with walkable size (1)→(2)→(3)→(4)→(5)→(6)→home Land coverage Land construction coverage is approx. over 80% Residents approx. 900 Site/Block Arrangement Open side (organically and internally grown) Externally no strict defined boundaries Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis Site/Block Arrangement Open side (organically and internally grown) Externally no strict defined boundaries Size and shape Max. 7 turns (approx. 180 m) from primary streets to private property. Regular and historical continued layout shape with walkable size (1)→(2)→(3)→(4)→(5)→(6)→home Land coverage Land construction coverage is approx. over 80% Residents approx. 900 Size and shape Max. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 7 turns (approx. 180 m) from primary streets to private property. Regular and historical continued layout shape with walkable size (1)→(2)→(3)→(4)→(5)→(6)→home Land coverage Land construction coverage is approx. over 80% Residents approx. 900 Sustainability 2021, 13, x FOR PEER REVIEW 12 of 31 Density and spatial flow Relatively, the higher density is located in the west which is connected to the primary street for efficient transportation and facilities, instead of the east that links to historical neighborhoods, where large facilities (such as shopping malls) and public transport cannot be built. The cul-de-sacs provide a good level of privacy and easy access to public areas in terms of block size. Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians Sustainability 2021, 13, x FOR PEER REVIEW 12 of 31 Density and spatial flow Relatively, the higher density is located in the west which is connected to the primary street for efficient transportation and facilities, instead of the east that links to historical neighborhoods, where large facilities (such as shopping malls) and public transport cannot be built. The cul-de-sacs provide a good level of privacy and easy access to public areas in terms of block size. Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians Density and spatial flow Relatively, the higher density is located in the west which is connected to the primary street for efficient transportation and facilities, instead of the east that links to historical neighborhoods, where large facilities (such as shopping malls) and public transport cannot be built. The cul-de-sacs provide a good level of privacy and easy access to public areas in terms of block size. Sustainability 2021, 13, x FOR PEER REVIEW 12 of 31 Density and spatial flow Relatively, the higher density is located in the west which is connected to the primary street for efficient transportation and facilities, instead of the east that links to historical neighborhoods, where large facilities (such as shopping malls) and public transport cannot be built. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 The cul-de-sacs provide a good level of privacy and easy access to public areas in terms of block size. Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians Table 5. Neighborhood layout analysis. Table 5. Neighborhood layout analysi Neighborhood Layout Analysis Table 5. Neighborhood layout analysis. Table 5. Neighborhood layout analysi Neighborhood Layout Analysis Neighborhood Layout Analysis Neighborhood Layout Analy pen side (organically and internally grown) Open side (organically and internally grown) Externally no strict defined boundaries pen side (organically and internally grown) ternally no strict defined boundaries y 2021, 13, x FOR PEER REVIEW 2021, 13, x FOR PEER REVIEW 2021, 13, x FOR PEER REVIEW nd coverage nd construction coverage is approx over 80% nd construction coverage is approx. over 80% sidents approx 900 Land construction coverage is approx. over 80% Residents approx. 900 nsity and spatial flow sidents approx. 900 ensity and spatial flow nsity and spatial flow Density and spatial flow latively, the higher dens e ative y, t e ig e de sity is ocated i t e west w ic is nnected to the primary street for efficient transportation an cilities, instead of the east that links to historical ighborhoods, where large facilities (such as shopping mall nd public transport cannot be built. y, g y nected to the primary street for efficient transportation and lities, instead of the east that links to historical ghborhoods, where large facilities (such as shopping malls d public transport cannot be built. Relatively, the higher density is located in the west which is connected to the primary street for efficient transportation and facilities, instead of the east that links to historical neighborhoods, where large facilities (such as shopping malls) and public transport cannot be built. nnected to the primary street for efficient transportation an ilities, instead of the east that links to historical ghborhoods, where large facilities (such as shopping malls d public transport cannot be built. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians 12 of 28 visually t 12 of 28 visually t Sustainability 2021, 13, 6212 (a) (b) Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a), and the right subfigure presents how multi-families occupied the courtyard houses currently (b). Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a), and the right subfigure presents how multi-families occupied the courtyard houses currently (b). ity 2021, 13, x FOR PEER REVIEW (a) ty 2021, 13, x FOR PEER REVIEW (b) (b) (a) EVIEW Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a), and the right subfigure presents how multi-families occupied the courtyard houses currently (b). Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a), and the right subfigure presents how multi-families occupied the courtyard houses currently (b). igure 9. The transformation of courtyard houses in relation to block. Original spatial design of the block (a), spat ationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), huto d courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses multi-families occupied (f). Figure 9. The transformation of courtyard houses in relation to block. Original spatial design of the block (a), spatial relationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), hutongs and courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses after multi-families occupied (f). igure 9. The transformation of courtyard houses in relation to block. Original spatial design of the block (a), spati ationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), huto courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses multi-families occupied (f). Figure 9. The transformation of courtyard houses in relation to block. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 e cul-de-sacs provide a good level of privacy and easy acce p p he cul-de-sacs provide a good level of privacy and easy acce public areas in terms of block size p p e cul-de-sacs provide a good level of privacy and easy acces public areas in terms of block size p p The cul-de-sacs provide a good level of privacy and easy access to public areas in terms of block size. e cul de sacs provide a good level of privacy and easy acce public areas in terms of block size. p Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians p Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians Street configurations The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates risks and safety issues for pedestrians he primary street directly leads the secondary street that rms efficient access. The cul-de-sacs link to the case VI plot at creates third-levels of spatial class. e primary street directly leads the secondary street that ms efficient access. The cul-de-sacs link to the case VI plot t creates third-levels of spatial class. e primary street directly leads the secondary street that ms efficient access. The cul-de-sacs link to the case VI plot at creates third-levels of spatial class. xed pedestrian and vehicle parking and access, which The primary street directly leads the secondary street that forms efficient access. The cul-de-sacs link to the case VI plot that creates third-levels of spatial class. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Original spatial design of the block (a), spatial relationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), hutongs and courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses after multi-families occupied (f). 13 of 28 ngs fter er Sustainability 2021, 13, 6212 relationsh and courty and courtyard h Table 6. Case 1 and Case 2—building structure analysis. g y Building Structu Building Structure A Table 6. Case 1 and Case 2 building structure analysis. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Soviet Influence Period: Case 3, Case 4, and Case 5 In this particular time (1949–1979), soviet socialism and collectivism have seriously influenced not only urban construction and development, but also socio-political ideologies through people’s life [75]. During this period, physical urban settings are the major change in China; a large number of traditional buildings were demolished and replaced by soviet style raised buildings, and traditional urban tissue is transited to suit new sociopolitical ideologies, modern facilities (train station, automobile, and motorway) and increased population [75]. The building typologies in this period are mainly presented as two or three storage soviet style buildings, with shared kitchen and bathrooms in each floor. However, in contemporary Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis Open Space Analysis Open Space Analysis Ca e 1 Open Space Analysis C 1 Open Space Analysis Open Space Analysi Open Space Analysis 1 Case 1 Case 1 Case 1 Number of Case o Bui Buildin height/ Bu he Building height/street width r w wid 1:1.2 Active front S hi Spat hierah Spatial movement nt fluence Period: Case 3 Case 4 and Case 5 uence Period: Case 3, Case 4, and Case 5 oviet Influence Period: Case 3, Case 4, and Case 5 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 fluence Period: Case 3, Case 4, and Case 5 2. Soviet Influence Period: Case 3, Case 4, and Case 5 nce Period: Case 3, Case 4, and Case 5 nfluence Period: Case 3, Case 4, and Case 5 Soviet Influence Period: Case 3, Case 4, and Case 5 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 fluence Period: Case 3, Case 4, and Case 5 Soviet Influence Period: Case 3, Case 4, and Case 5 uence Period: Case 3, Case 4, and Case 5 oviet Influence eriod Case 3, Case , and Case 5 fl In this particular time (1949–1979), soviet socialism and collectivism have seriously influenced not only urban construction and development, but also socio-political ideologies through people’s life [75]. During this period, physical urban settings are the major change in China; a large number of traditional buildings were demolished and replaced by soviet style raised buildings, and traditional urban tissue is transited to suit new sociopolitical ideologies, modern facilities (train station, automobile, and motorway) and increased population [75]. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Building Structure Analysis Number of Case Case 1 Case 2 Building Structure Analysis Number of Case Case 1 Case 2 Building Structure Analysis Number of Case Case 1 Case 2 Layout OR PEER REVIEW 14 of 3 Layout Functions Number of floors Spatial sequence Access efficiency to open space EER REVIEW 14 of 31 Layout Functions Number of floors Spatial sequence Access efficiency to open space Functions OR PEER REVIEW 14 of 3 Layout Functions Number of floors Spatial sequence Access efficiency to open space ER REVIEW 14 of 31 Layout Functions Number of floors Spatial sequence Access efficiency to open space Number of floors OR PEER REVIEW 14 of 3 Layout Functions Number of floors Spatial sequence Access efficiency to open space EER REVIEW 14 of 31 Layout Functions Number of floors Spatial sequence Access efficiency to open space Spatial sequence OR PEER REVIEW 14 of 3 Layout Functions Number of floors Spatial sequence Access efficiency to open space EER REVIEW 14 of 31 Layout Functions Number of floors Spatial sequence Access efficiency to open space Access efficiency to open space OR PEER REVIEW 14 of 3 Layout Functions Number of floors Spatial sequence Access efficiency to open space EER REVIEW 14 of 31 Layout Functions Number of floors Spatial sequence Access efficiency to open space Building Structure Analysis Case 1 Case 1 Number of Case of Case of Case 14 of 14 of 31 14 of 14 of 31 14 o 14 of 31 14 of 14 of 31 14 o 14 of 31 F ti ti Functions Functions nctions u io Number Number Number of floors Number of floors Number of floors of floors of floors Spatial Spatial Spatial sequence Spatial sequence Spatial sequence sequence sequence Access Access Access efficiency Access efficiency Access efficiency to o e Access fficiency to o e Access efficiency to open space efficiency to open efficiency to open 14 of 28 Sustainability 2021, 13, 6212 Number of Case Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis Number of Case Case 1 Case 2 Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis Number of Case Case 1 Case 2 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Table 7. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Case 1 and Case 2—open space analysis. Open Space Analysis Number of Case Case 1 Case 2 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Building/plot arrangement Open Space Analysis Number of Case Case 1 Case 2 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Open Space Analysis Number of Case Case 1 Case 2 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis Number of Case Case 1 Case 2 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis Number of Case Case 1 Case 2 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Building height/street width 1:1.2 1:1 Active front Number of Case Case 1 Case 2 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 of Case Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Spatial hierarchy Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Building /plot arrange ment Building height/st reet width 1 : 1.2 1 : 1 Active front Spatial hierarch y Spatial moveme nt 4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 Spatial movement 4.2. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Building Building Structure Analysis p p p p g y p g and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis Building Building Structure Analysis Building Building Structure Analysis Building Building Structure Analysis C 3 C 4 C 5 Building Building Structure Analysis 3 C 4 C 5 Building Building Structure Analysis 3 C 4 C 5 ase 3 Case 4 Case 5 ase 3 Case 4 Case 5 se 3 Case 4 Case 5 Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis Building Building Structure Analysis Building Building Structure Analysis Building Building Structure Analysis 3 C 4 C 5 Building Building Structure Analysis 3 C 4 C 5 Building Building Structure Analysis 3 C 4 C 5 ase 3 Case 4 Case 5 ase 3 Case 4 Case 5 e 3 Case 4 Case 5 Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis Building Building Structure Analysis Building Building Structure Analysis Building Building Structure Analysis C C 4 Building Building Structure Analysis 3 C 4 Building Building Structure Analysis 3 C 4 ase 3 Case 4 ase 3 Case 4 se 3 Case 4 Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 The building typologies in this period are mainly presented as two or three storage soviet style buildings, with shared kitchen and bathrooms in each floor. However, in contemporary use (Tables 8 and 9), many of these buildings are regenerated; the representative examples are: Sustainability 2021, 13, 6212 15 of 28 ny of the y of thes of these ny of the al buildi y of thes l buildin of these building al buildin opulatio l buildin opulation building ulations 15 of 28 ny of the y of thes of these ny of the al buildi y of thes l buildin of these building al buildin opulatio l buildin opulation building ulations case 3—the original building has extended its height and length with changed façade to adapt to increased populations and transitions of needs to suit new lifestyles and technologies such as automobile use; case 4—develops its own semi-private space for improving activity needs and parking; and case 5—keeps its original design. and transitions of needs to suit new lifestyles and technologies such as automobile us case 4—develops its own semi-private space for improving activity needs and parkin and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile us case 4—develops its own semi-private space for improving activity needs and parkin and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile use; case 4—develops its own semi-private space for improving activity needs and parking; and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parkin and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking; and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. p p p p g y p and case 5—keeps its original design. Table 8. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis p p p p g y p and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis p p p p g y p g and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis case 3—the original building has extended its height and length with changed façade to adapt to increased populations and transitions of needs to suit new lifestyles and technologies such as automobile use; case 4—develops its own semi-private space for improving activity needs and parking; and case 5—keeps its original design. and transitions of needs to suit new lifestyles and technologies such as automobile us case 4—develops its own semi-private space for improving activity needs and parkin and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile us case 4—develops its own semi-private space for improving activity needs and parkin and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile use; case 4—develops its own semi-private space for improving activity needs and parking; and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parkin and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking; and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. p p p p g y p and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis p p p p g y p and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis Open Space Analysis Open Space Analysis Open Space Analysis Open Space Analysis Open Space Analysis Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Building/plot arrangement Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Open Space Analysis Number of Case Case 3 Case 4 Case 5 Building/plot arrangement Building height/street width 1 : 0.1 1 : 1 1 : 0.3 Building height/street width 1:0.1 1:1 1:0.3 Active front Sustainability 2021, 13, x FOR PEER REVIEW 18 of 31 Active front Spatial hierarchy Spatial movement 4.3. Post-reform and Contemporary Period: Case 6 Sustainability 2021, 13, x FOR PEER REVIEW 18 of 31 Active front Spatial hierarchy Spatial movement 4.3. Post-reform and Contemporary Period: Case 6 Since 1978 the country has transformed from a planned economy to an open market Sustainability 2021, 13, x FOR PEER REVIEW 18 of 31 Active front Spatial hierarchy Spatial movement 4.3. Post-reform and Contemporary Period: Case 6 Spatial hierarchy Sustainability 2021, 13, x FOR PEER REVIEW 18 of 31 Active front Spatial hierarchy Spatial movement 4.3. Post-reform and Contemporary Period: Case 6 Si 1978 h h f d f l d k Sustainability 2021, 13, x FOR PEER REVIEW 18 of 31 Active front Spatial hierarchy Spatial movement 4.3. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Cont. Table 8. Cont. Building Building Structure Analysis Number of Case Case 3 Case 4 Case 5 Access efficiency to open space Spatial squence Access efficiency to open space Spatial squence Access efficiency to open space Spatial squence Access efficiency to open space squence Access efficiency to open space Table 9 Case 3 Case 4 and Case 5—open space analysis squence Access efficiency to open space Table 9 Case 3 Case 4 and Case 5—open space analysis squence Access efficiency to open space Table 9 Case 3 Case 4 and Case 5—open space analysis Access efficiency to open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Access efficiency to open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Access efficiency to open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Access efficiency to open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Access efficiency to open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Access efficiency to open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Building Building Structure Analysis Access efficiency to open space Access efficiency to open space Access efficiency to open space Access efficiency to open space Access fficiency to pen space Access efficiency to open space Access fficiency to open space ficiency to pen space fficiency to open space efficiency to open space efficiency to open space efficiency to open space fficiency to open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis Open Space Analysis Open Space Analysis Open Space Analysis Open Space Analysis Open Space Analysis Table 9. Case 3, Case 4 and Case 5—open space analy Table 9. Case 3, Case 4 and Case 5—open space analys Table 9. Case 3, Case 4 and Case 5—open space analysis Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Building Building Structure Analysis Number of Case Case 3 Case 4 Case 5 Building Building Structure Analysis Number of Case Case 3 Case 4 Case 5 Layout Functions Building Building Structure Analysis Number of Case Case 3 Case 4 Case 5 Layout Functions Building Building Structure Analysis Number of Case Case 3 Case 4 Case 5 Layout Functions Layout Number of Case Case 3 Case 4 Case 5 Layout Functions Number of Case Case 3 Case 4 Case 5 Layout Functions Number of Case Case 3 Case 4 Case 5 Layout Functions Functions Case Case 3 Case 4 Case 5 Layout Functions Case Case 3 Case 4 Case 5 Layout Functions Case Case 3 Case 4 Case 5 Layout Functions Number of floors Sustainability 2021, 13, x FOR PEER REVIEW 17 of 3 Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 31 Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 31 Number of floors Spatial squence Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 3 Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 31 Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 31 Number of floors Spatial squence A Number of Case Case Case Case L t L t L t Layout Layout Layout Layout Layout Layout Layout Functions Functions Functions Functions Functions Functions Functions Functions Functions Functions Number of floors Sustainability 2021, 13, x FOR PEER REVIEW 17 of Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 3 Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 31 Number of floors Spatial squence Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 3 Number of floors Spatial squence Sustainability 2021, 13, x FOR PEER REVIEW 17 of 31 Number of floors Spatial squence F ti F ti F ti Functions Functions Functions Functions S t i bilit 2 S t i bilit 2 t i bilit 202 Sustainability Sustainability 2 ustainability 20 Number of floors Number of floors Number of floors Number of floors floors floors floors Spatial Spatial Spatial Spatial squence Spatial squence Spatial squence Spatial squence 16 of 28 Sustainability 2021, 13, 6212 Table 8. Table 5 Neighborhood layout ana 4.1. Imperial Period: Case 1 and Case 2 Post-reform and Contemporary Period: Case 6 Si 1978 th t h t f d f l d t k t Sustainability 2021, 13, x FOR PEER REVIEW 18 of 31 Active front Spatial hierarchy Spatial movement 4.3. Post-reform and Contemporary Period: Case 6 Since 1978, the country has transformed from a planned economy to an open market, Spatial movement 1 : 1 1 : 1 1 : 1 1 : 1 1 : 1 1 : 1 1:1 1 : 0 3 1 : 0 3 1 : 0 3 1 : 0 3 1 : 0 3 1 : 0 3 1:0.3 18 of 31 18 of 31 18 of 31 18 of 31 18 of 3 18 of 31 1 1 1 1 1 1 : 1 1 1 1 1 1:1 et 1 : 0.1 1 : 1 t 1 : 0.1 1 : 1 1 : 0.1 1 : 1 1 : 0.1 1 : 1 1 1 : 0.1 1 : 1 1 : 0.1 1 : 1 1 nt Active front Active front Active Active fron Active fron Ac i e o Spatial hierarchy Spatial movement Spatial hierarchy Spatial movement Spatial hierarchy Spatial movement Spatial hierarchy Spatial hierarchy Spatial movement Spatial hierarchy Spatial movement p hierarchy Spatial movement Spatial movement 17 of 28 17 of 28 Sustainability 2021, 13, 6212 4.3. Post-Reform and Contemporary Period: Case 6 4.3. Post-Reform and Contemporary Period: Case 6 Since 1978, the country has transformed from a planned economy to an open market, and socio-political and economic ideology is changing under the influence of globalization. Functional apartments with a good level of privacy have been introduced since this time, and the living style has been transformed from shared facilities (e.g., bathroom and kitchen) to privately owned [75] (Figure 10). Case 6 is an example to present the design of the building in this period (Table 10). The building is international style with four storages, and due to lack of professional design with appropriate spatial relation to the surroundings, the spaces between nearby houses are extremely narrow. In contemporary use, residents occupied part of public areas and built extensional rooms as storage for bicycle parking at the beginning, and pure storage later. REVIEW 18 of 31 (a) (b) (c) Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the b ildi d i (b) d d i ill t t th fl l f th b ildi ( ) Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the ustainability 2021, 13, x FOR PEER REVIEW 20 o Sustainability 2021, 13, x FOR PEER REVIEW 20 (a) (b) (c) Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the building design (b); and down image illustrates the floor plan of the building (c). Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the building design (b); and down image illustrates the floor plan of the building (c). ustainability 2021, 13, x FOR PEER REVIEW 20 of ustainability 2021, 13, x FOR PEER REVIEW 20 o (b) (a) (b) (c) Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the building design (b); and down image illustrates the floor plan of the building (c). Figure 10. Modern apartment and its layout. 4.3. Post-Reform and Contemporary Period: Case 6 Building Structure Analysis Open Space Analysis Case 6 Case 6 Layout Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Building/plot arrangement Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Functions Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Building height/street width 1:0.2 Number of floors Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Active front Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Spatial sequence Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Spatial hierarchy Sustainability 2021, 13, x FOR PEER REVIEW 21 of 31 Access efficiency to open space Spatial movement In total, six cases were identified based on morphological transformation analysis to cover all types of buildings and influences on the open spaces in the neighborhood (Figure 11). Access efficiency to open space Sustainability 2021, 13, x FOR PEER REVIEW 21 of 31 Access efficiency to open space Spatial movement In total, six cases were identified based on morphological transformation analysis to ll f b ld d fl h h hb h d Spatial movement In total, six cases were identified based on morphological transformation analysis to cover all types of buildings and influences on the open spaces in the neighborhood (Figure 11). Table 10. Cont. 4.3. Post-Reform and Contemporary Period: Case 6 Top left image shows the apartment layout (a); top right image presents the b ildi d i (b) d d i ill t t th fl l f th b ildi ( ) ustainability 2021, 13, x FOR PEER REVIEW 20 o Sustainability 2021, 13, x FOR PEER REVIEW 20 (b) (c) Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the building design (b); and down image illustrates the floor plan of the building (c). Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the building design (b); and down image illustrates the floor plan of the building (c). Table 10. Case 6—building structure analysis and open space analysis. Table 10. Case 6—building structure analysis and open space analysis. Table 10. Case 6—building structure analysis and open space analysis Table 10. Case 6—building structure analysis and open space analysis. Building Structure Analysis Open Space Analysis Case 6 Case 6 g y p p y Building Structure Analysis Open Space Analysis Case 6 Case 6 g y p p y Building Structure Analysis Open Space Analysis Case 6 Case 6 Building Structure Analysis Open Space Analysis Case 6 Case 6 Building Structure Analysis Open Space Analysis Case 6 Case 6 Building Structure Analysis Open Space Analysis Case 6 Case 6 18 of 28 Sustainability 2021, 13, 6212 Sustainability 2021, 13, 6212 18 of 28 Table 10. Cont. 4.3. Post-Reform and Contemporary Period: Case 6 Building Structure Analysis Open Space Analysis Case 6 Case 6 Layout Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Building/plot arrangement Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Functions Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Building height/street width 1:0.2 Number of floors Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Active front Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Spatial sequence Layout Building/plo t arrangement Functions Building height/street width 1 : 0.2 Number of floors Active front Spatial sequence Spatial hierarchy Spatial hierarchy Sustainability 2021, 13, x FOR PEER REVIEW 21 of 31 Access efficiency to open space Spatial movement In total, six cases were identified based on morphological transformation analysis to cover all types of buildings and influences on the open spaces in the neighborhood (Figure 11) Access efficiency to open space Sustainability 2021, 13, x FOR PEER REVIEW 21 of 31 Access efficiency to open space Spatial movement Spatial movement In total, six cases were identified based on morphological transformation analysis to cover all types of buildings and influences on the open spaces in the neighborhood (Figure 11). Table 10. Cont. 5. Analysis of Changes of Spatial Characteristics Between Cases 5. Analysis of Changes of Spatial Characteristics between Cases In this section, a third research strategy is implemented, in which the analytical framework is continually employed to analyze the continuities of spatial characteristics between cases. Based on the analysis of all six cases, the study identified a degree of con- tinuity of spatial characteristics in building structure and open space level in chronologi- cal order, which illustrates which spatial characteristics are continued, partly continued or changed from the previous morphological period to the next one. It is then clear to see, based on the transformation of socio-political and economic ideology, how different types of forms have changed local needs, and how local needs have changed these spatial char- acteristics. During the process, many spatial characteristics are discontinued due to tran- sitions of needs, but it also shows available spatial characteristics that can be continued to sustain socio-cultural sustainability in future development In this section, a third research strategy is implemented, in which the analytical frame- work is continually employed to analyze the continuities of spatial characteristics between cases. Based on the analysis of all six cases, the study identified a degree of continuity of spatial characteristics in building structure and open space level in chronological order, which illustrates which spatial characteristics are continued, partly continued or changed from the previous morphological period to the next one. It is then clear to see, based on the transformation of socio-political and economic ideology, how different types of forms have changed local needs, and how local needs have changed these spatial characteristics. During the process, many spatial characteristics are discontinued due to transitions of needs, but it also shows available spatial characteristics that can be continued to sustain socio-cultural sustainability in future development. sustain socio cultural sustainability in future development. As explained above, in the third research step a method of calculation is developed as a measurement to evaluate the degree of continuity and changes of spatial characteris- tics: continue, partly continue and change. The formula is developed in a two-step pro- cess: calculation of originality of spatial characteristics, and calculation of continuity of these spatial characteristics. As shown in Figure 12, each case in each spatial characteristic first needs to define the originality of its typology compared to its original design, whether it maintains original, or partly original or changed to another type (in Tables 11 and 12), to define the spatial characteristics in contemporary use. 4.3. Post-Reform and Contemporary Period: Case 6 Building Structure Analysis Open Space Analysis Case 6 Case 6 Layout Layout Building/plo t arrangement Building/plot arrangement Layout Building/plo t arrangement Layout Building/plo t arrangement Layout Building/plo t arrangement Layout Building/plo t arrangement Layout Building/plo t arrangement Layout Building/plo t arrangement Functions unctions nctions Functions Functions Functions Functions Functions Building height/street Building height/street Building height/street Building height/street width Building height/street width height/street width height/street width width ti i Functions unctions EER REVIEW A ti f Active fron Active front Active fro A i e o Number of Number of floors Number of floors Number of floors floors Spatial sequence Spatial hierarchy Spatial sequence Spatial hierarchy Spatial sequence Spatial hierarchy Spatial sequence Spatial hierarchy Spatial sequence Spatial hierarchy Spatial sequence Spatial hierarchy Spatial sequence Spatial sequence Spatial hierarchy Spatial hierarchy Access efficiency to open space Spatial movement In total, six cases were identified based on morphological transformation analysis to Access efficiency to open space Sustainability 2021, 13, x FOR PEER REVIEW 21 of 31 Access efficiency to open space Spatial movement Spatial movement In total, six cases were identified based on morphological transformation analysis to cover Spatial Spatial Spatial hierarchy Spatial hierarchy Spatial Spatial Spatial sequence Spatial sequence q efficiency to open space Access efficiency to open space Access efficiency to open space Access efficiency to open space Access efficiency to open space Spatial movement Spatial movement In total, six cases were identified based on morphological transformation analysis to cover all types of buildings and influences on the open spaces in the neighborhood (Figure 11) In total, six cases were identified based on morphological transformation analysis to cover all types of buildings and influences on the open spaces in the neighborhood (Figure 11). 19 of 28 alysis to (Fi Sustainability 2021, 13, 6212 Figure 11. Locations of cases and their categories. Figure 11. Locations of cases and their categories. 11. Locations of cases and their categories. Figure 11. Locations of cases and their categories. 5. Analysis of Changes of Spatial Characteristics Between Cases 5. Analysis of Changes of Spatial Characteristics between Cases Open Space Analysis Morphological Periods Period 1 Period 2 Period 3 Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Building/plot arrangement 8 8 5 9 9 9 Building height/street width 6 9 6 9 9 6 Active frontage 6 9 6 9 9 6 Spatial hierarchy 9 3 9 9 9 9 Spatial movement 6 3 9 9 9 9 a e i i ( ) Table 11. Calculation results of originality between cases in building analysis by using formula. tinuity score (O) is calculated using the following formula:  =   = m 1 ro 1 j j m k O (1) represents the originality of physical characteristics, k is a constant number represents the total number of sub-items in each spatial characteristic, o rep- originalities of the sub-item, and r represents the score of originalities of the hen r = 3, it means the case maintains its original design, when r = 2, it means tly changed its design, and when r = 1, it means the case has completely g y g y y g Building Analysis Morphological Periods Period 1 Period 2 Period 3 Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Layout 9 3 9 9 9 9 Functions 6 3 9 9 9 9 Number of floor 6 6 3 9 9 9 Spatial sequences 9 3 9 9 9 9 Access efficiency 6 3 9 9 9 9 riginal design. J represents the number of sub-item in sequence in each spa istics Table 12. Calculation results of originality between cases in open space analysis. original design. J represents the number of sub item in sequence in each spa istics. ‘O’ scores indicate original design, while lower ‘O’ scores represent change. m possible score is thus 9 [= 3 × 1/6 × (3 + 3+3 + 3+3 + 3)] if all spatial charac- maintained in its original formation; while the minimum possible score is 3 [= if all spatial characteristics are changed. Therefore, If 7 ≤ C < 9, the originality cal characteristic is maintained as original; if 5 ≤ C < 7, the originality of the racteristic is defined as partly original; if 3 ≤ C < 5, the originality of the phys- ristic is indicated as changed. 5. Analysis of Changes of Spatial Characteristics Between Cases 5. Analysis of Changes of Spatial Characteristics between Cases Then, formula 1 is used to calcu- late the score of originality (Represented by O) and justify the category of spatial charac- teristics based on the originality of each case (Table 11 and Table 12). Secondly, when the spatial characteristics in contemporary uses are defined, the originality score of calcula- tion from formula 1 is used to clarify its categories: the clear cycle in Table 13 and Table 14 represents the spatial characteristics of each case maintained in the original, grey cycle symbols are partly original, and black cycle means the physical characteristics have changed. Each originality score is used in formula 2 to calculate the continuity of these spatial characteristics between cases. The analysis results (Table 13 and Table 14) are then As explained above, in the third research step a method of calculation is developed as a measurement to evaluate the degree of continuity and changes of spatial characteristics: continue, partly continue and change. The formula is developed in a two-step process: calculation of originality of spatial characteristics, and calculation of continuity of these spatial characteristics. As shown in Figure 12, each case in each spatial characteristic first needs to define the originality of its typology compared to its original design, whether it maintains original, or partly original or changed to another type (in Tables 11 and 12), to define the spatial characteristics in contemporary use. Then, formula 1 is used to calculate the score of originality (Represented by O) and justify the category of spatial characteristics based on the originality of each case (Tables 11 and 12). Secondly, when the spatial characteristics in contemporary uses are defined, the originality score of calculation from formula 1 is used to clarify its categories: the clear cycle in Tables 13 and 14 represents the spatial characteristics of each case maintained in the original, grey cycle symbols are partly original, and black cycle means the physical characteristics have changed. Each originality score is used in formula 2 to calculate the continuity of these spatial characteristics between cases. The analysis results (Tables 13 and 14) are then clear—each physical characteristic clearly presents the form of transformation at different morphological layers through different periods. 20 of 28 Sustainability 2021, 13, 6212 clear—e Figure 12. The interrelationship between originalities of physical characteristics (O) and continuity Figure 12. The interrelationship between originalities of physical characteristics (O) and continuity of spatial characteristics (C). Figure 12. 5. Analysis of Changes of Spatial Characteristics Between Cases 5. Analysis of Changes of Spatial Characteristics between Cases The interrelationship between originalities of physical characteristics (O) and continuity Figure 12. The interrelationship between originalities of physical characteristics (O) and continuity of spatial characteristics (C). of spatial characteristics (C). The continuity score (O) is calculated using the following formula:  =   = m 1 ro 1 j j m k O (1) The O represents the originality of physical characteristics, k is a constant number equal to 3, m represents the total number of sub-items in each spatial characteristic, o rep- resents the originalities of the sub-item, and r represents the score of originalities of the sub-item. When r = 3, it means the case maintains its original design, when r = 2, it means the case partly changed its design, and when r = 1, it means the case has completely changed its original design. J represents the number of sub-item in sequence in each spa- tial characteristics. Higher ‘O’ scores indicate original design, while lower ‘O’ scores represent change. The maximum possible score is thus 9 [= 3 × 1/6 × (3 + 3+3 + 3+3 + 3)] if all spatial charac- teristics are maintained in its original formation; while the minimum possible score is 3 [= 3 × 1/1 × (1)] if all spatial characteristics are changed. Therefore, If 7 ≤ C < 9, the originality of the physical characteristic is maintained as original; if 5 ≤ C < 7, the originality of the physical characteristic is defined as partly original; if 3 ≤ C < 5, the originality of the phys- ical characteristic is indicated as changed. By using the score of originality, Formula 2 calculates the score of continuity of spa- tial characteristics (C) When the analysis results between cases present continuity, use the formula: Table 11. Calculation results of originality between cases in building analysis by using formula. Building Analysis Morphological Periods Period 1 Period 2 Period 3 Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Layout 9 3 9 9 9 9 Functions 6 3 9 9 9 9 Number of floor 6 6 3 9 9 9 Spatial sequences 9 3 9 9 9 9 Access efficiency 6 3 9 9 9 9 Table 12. Calculation results of originality between cases in open space analysis. 5. Analysis of Changes of Spatial Characteristics Between Cases 5. Analysis of Changes of Spatial Characteristics between Cases g the score of originality, Formula 2 calculates the score of continuity of spa- istics (C) he analysis results between cases present continuity, use the formula: Table 12. Calculation results of originality between cases in open space analysis. Open Space Analysis Morphological Periods Period 1 Period 2 Period 3 Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Building/plot arrangement 8 8 5 9 9 9 Building height/street width 6 9 6 9 9 6 Active frontage 6 9 6 9 9 6 Spatial hierarchy 9 3 9 9 9 9 Spatial movement 6 3 9 9 9 9 21 of 28 25 of 31 Sustainability 2021, 13, 6212 Sustainability 2021, 13, x FO Table 13. Building structure analysis results. Table 13. Building structure analysis results. Table 13. Building structure analysis results. Table 13. Building structure analysis results Table 13. Building structure analysis results. Table 13. Building structure analysis results structure analysis results. ng structure analysis results. Table 13. Build Table 13. Bu 22 of 28 26 of 3 Sustainability 2021, 13, 6212 Sustainability 2021, 13, x FO Table 14. Open space analysis results. Table 14. Open space analysis result Table 14. Open space analysis results. Table 14. Open space analysis results ce analysis results. pace analysis results. p Sustainability 2021, 13, 6212 23 of 28 The continuity score (O) is calculated using the following formula: O = k × 1 m × m ∑ j=1 orj (1) (1) The O represents the originality of physical characteristics, k is a constant number equal to 3, m represents the total number of sub-items in each spatial characteristic, o represents the originalities of the sub-item, and r represents the score of originalities of the sub-item. When r = 3, it means the case maintains its original design, when r = 2, it means the case partly changed its design, and when r = 1, it means the case has completely changed its original design. J represents the number of sub-item in sequence in each spatial characteristics. g p q p Higher ‘O’ scores indicate original design, while lower ‘O’ scores represent change. 5. Analysis of Changes of Spatial Characteristics Between Cases 5. Analysis of Changes of Spatial Characteristics between Cases The maximum possible score is thus 9 [=3 × 1/6 × (3 + 3 + 3 + 3 + 3 + 3)] if all spatial characteristics are maintained in its original formation; while the minimum possible score is 3 [=3 × 1/1 × (1)] if all spatial characteristics are changed. Therefore, If 7 ≤C < 9, the originality of the physical characteristic is maintained as original; if 5 ≤C < 7, the originality of the physical characteristic is defined as partly original; if 3 ≤C < 5, the originality of the physical characteristic is indicated as changed. g y p y g By using the score of originality, Formula 2 calculates the score of continuity of spatial characteristics (C). When the analysis results between cases present continuity, use the formula: C =  Nc + − O  (2) (2) When the analysis results between cases present partial continuity, use the formula: When the analysis results between cases present partial continuity, use the formula: C =  Npc + − O  (3) (3) When the analysis results between cases present discontinuity, use the formula: When the analysis results between cases present discontinuity, use the formula: C =  Nd + − O  (4) (4) C represents the continuity of spatial characteristics, Nc represents continue and equals 25, Npc presents partly continue and equals 15, Nd means discontinue and equals 5 and −represents interrelations between two compared cases. p Higher ‘C’ scores represent continuity of physical characteristic, while lower “C” scores indicate discontinuity of physical characteristic. The maximum possible score is therefore 31 [= 25 + |9−3|] if all spatial characteristics are continued between cases; while the minimum possible score is 5 [5 + |9−9|] if all spatial characteristics are discontinued. Thus, the categories are divided—if 5 ≤C ≤11, the physical characteristic maintains continuity; if 11 < C ≤21, the physical characteristic has partly continued; if 21 < C ≤31, the physical characteristic discontinued. The transformations among six cases were found to be at different degrees at the levels of building structure analysis and open space analysis (Figure 13). With regard to building structure level, five spatial characteristics were found to strictly continue through morphological transformation between case 4–case 5. The relations between the rest of cases were partly continued. 6. Key Findings Based on th 6. Key Findings Based on the analysis above, the key findings are very clear. Through the transfor mation of the urban development and changes of political-socio-economic ideologies both historically and due to current globalization influences, the layout, design and func- tions of buildings are transformed in each period to follow the changes and influences of these ideologies and fulfill permanent or contemporary needs. Due to transformations of political-economic-socio-cultural ideologies, the spatial structure of the neighborhood is dramatically changed through time. The political ideology is key throughout the design of the buildings and planning of the city in imperial times to emphasize socio-politica priority, in which the higher social class was located in the center of the block and along the main streets, while lower social classes gradually decentralized to the edge of the block and mostly occupied secondary streets and small lanes. The same design principle of so- cio-political hierarchy applied in the spatial design of houses to represent the social class of each family, and thus, the spatial characteristics of the buildings were originally de- signed to address socio-political ideologies, and their spatial structure involved areas in the front and back (where servants and female members of the families lived) that gradu ally centralized to the center (where senior or male members of the family lived). I i d t liti l id l till l d th k l th h t th d l Based on the analysis above, the key findings are very clear. Through the transfor- mation of the urban development and changes of political-socio-economic ideologies, both historically and due to current globalization influences, the layout, design and func- tions of buildings are transformed in each period to follow the changes and influences of these ideologies and fulfill permanent or contemporary needs. Due to transformations of political-economic-socio-cultural ideologies, the spatial structure of the neighborhood is dramatically changed through time. The political ideology is key throughout the design of the buildings and planning of the city in imperial times to emphasize socio-political priority, in which the higher social class was located in the center of the block and along the main streets, while lower social classes gradually decentralized to the edge of the block and mostly occupied secondary streets and small lanes. 5. Analysis of Changes of Spatial Characteristics Between Cases 5. Analysis of Changes of Spatial Characteristics between Cases While at open space level, there was discontinuity found between case 2-case 3, and case 2-case 5, in which spatial characteristics were muted completely. In addition, spatial characteristics were continually maintained between case 4–case 5. In the understanding of continuity of spatial characteristics, this not only means the spatial characteristics physically and directly continued to another (such as spatial characteristics found in the same morphological period), but also presents in different types of forms with shared common design principles. For example, the spatial relationship Sustainability 2021, 13, 6212 24 of 28 y means 24 of 28 y means of public→semi-private→private was found in imperial case 1, but it was also found at cases 4, 5 and 6. Although their typologies were different, at a spatial relationship scale the physical characteristic was continued. types of forms with shared common design principles. For example, the spatial relation- ship of public→semi-private→private was found in imperial case 1, but it was also found at cases 4, 5 and 6. Although their typologies were different, at a spatial relationship scale the physical characteristic was continued (a) (b) Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes between periods at building scale; and (b) presents the spatial changes between periods at open space scale. 6 K Fi di Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes between periods at building scale; and (b) presents the spatial changes between periods at open space scale. (b) (a) (a) (b) Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes between periods at building scale; and (b) presents the spatial changes between periods at open space scale. Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes between periods at building scale; and (b) presents the spatial changes between periods at open space scale. 6. Key Findings Based on th 6. Key Findings The same design principle of socio-political hierarchy applied in the spatial design of houses to represent the social class of each family, and thus, the spatial characteristics of the buildings were originally designed to address socio-political ideologies, and their spatial structure involved areas in the front and back (where servants and female members of the families lived) that gradually centralized to the center (where senior or male members of the family lived). In period two, political ideology still played the key role throughout the development of the city, although the design ideas were different from imperial times. The socialist ideas and collectivism were largely applied to regeneration of historical neighborhoods which transformed the traditional private houses into shared properties to address the core socio-political ideology—social equity—and the spatial layout of houses was then organically regenerated by each family who occupied the property, without regulatory control. As a consequence, the regeneration of traditional courtyard houses in the area In period two, political ideology still played the key role throughout the development of the city, although the design ideas were different from imperial times. The socialist ideas and collectivism were largely applied to regeneration of historical neighborhoods, which transformed the traditional private houses into shared properties to address the core socio- political ideology—social equity—and the spatial layout of houses was then organically regenerated by each family who occupied the property, without regulatory control. As a consequence, the regeneration of traditional courtyard houses in the area converted from one family owned homes to multi-family occupied residences (case 1 and 2), and part of originally residential buildings are now being turned from pure residential to mixed commercial functions (case 2), in order to satisfy increased commercial activities and needs. In the meantime, in terms of its spatial structure, it first transformed internal shared space and then expanded horizontally to external public open spaces, and grew up vertically to respond continually increased population (case 1 and 2), which also caused the internal road network of the block to be transformed from open access to blind alley. On the other hand, soviet style modern buildings for laborers started to be built freely by work units Sustainability 2021, 13, 6212 25 of 28 25 of 28 in the neighborhood without planning and spatial control, but also to emphasize socialist ideas and the collectivist lifestyle (cases 3, 4 and 5). 6. Key Findings Based on th 6. Key Findings Due to the lack of building regulations to control the construction in historical neighborhoods, the transformation of the buildings and even the entire neighborhood was seen as a result of negotiations between neighbors. Since post-reform, the economic ideologies of the global market have started to replace the socio-political ideologies that led the development of the neighborhood. The impact of mass global culture brought enormous socio-cultural conflict, changing urban development, and resulting in higher density international style modern buildings (case 6). Large scale individual modern architecture began in Chinese cities. Due to lack of building and planning regulation control and design, these high raised modern buildings created huge visual diversity in narrow lanes, which further limited the external spaces, and largely changed the way of living and pattern of use, both internal and external, of buildings. In contemporary use of all cases, the interesting part is after the transformation of political-economic-socio-cultural ideologies and forms of building typologies, the spatial sequence remains the same as vernacular design, which follows the public-semi-public- private pattern. Even if the houses have been changed from private to shared ownership, the residents will have built up a transitional semi-private space themselves slowly through time, which remains the preferred spatial sequence (case 1). Therefore, the spatial sequence, plot arrangement and spatial movement of most cases continually adapt to local needs, and maintain memories and meaning associated with local place. On the other hand, the other aspects only remain partly adapted to the local preferences in use, and whether they can be continued depends on transformation of political-socio-economic ideologies, and this explains the most important mutation between period 1 and 2. 7. Conclusions This study has developed an analytical framework to investigate changes of spatial characteristics in adaptation to the changing of local needs for improving socio-cultural sustainability in historical neighborhoods in the context of Beijing, China. The key contribu- tions of the research are twofold: firstly, due to lack of studies on adaptation of local needs from a physical perspective for sustaining socio-cultural sustainability in the context of China, particularly Beijing, this paper identifies continuities and discontinuities of spatial characteristics, which are as follows: (1) Continuities of spatial characteristics—continually supporting transformation of local needs through time and mutations of political-economic-socio-cultural ideologies. The analysis results could be used to support regulations and policies that fill the gaps of legislation in spatial design for sustaining socio-cultural sustainability. (2) Discontinuities of spatial characteristics—discover the gaps to adapt local socio- cultural needs in spatial design. On the other hand, some of them may also see new lifestyles identified in case studies, such as automobile use, and demands for in parking facilities. Secondly, the proposed method for analyzing physical forms of cases improves clarity and consistency in defining and analyzing typological cases at an articulated scale, pair- wise comparison and categorization of the three degrees of morphological changes. This analytical method is applicable to other cities and countries that improve efficiency to quickly define continuities and discontinuities of spatial characteristics for maintaining or improving, which improve socio-cultural sustainability of a neighborhood. The limitations of the study might involve three aspects. Firstly, one may ask to what degree each spatial characteristic analytical framework contributes to local needs in terms of socio-cultural sustainability, and whether there are other factors affecting these needs that influence socio-cultural sustainability. Secondly, the analytical framework was only tested in a historical neighborhood (covers vernacular, soviet style and international style raised buildings, but high raised towers are not available in this area) in Beijing. Although the results include clear identifications of continuity, partial continuity or discontinuity Sustainability 2021, 13, 6212 26 of 28 26 of 28 of spatial characteristics in adaptation of local needs, it still needs more evaluations of variant types of neighborhoods in different context. Finally, the decision made for a case in regard to continuity and mutation in its spatial transformation was made based on score calculation. However, if the calculation range was set to a larger number, then partial continuity might reduce and continuity would increase. 7. Conclusions y g y In order to reduce limitations of the study, further research with a bigger sample size with more house types in different contexts could be explored to provide a fuller picture of how changes of spatial characteristics could improve socio-cultural sustainability by evaluating the adaptation to local needs. The degree of each spatial characteristic’s contribution to the impact of spatial changes in relation to adaptation of local needs would also be interesting to study. Larger score range of calculation of continuity and discontinuity of spatial characteristics should be tested with a larger sample to justify the stability and applicability of the analytical framework. Author Contributions: Conceptualization, H.L. and B.L.; methodology, H.L.; software, H.L.; val- idation, H.L. and B.L.; formal analysis, H.L.; investigation, H.L.; resources, H.L.; data curation, H.L.; writing—original draft preparation, H.L. and B.L.; writing—review and editing, H.L. and B.L.; visualization, H.L.; supervision, B.L.; project administration, B.L.; funding acquisition, B.L. All authors have read and agreed to the published version of the manuscript. Funding: This research is supported by National Natural Science Foundation of China: 41871154. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy concerns. Acknowledgments: Great appreciation to Lu Yi, who offers great assist in development of formulas in the study. Acknowledgments: Great appreciation to Lu Yi, who offers great assist in development of formulas in the study. Conflicts of Interest: The authors declare no conflict of interest. nflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Krier, R.; Rowe, C. Urban Space; Academy editions London: London, UK, 1979. 1. Krier, R.; Rowe, C. Urban Space; Academy editions London: London, UK, 1979. 2. Relph, E. Place and Placelessness; Pion: London, UK, 1976. p 3. Zheng, J. Conservation Planning For Heritage Sites: A Critical Review and Case Studies; Courtauld Institute of Art (University of London): London, UK, 2007. 3. Zheng, J. Conservation Planning For Heritage Sites: A Critical Review and Case Studies; Courtauld Institute of Art (University of London): London, UK, 2007. 4. Chen, F. 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Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes
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orts | (2023) 13:17734 | https://doi.org/10.1038/s41598-023-44880-3 Contributions of viral oncog of HPV‑18 and hypoxia to ox stress and genetic damage in human keratinocytes Jimena Hochmann 1,2*, Magdalena Millán 1, Paola Hernández 3, Laura La Natali D’ Aiuto 1,5, Alejandro Silva 6, Juan Llaguno 6, Julia Alonso 6, Ariel Fe Vanesa Pereira‑Prado 2, José Sotelo‑Silveira 1,7, Ronell Bologna‑Molina 2 & Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is high the development of cervical and other cancers. Malignant transformation requir E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxid hypoxia are also key factors in cervical malignant transformation. Increased leve of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenviro genetic instability and invasiveness. In this work, we studied the combined effe and hypoxia in increasing oxidative stress and promoting DNA damage and nuc alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18 levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT pare higher genetic damage in hypoxia as measured by γH2AX and comet assays. In E7-18 increased its nuclear dry mass and both cell types displayed marked heter mass distribution and increased nuclear foci. Our results show contributions of b and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, altered microenvironment combines with oncogenic transformation to promote Abbreviations (HR-HPVs) High-Risk Human Papillomaviruses (EGFR) Epidermal Growth Factor (OS) Oxidative stress (ROS) Reactive Oxygen Species (·NO) Nitric oxide (RNS) Reactive Nitrogen Species (eNOS) Endothelial Nitric Oxide Synthase (HIF-1/2) Hypoxia Inducible Factor 1/2 (SCC) Squamous cell carcinomas (E6AP) E6-associatedprotein (pRb) Protein retinoblastoma (NOSs) Nitric oxide synthases (8-oxodG) 8-Oxo-7,8-dihydro-20 -deoxyguanosine OPEN 1Departamento de Genómica, Instituto de Investigaciones Biológicas Clement Uruguay. 2Departamento de Diagnóstico en Patología y Medicina Bucal, Facultad de de la República, General Las Heras 1925, Montevideo, Uruguay. 3Departamento d Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. 4Grupo de Biofisic de Ciencias Biológicas, Centro Universitario Regional Litoral Norte ‑Sede Salto, Uni (CENUR LN, UdelaR), Montevideo, Uruguay. 5Departamento de Biología Odontológica, Universidad de la República, General Las Heras 1925, Montevideo, Uruguay. 6Institu Ingeniería, Universidad de la República, Montevideo, Uruguay. 7Sección Biología Celu Universidad de la República, Montevideo, Uruguay. *email: jhochmann@fcien.e gmail.com www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Contributions of viral oncogenes of HPV‑18 and hypoxia to oxidative stress and genetic damage in human keratinocytes OPEN Jimena Hochmann 1,2*, Magdalena Millán 1, Paola Hernández 3, Laura Lafon‑Hughes 3,4, Natali D’ Aiuto 1,5, Alejandro Silva 6, Juan Llaguno 6, Julia Alonso 6, Ariel Fernández 6, Vanesa Pereira‑Prado 2, José Sotelo‑Silveira 1,7, Ronell Bologna‑Molina 2 & Miguel Arocena 1,5* Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the development of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/ E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression. Abbreviations (HR-HPVs) High-Risk Human Papillomaviruses (EGFR) Epidermal Growth Factor (OS) Oxidative stress (ROS) Reactive Oxygen Species (·NO) Nitric oxide (RNS) Reactive Nitrogen Species (eNOS) Endothelial Nitric Oxide Synthase (HIF-1/2) Hypoxia Inducible Factor 1/2 (SCC) Squamous cell carcinomas (E6AP) E6-associatedprotein (pRb) Protein retinoblastoma (NOSs) Nitric oxide synthases (8-oxodG) 8-Oxo-7,8-dihydro-20 -deoxyguanosine 1Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. 2Departamento de Diagnóstico en Patología y Medicina Bucal, Facultad de Odontología, Universidad de la República, General Las Heras 1925, Montevideo, Uruguay. 3Departamento de Genética, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. 4Grupo de Biofisicoquímica, Departamento de Ciencias Biológicas, Centro Universitario Regional Litoral Norte ‑Sede Salto, Universidad de la República (CENUR LN, UdelaR), Montevideo, Uruguay. 5Departamento de Biología Odontológica, Facultad de Odontología, Universidad de la República, General Las Heras 1925, Montevideo, Uruguay. 6Instituto de Física, Facultad de Ingeniería, Universidad de la República, Montevideo, Uruguay. 7Sección Biología Celular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. Contributions of viral oncogenes of HPV‑18 and hypoxia to oxidative stress and genetic damage in human keratinocytes OPEN *email: jhochmann@fcien.edu.uy; m.arocena.sutz@ gmail.com | https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 www.nature.com/scientificreports/ (HaCaT) Spontaneously immortalized human keratinocyte (BCRJ) Banco de células do Rio de Janeiro (DMEM) Dulbecco’s Modified Eagle’s Medium (FBS) Fetal bovine serum (Bcl-xL) B-cell lymphoma extra-large (IL-8) Interleukin-8 (BCRJ) Banco de Células do Rio de Janeiro (DMEM) Dulbecco’s Modified Eagle’s Medium (FBS) Fetal bovine serum (Bcl-xL) B-cell lymphoma extra-large (IL-8) Interleukin-8 Human papilloma viruses (HPVs) are non-enveloped small DNA viruses that infect keratinocytes of the skin and mucosa at different anatomic locations. The main risk factor for the development of cervical cancer and its precursor lesions worldwide is the persistent infection with a high-oncogenic risk HPV type (HR-HPV)1. HPV- 16 and HPV-18 are associated with about 70% of all cervical cancers. HPV16 is the most frequent one, and it is associated with squamous cell carcinomas (SCC), while HPV18 is the second most frequent and often detected in ­adenocarcinoma2. The high-risk types of HPV encode three viral oncoproteins, E5, E6 and E7 that bind to and modulate numerous cellular proteins. These oncoproteins serve as the major initiators of cell transformation, interacting with cellular proteins controlling key aspects of cell proliferation. For instance, E5 interacts with members of the epidermal growth factor receptor family (EGFRs), causing an enhancement of ligand-dependent activation of the EGFR, and EGF-dependent proliferation of cultured human ­keratinocytes3,4. As for E6 from high risk HPVs, its main target is the tumor suppressor p53. E6 forms a ternary complex with E6-associated protein (E6AP) and p53 and induce the latter degradation by the ubiquitin-mediated proteolysis ­pathway5,6. E7 from high risk HPV binds and induces the degradation of the tumor suppressor protein retinoblastoma (pRb), an essential regulator cell cycle, which disrupts the interaction of pRb with E2F transcription factor, releasing E2F and promoting the transcription of genes involved in S phase ­induction7. p g p g p Multiple steps are involved in the progression from HPV infection to cellular transformation to cervical cancer. Among the main factors that promote malignant transformation are chronic inflammation and oxidative stress (OS). Chronic inflammation contributes to approximately 25% of human ­cancers8, and induces reactive oxygen species (ROS) and nitrogen species (RNS) in inflammatory and epithelial cells that can damage DNA leading to mutagenic ­lesions9. An increase in ROS production can potentially promote cervical ­carcinogenesis10,11. Nitric oxide (·NO) is another contributor to oxidative ­stress12. Contributions of viral oncogenes of HPV‑18 and hypoxia to oxidative stress and genetic damage in human keratinocytes OPEN Previous studies have shown that women with cervical cytological changes caused by HPV infections exhibited increased cervical nitric oxide ­release13. Several studies have shown a link between hypoxia and increased oxidative ­stress14,15 and, on the other hand, oxidative stress has been linked to increased tumor genetic instability and ­invasiveness16,17. For instance, ROS and RNS can damage DNA to form mutagenic lesions, such as 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG) and 8-nitroguanine18,19. On the other hand, DNA damage is a key event in HPV-induced carcinogenesis, which can lead to the development of genomic instability. Recently, it was observed that HR-HPV oncoproteins activate members of the DNA damage repair pathways, and this is critical for viral ­replication20. However, it has been shown that a persistent expression of E6/E7 from high risk HPVs in normal cells could result in DNA damage and chromosomal aberrations, and various mechanisms have been proposed to explain these observations, such as replication stress and centrosome ­amplifications20,21. p pi In this work, we studied the relationship between hypoxia, oxidative stress, and DNA damage in HaCaT E5/ E6/E7 HPV-18 cells, which are human immortalized keratinocytes transduced with the three viral oncogenes (E5, E6 and E7) of Human Papilloma Virus type 18 (HPV-18), previously developed by our group and which constitute a cellular model of an intermediate stage in viral ­oncogenesis22. When cultured in an in vitro model of the hypoxic, tumoral microenvironment, which we have previously ­characterized23,24, HaCaT E5/E6/E7-18 cells markedly increase ROS and RNS production, and they simultaneously display increased DNA damage and altered nuclear architecture. Our results show that both hypoxia and oncogenic transformation contribute to increase oxidative stress and DNA damage, cooperating therefore in the path towards malignant transformation in HPV-induced carcinogenesis. ROS and RNS detection in normoxia and hypoxia yp We evaluated the levels of intracellular ROS in HaCaT parental and HaCaT E5/E6/E7-18 live cells in normoxia by adding the ROS Detection Reagent Deep Red (640/675 nm excitation/emission) to cells and through visualization by confocal microscopy. We observed that cells containing viral oncogenes of HPV-18 (E5/E6/E7-18) exhibited significantly higher intracellular ROS compared to HaCaT parental cells (Fig. 1A,B). Furthermore, in agreement with our previous results, we detected higher expression levels of Superoxide Dismutase 2 (SOD2) in HaCaT E5/E6/E7-18 compared to HaCaT parental cells in both normoxia and hypoxia. In addition, it is interesting to point out that hypoxia seems not to alter SOD2 expression (Fig. 1C). p yp p g On the other hand, we also measured ·NO release by Griess method, and we observed that the production of ·NO in both HaCaT parental and E5/E6/E7-18 cells cultured under treatment with coverslips for 24 h was increased compared to cells cultured in normoxia. In addition, ·NO content was significantly higher in HaCaT E5/E6/E7-18 cultured in hypoxia compared to HaCaT parental cells (Fig. 1D).We also observed higher expression of Endothelial Nitric Oxide Synthase (eNOS) in HaCaT E5/E6/E7-18 cells cultures in normoxia and hypoxia compared to HaCaT parental cells, although it was not statistically significant (data not shown). https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 1. Detection of ROS and ·NO in HaCaT parental and HaCaT E5/E6/E7-18 cells. (A) Representative images of ROS Detection Reagent Deep Red signal. Scale bar: 20 μm. (B) Quantification of ROS intensity (mean ± SD) in normoxia for HaCaT parental and HaCaT E5/E6/E7-18 cells. Student`s Test was conducted to compare two means. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (C) Relative expression levels of SOD2 expressed as mean spot pixel density is shown for HaCaT parental and HaCaT E5/E6/E7-18 cells as assessed by the Human Cell Stress Array Kit as suggested by the manufacturer (R&D Systems, MN, USA, ARY018). Averages ± standard deviations (SDs) of two independent experiments are shown. A two-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Dunnett post-hoc test for intra-group comparison. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (D) Nitrate/nitrite fold increase produced in HaCaT parental and HaCaT E5/E6/E7-18 cells in normoxia and coverslip induced hypoxia. Three independent experiments were conducted in triplicate. Mean ± SD is shown for each cell line. ROS and RNS detection in normoxia and hypoxia A one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. Figure 1. Detection of ROS and ·NO in HaCaT parental and HaCaT E5/E6/E7-18 cells. (A) Representative images of ROS Detection Reagent Deep Red signal. Scale bar: 20 μm. (B) Quantification of ROS intensity (mean ± SD) in normoxia for HaCaT parental and HaCaT E5/E6/E7-18 cells. Student`s Test was conducted to compare two means. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (C) Relative expression levels of SOD2 expressed as mean spot pixel density is shown for HaCaT parental and HaCaT E5/E6/E7-18 cells as assessed by the Human Cell Stress Array Kit as suggested by the manufacturer (R&D Systems, MN, USA, ARY018). Averages ± standard deviations (SDs) of two independent experiments are shown. A two-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Dunnett post-hoc test for intra-group comparison. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (D) Nitrate/nitrite fold increase produced in HaCaT parental and HaCaT E5/E6/E7-18 cells in normoxia and coverslip induced hypoxia. Three independent experiments were conducted in triplicate. Mean ± SD is shown for each cell line. A one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. DNA damage in normoxia and hypoxia g yp Next, we assessed the contributions of both hypoxia and HPV-18 viral oncogenes in promoting DNA damage. First, we evaluated γH2AX levels in HaCaT parental and E5/E6/E7-18 cells both in normoxia and hypoxia. As shown in Fig. 2, both HaCaT parental and HaCaT E5/E6/E7-18 cells had significantly higher γH2AX fluores- cence intensity in hypoxia compared to normoxia. However, no significant differences in γH2AX intensity were observed between HaCaT parental and HaCaT E5/E6/E7-18 cells either in normoxia or hypoxia, which suggests that hypoxia leads to greater γH2AX accumulation than viral oncogene expression. To further evaluate the level of endogenous DNA damage produced, we employed the alkaline comet assay (Fig. 3A). We observed a signifi- cant increase in the damage index in HaCaT E5/E6/E7-18 cells in hypoxia compared to normoxia, whereas the increase in the damage index in HaCaT parental cells in hypoxia compared to normoxia was not statistically significant (Fig. 3B). This suggests that hypoxia and viral oncogenes make a greater contribution to DNA strand breaks as measured by the comet assay than hypoxia alone. Nuclear dry mass density, dry mass and volume in normoxia and hypoxia We then sought to analyze global changes in nuclear structure caused by hypoxia and viral oncogenes. To this end, we used the quantitative phase microscope Nanolive 3D Cell Explorer-Fluo, which allows to measure refractive Nuclear dry mass density, dry mass and volume in normoxia and hypoxia y y y yp We then sought to analyze global changes in nuclear structure caused by hypoxia and viral oncogenes. To this end we used the quantitative phase microscope Nanolive 3D Cell Explorer-Fluo, which allows to measure refractive https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 2. Detection of γH2AX signal by confocal microscopy. (A) Representative images of γH2AX signal in parental HaCaT and HaCaT E5/E6/E7-18 cells in normoxia and under treatment with coverslips for 24 h. Scale bar: 20 μm. (B) Quantification of γH2AX intensity (mean ± SD) in normoxia and coverslip induced hypoxia for HaCaT parental and HaCaT E5/E6/E7-18 cells. Three independent experiments were conducted. Mean ± SD is shown for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. Figure 2. Detection of γH2AX signal by confocal microscopy. (A) Representative images of γH2AX signal in parental HaCaT and HaCaT E5/E6/E7-18 cells in normoxia and under treatment with coverslips for 24 h. Scale bar: 20 μm. (B) Quantification of γH2AX intensity (mean ± SD) in normoxia and coverslip induced hypoxia for HaCaT parental and HaCaT E5/E6/E7-18 cells. Three independent experiments were conducted. Mean ± SD is shown for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. index distributions inside cells, from which dry mass density distributions can be obtained, and which pro- duces z-stacks from which cellular and subcellular volumes can be estimated. In hypoxia, for both cell lines, we observed marked changes in nuclei visualized by quantitative phase microscopy, with the appearance of brighter dots throughout the nuclear volume, suggesting that areas with increased dry mass density form inside nuclei in cells exposed to hypoxia (Fig. 4A and supplementary videos 1 and 2). We then obtained nuclear volume and nuclear dry mass density values, observing a small decrease in nuclear volume in hypoxia (Fig. 4B, not statistically significant), accompanied by a significant increase in nuclear mass density in hypoxia (Fig. Nuclear dry mass density, dry mass and volume in normoxia and hypoxia 4C), suggesting that nuclei in hypoxia do not decrease their dry mass content even if they decrease in volume. Interestingly, nuclear dry mass content actually increases significantly in hypoxia for HaCaT E5/E6/E7-18 cells (Fig. 4D). However, the most marked change we observed was in nuclear dry mass density variance, which greatly increased in hypoxia for both HaCaT parental and HaCaT E5/E6/E7-18 cells (Fig. 4E), indicating a more heterogeneous dry mass distribution in nuclei in hypoxia, and suggesting that hypoxia results in marked changes in nuclear architecture. index distributions inside cells, from which dry mass density distributions can be obtained, and which pro- duces z-stacks from which cellular and subcellular volumes can be estimated. In hypoxia, for both cell lines, we observed marked changes in nuclei visualized by quantitative phase microscopy, with the appearance of brighter dots throughout the nuclear volume, suggesting that areas with increased dry mass density form inside nuclei in cells exposed to hypoxia (Fig. 4A and supplementary videos 1 and 2). We then obtained nuclear volume and nuclear dry mass density values, observing a small decrease in nuclear volume in hypoxia (Fig. 4B, not statistically significant), accompanied by a significant increase in nuclear mass density in hypoxia (Fig. 4C), suggesting that nuclei in hypoxia do not decrease their dry mass content even if they decrease in volume. Interestingly, nuclear dry mass content actually increases significantly in hypoxia for HaCaT E5/E6/E7-18 cells (Fig. 4D). However, the most marked change we observed was in nuclear dry mass density variance, which greatly increased in hypoxia for both HaCaT parental and HaCaT E5/E6/E7-18 cells (Fig. 4E), indicating a more heterogeneous dry mass distribution in nuclei in hypoxia, and suggesting that hypoxia results in marked changes in nuclear architecture. Nuclear foci quantitative image analysis q g y In hypoxia, the number of nuclear foci detected with Hoechst 33342 staining was significantly higher than in normoxia for both HaCaT parental and E5/E6/E7-18 cells, indicating an altered chromatin distribution after exposure to a hypoxic microenvironment, and confirming our previous results with quantitative phase imaging (Fig. 5). Discussion Furthermore, hypoxia increased DNA strand breaks in both HaCaT parental and HaCaT E5/E6/E7-18 cells, while the extent of DNA damage was greatest in HaCaT E5/E6/E7-18 cells exposed to hypoxia, as measured by the comet assay, showing that HPV- 18 oncogenes and hypoxia in combination contribute towards increased genotoxicity in addition to increased oxidative stress. compared to HaCaT parental cells. In addition, HaCaT E5/E6/E7-18 cells increased ·NO production in hypoxia and had significantly higher ·NO levels in this condition compared to HaCaT parental cells. Therefore, both HPV-18 oncogenes and hypoxia contribute to an increase in oxidative stress. Furthermore, hypoxia increased DNA strand breaks in both HaCaT parental and HaCaT E5/E6/E7-18 cells, while the extent of DNA damage was greatest in HaCaT E5/E6/E7-18 cells exposed to hypoxia, as measured by the comet assay, showing that HPV- 18 oncogenes and hypoxia in combination contribute towards increased genotoxicity in addition to increased oxidative stress. HPV oncogenes have been shown to cause increased oxidative stress levels, DNA damage and resistance to oxidative stress induced cell death: the E6 small isoform E6* increases oxidative stress and induces DNA ­damage25, whereas E7 modulates the expression of catalase, B-cell lymphoma extra-large (Bcl-xL), interleukin-8 (IL-8), Fas and Bad, resulting in resistance to oxidative stress-induced cell death in human keratinocyte ­cells26. More recently, it was reported that HPV-16 E6 and E7 induce oxidative stress and DNA damage in head and neck cancer ­cells27. We have also previously observed by flow cytometry that E5 in cooperation with E6 and E7 of HPV-18 promotes intracellular ROS ­production22, which we have now confirmed by confocal microscopy as well. In the present study, we have also observed increased expression in HaCaT E5/E6/E7-18 cells of SOD2. This enzyme catalyzes the dismutation of the superoxide ­(O−2) radical into ordinary molecular oxygen ­(O2) and hydrogen peroxide, it has key roles in controlling the redox status of tumor cells and is upregulated in several HPV-associated tumors, including penile and cervical ­carcinomas28,29, which is consistent with our results. We have also observed an increase, albeit not significant, in eNOS expression in HaCaT E5/E6/E7-18 cells, which is consistent with the increase in ·NO production by HaCaT E5/E6/E7-18 cells in hypoxia compared to HaCaT parental cells in the same condition. Discussion In this work, we have studied the contributions of hypoxia and HPV-18 viral oncogenes to promoting oxidative stress and DNA damage, and therefore advancing the process of malignant transformation. HaCaT cells trans- duced with the E5, E6 and E7 HPV-18 oncogenes exhibited significantly higher intracellular ROS production https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 3. Comet assay in HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative comet images of HaCaT parental and HaCaT E5/E6/E7-18 cell lines stained with DAPI, in normoxia and in hypoxia visualized by epifluorescence microscopy following alkaline electrophoresis. The length of the comet tail is an indicator of the level of DNA damage (red arrows). (B) DNA damage index of HaCaT parental and HaCaT E5/E6/E7-18 cells measured in normoxia and coverslip-induced hypoxia. Two independent experiments were conducted in triplicate. Mean ± SD is shown for each cell line and condition. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Figure 3. Comet assay in HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative comet images of HaCaT parental and HaCaT E5/E6/E7-18 cell lines stained with DAPI, in normoxia and in hypoxia visualized by epifluorescence microscopy following alkaline electrophoresis. The length of the comet tail is an indicator of the level of DNA damage (red arrows). (B) DNA damage index of HaCaT parental and HaCaT E5/E6/E7-18 cells measured in normoxia and coverslip-induced hypoxia. Two independent experiments were conducted in triplicate. Mean ± SD is shown for each cell line and condition. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. compared to HaCaT parental cells. In addition, HaCaT E5/E6/E7-18 cells increased ·NO production in hypoxia and had significantly higher ·NO levels in this condition compared to HaCaT parental cells. Therefore, both HPV-18 oncogenes and hypoxia contribute to an increase in oxidative stress. Discussion Increased levels of ·NO have been observed in women with cervical cytologi- cal changes likely related to ­HPV13, and this free radical can contribute to the induction of genotoxic lesions and to aspects of tumor progression such as cell growth, invasion and ­angiogenesis30, while also being an important component of the microenvironment in tumors such as cervical cancer, where it modifies immune responses and tumor ­behavior31,32. Therefore, the increase in ·NO produced by the combination of a hypoxic microen- vironment and HPV oncogenes that we observed could be an important factor mediating the contribution of ·NO towards malignant transformation in HPV-related cancers, such as cervical cancers. More generally, our results suggest that the combination of microenvironmental hypoxia and HPV oncogenes can lead to increased oxidative stress during initial stages of tumor progression, likely contributing to advancing this process in HPV- associated ­cancers33.f HPV oncogenes induce both oxidative stress and DNA ­damage20,21 in different tumor types frequently asso- ciated with HPV infection, such as head and neck ­cancer34. Also, expression of HPV oncogenes E6 and E7 in https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ ntificreports/ Figure 4. Quantitative phase imaging of HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative quantitative phase images of HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip- induced hypoxia. Scale bar: 10 μm. (B) Nuclear volume in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (C) Nuclear dry mass density (g/dL) in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (D) Nuclear dry mass (picograms) in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (E) Variance of nuclear dry mass density in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip- induced hypoxia. For Figs. 4B, C, D and E, three independent experiments were conducted. Mean ± SD is shown for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. Figure 4. Quantitative phase imaging of HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative quantitative phase images of HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip- induced hypoxia. Scale bar: 10 μm. Discussion (B) Nuclear volume in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (C) Nuclear dry mass density (g/dL) in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (D) Nuclear dry mass (picograms) in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (E) Variance of nuclear dry mass density in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip- induced hypoxia. For Figs. 4B, C, D and E, three independent experiments were conducted. Mean ± SD is shown for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. normal cells results in DNA damage and genomic ­instability21, one of the hallmarks of HPV-induced carcino- genesis. Hypoxia has been associated with DNA damage as well, as for instance moderate hypoxia can cause indirectly DNA damage in the form of replication stress, as evidenced by S-phase arrest, ATR activation and phosphorylation of H2AX-Ser139 (γ-H2AX), p53-Ser15, Chk1- Ser345, and Chk2-Thr6835,36. In the present study, we evaluated DNA damage by assessing γH2AX nuclear accumulation and by the comet assay. γH2AX nuclear accumulation was markedly increased by hypoxia, but we did not observe differences between HaCaT E5/E6/E7-18 cells and HaCaT parental cells in normoxia. On the other hand, the DNA damage index measured by the alkaline comet assay increased in hypoxia for HaCaT E5/E6/E7-18 cells, but not for HaCaT parental cells. The γH2AX immunoassay is a sensitive technique to detect DNA double-strand ­breaks37, while the alkaline comet assay does not discriminate between alkali-labile sites and single or double-strand ­breaks38,39. Therefore, these results suggest, firstly, that the hypoxia condition is the main responsible for inducing DNA double-strand breaks and that HPV-18 viral oncogenes alone had no significant implications in this type of DNA damage. On the other hand, the combination of HPV oncogenes and hypoxia increases the DNA damage detected by the alkaline comet assay in HaCaT E5/E6/E7-18 cells, which, taken together with the evidence of increased oxida- tive stress in hypoxia in these cells, suggests that hypoxia and HPV oncogenes combine to induce DNA damage consisting of single and double strand breaks and alkali-labile sites via higher oxidative damage. Discussion normal cells results in DNA damage and genomic ­instability21, one of the hallmarks of HPV-induced carcino- genesis. Hypoxia has been associated with DNA damage as well, as for instance moderate hypoxia can cause indirectly DNA damage in the form of replication stress, as evidenced by S-phase arrest, ATR activation and phosphorylation of H2AX-Ser139 (γ-H2AX), p53-Ser15, Chk1- Ser345, and Chk2-Thr6835,36. In the present study, we evaluated DNA damage by assessing γH2AX nuclear accumulation and by the comet assay. γH2AX nuclear accumulation was markedly increased by hypoxia, but we did not observe differences between HaCaT E5/E6/E7-18 cells and HaCaT parental cells in normoxia. On the other hand, the DNA damage index measured by the alkaline comet assay increased in hypoxia for HaCaT E5/E6/E7-18 cells, but not for HaCaT parental cells. The γH2AX immunoassay is a sensitive technique to detect DNA double-strand ­breaks37, while the alkaline comet assay does not discriminate between alkali-labile sites and single or double-strand ­breaks38,39. Therefore, these results suggest, firstly, that the hypoxia condition is the main responsible for inducing DNA double-strand breaks and that HPV-18 viral oncogenes alone had no significant implications in this type of DNA damage. On the other hand, the combination of HPV oncogenes and hypoxia increases the DNA damage detected by the alkaline comet assay in HaCaT E5/E6/E7-18 cells, which, taken together with the evidence of increased oxida- tive stress in hypoxia in these cells, suggests that hypoxia and HPV oncogenes combine to induce DNA damage consisting of single and double strand breaks and alkali-labile sites via higher oxidative damage. https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 5. Nuclear foci quantitative image analysis (A) Segmentation of nuclear foci of a representative image of HaCaT E5/E6/E7-18 cells. a) Hoechst 33342 greyscale image. b) Threshold segmentation. c) StarDist 2D segmentation. Note that using this method the result is more precise than using only threshold, for example in the shape of the pink nucleus. d) Nuclear profile recognition and numeration. Note that on edge nucleus were excluded and only complete profiles were segmented. e) Find maxima single point tool for foci analysis. Isolated single points outside the nucleus were not considered. f) Combination of D and E for foci measurement. (B) Quantification of nuclear foci. Discussion For this analysis Mann–Whitney test and one-way analysis of variance ANOVA test was conducted to assess statistical significance among groups (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. Figure 5. Nuclear foci quantitative image analysis (A) Segmentation of nuclear foci of a representative image of HaCaT E5/E6/E7-18 cells. a) Hoechst 33342 greyscale image. b) Threshold segmentation. c) StarDist 2D segmentation. Note that using this method the result is more precise than using only threshold, for example in the shape of the pink nucleus. d) Nuclear profile recognition and numeration. Note that on edge nucleus were excluded and only complete profiles were segmented. e) Find maxima single point tool for foci analysis. Isolated single points outside the nucleus were not considered. f) Combination of D and E for foci measurement. (B) Quantification of nuclear foci. For this analysis Mann–Whitney test and one-way analysis of variance ANOVA test was conducted to assess statistical significance among groups (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. Moreover, it is important to consider that early HPV infection produces DNA damage and upregulates DNA damage response genes, as shown for instance in a model of HPV infection using organotypic raft cultures of human cervical ­keratinocytes40. The combination of HPV infection and a hypoxic microenvironment could therefore result in increased selective pressure on the DNA damage repair capability of cells, leading to a cellular phenotype tolerant of higher DNA damage burdens and eventually to the emergence of resistant and aggressive tumors. Genetic damage can cause profound alterations in nuclear ­architecture41. In this study, we used quantitative phase imaging to assess nuclear dry mass distribution as an indirect way to evaluate changes in nuclear archi- tecture caused by hypoxia and HPV oncogenes. Our results indicate that nuclear dry mass density increases significantly with hypoxia, whereas nuclear volume tends to decrease, although not significantly. This would imply that the increase in nuclear dry mass density observed in hypoxia is mainly due to a decrease in nuclear volume and not an increase in nuclear dry mass. This appears to be the case for HaCaT parental cells, which do not show an increase in nuclear dry mass in hypoxia, whereas nuclear dry mass in HaCaT E5/E6/E7-18 cells increases in hypoxia compared to normoxia and compared to HaCaT parental cells in hypoxia. www.nature.com/scientificreports/ redistribution in hypoxia, and therefore supporting the notion that hypoxia results in profound rearrangements of nuclear components. As future perspectives of this work, it will be important to identify if these foci contain components of the DNA damage repair machinery, and in particular which DNA damage repair pathways are activated in response to the combination of hypoxia and HPV oncogenes.i redistribution in hypoxia, and therefore supporting the notion that hypoxia results in profound rearrangements of nuclear components. As future perspectives of this work, it will be important to identify if these foci contain components of the DNA damage repair machinery, and in particular which DNA damage repair pathways are activated in response to the combination of hypoxia and HPV oncogenes.i p yp g In conclusion, this is the first report that studies the contributions of hypoxia and the three viral oncogenes of HPV-18 to oxidative stress and DNA damage, and our results suggest that the interplay between microenvi- ronmental hypoxia and HPV oncogenes is likely to cause increased oxidative stress and DNA damage, as well as an altered nuclear architecture, therefore propelling forward the process of HPV induced carcinogenesis. Nitric oxide‑releasing activity g In order to measure the ·NO production in our cells, we used the Griess reaction as we described ­previously45. This assay consists in measuring the nitrate/nitrite content in the cell culture medium. We performed this assay in normoxic and hypoxic conditions previously described. For the latter condition, cells were cultured under coverslips for 24 h, and after this period, cell supernatants were collected and analyzed. Briefly, 50 µl of culture medium were transferred to a new multiwell plate and incubated first with 1% sulfanilamide solution and then with 0.1% N-1-naphtilethylenrdiamine dihydrochloride protected from light. In parallel, a reference curve with ­NaNO2 was performed at serial dilutions between 0 and 100 µmol/l in 50 µl of culture medium. The absorbance was measured at 540 nm using a microplate spectrophotometer (Varioskan Flash Microplate spectrophotometer; Thermo Fisher, Vantaa, Finland). Induction of hypoxia yp In order to induce hypoxia to HaCaT parental and HaCaT E5/E6/E7-18 cells, we used the variant of coverslip- induced hypoxia method previously described 23. Briefly, this assay consists in culturing cells in the 10 mm radius wells of glass bottom dishes of 35 mm diameter (Cellvis, CA, USA) at increasing distances from an oxygen source. For this, after the cells were adhered to the wells, they were covered for 24 h with square acrylic coverslips of 24 mm of width and 2 mm of thickness with a square hole in the middle. In this way, an oxygen gradient is gener- ated, with cells in the periphery of the chamber located farthest from the coverslip hole (the sole oxygen source). γH2AX immunocytochemistryh The DNA damage was evaluated by quantification of γH2AX nuclear immunofluorescence. For this, HaCaT parental and HaCaT E5/E6/E7-18 cells were seeded in a 24-well plate at density of 2 × ­105 per well and incubated for 24 h in a humidified 5% ­CO2 atmosphere at 37 °C. For the hypoxia condition, cells were cultured under coverslips for 24 h before the experiment. After that, culture medium was removed and cells were washed once with PBS. Then, cells were fixed for 15 min in 4% paraformaldehyde, rinsed in PBS three times, 5 min each, and permeabilized with blocking buffer (0.05% Triton X-100, 1% BSA in PBS) for 45 min at room temperature. Afterwards, permeabilized cells were incubated with the anti-γH2AX antibody diluted in blocking buffer (1/500, ab26350; Abcam, Cambridge, Massachusetts, USA) overnight at 4 °C. Finally, cells were washed with PBS three times for 5 min each, and incubated for 45 min with the secondary antibody (1:1000, goat anti-mouse conjugated with Alexa488; Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and with DAPI (300 nmol/l; Invitrogen) to stain nuclei. Finally, cells were visualized using a Zeiss LSM800 confocal microscope. The γH2AX nuclear fluorescence was quantified related to DAPI signal for each condition, using the ImageJ software v. 1.43n4. Materials and methods Cell lines Spontaneously immortalized human keratinocyte (HaCaT) cells were purchased from Banco de Células do Rio de Janeiro (BCRJ), Brazil (batch number 001071, certificate of analysis provided by the supplier) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) low glucose medium (Capricorn, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Massachusetts, USA). HaCaT cells were transduced with three viral oncogenes (E5/E6/E7-18) of HPV-18 using lentiviral packaging systems. For this, HaCaT E5/E6/ E7 HPV-18 cells were previously prepared by co-infection with a retroviral vector carrying the MSCV-N-puro- 18E5 plasmid (Addgene # 37,882, MA, USA) and with a pLXSN retroviral vector that contained cloned both E6/ E7 of HPV-18 genes that was kindly provided by Dra, Sichero from Instituto do Câncer do Estado de São Paulo. The preparation and characterization of these cell lines was detailed in Hochmann et al.22. Intracellular ROS production p We evaluated the intracellular ROS production in live HaCaT parental and HaCaT E5/E6/E7-18 cells using ROS Detection Reagent Deep Red (Sigma, MO, USA, 1:1000 dilution from stock solution). Once cells reached a confluence of 60 per cent approximately, they were incubated with the reagent for one hour at 37 °C following manufacturer instructions. Then cells were visualized by confocal microscopy using a Zeiss LSM800 confocal microscope (Zeiss, Oberkochen, Germany) equipped with a Plan Apo N 63X oil NA 1.4 lens. Quantification of ROS intensity was evaluated using Image J software v. 1.43n4. Discussion yp p p p yp A previous study has shown that nuclear dry mass is similar in SW480 and SW620 cell lines (derived from the same patient before and after metastatic spread), but that nuclear volume is smaller in metastatic SW620 ­cells42, suggesting that nuclear dry mass tends to be more conserved than nuclear volume. However, another study found that nuclear dry mass increases after ionizing radiation, in parallel with an increase in fluorescence intensity of γH2AX nuclear ­foci43, suggesting that an increase in nuclear dry mass might be related to the mobilization of DNA repair machinery. The increase in nuclear dry mass in HaCaT E5/E6/E7-18 cells in hypoxia might therefore be related to an increased DNA repair response, which could be caused in turn by the increased DNA damage observed in this condition using the comet assay. We also observed a marked increase in nuclear dry mass density variance in hypoxia, indicating that the het- erogeneity of the nuclear dry mass distribution increases in this condition, which implies that marked changes in nuclear architecture are taking place during hypoxia. Moreover, increased variance in dry mass distribution is linked to a decrease in cell ­stiffness44, whereas DNA damage has been shown to decrease nuclear ­stiffness44. There- fore, the marked increase in the variance of nuclear dry mass density that we observe in hypoxia would indicate that nuclear stiffness decreases in this condition, which could be related in turn to the increase in DNA damage observed. In summary, we have observed that both hypoxia and HPV oncogenes alter key physical nuclear parameters, which is likely related to the contributions of both factors to increased DNA damage. Moreover, as shown by Hoechst 33342 staining of live cells, hypoxia increases the number of nuclear foci, indicating chromatin Scientific Reports | (2023) 13:17734 | https://doi.org/10.1038/s41598-023-44880-3 www.nature.com/scientificreports/ Quantitative phase imaging and nuclear dry mass density calculation HaCaT parental and E5/E6/E7-18 cells were imaged in normoxia or under thin glass coverslips to induce hypoxia using the quantitative phase microscope Nanolive 3D Cell Explorer-Fluo, which produces z-stacks of 96 slices with a depth of field of 30 μm. Cells were imaged for 24 h, and the refractive index distribution inside cell nuclei in normoxia and after 24 h under coverslips was obtained from the best focused slice, as each pixel in the quan- titative phase images corresponds to a refractive index value. Next, refractive index values were converted to dry mass density values according to the ­equation46: Drymassdensity  g/dL  = 665. RI RIH2O −1 where RI is refractive index and ­RIH2O is the refractive index of water, equal to 1.33. Nuclei were segmented manu- ally from quantitative phase images, and the average nuclear dry mass density and its variance were calculated using Python scripts. 30 nuclei were analyzed for each condition, and their volume was calculated from their areas in the slices in focus from the z-stacks. From the nuclear volume, and the nuclear dry mass density measured in the best focused slice, the estimated nuclear dry mass (expressed in picograms) was obtained. Expression level of eNOS and SOD2h The relative intensities of eNOS and SOD2 proteins were evaluated using the Human XL Oncology Array Kit (R&D Systems, MN, USA, ARY026), and Human Cell Stress Array Kit (R&D Systems, MN, USA, ARY018) respectively following manufacturer instructions. Protein levels were quantified using the ImageQuant TL soft- ware (GE Healthcare, Buckinghamshire, UK), and the individual Western blot membranes were normalized according to the pixel densities of the 6 reference spots. Nuclear foci quantitative image analysis q g y HaCaT parental and E5/E6/E7-18 live cells were stained with Hoechst 33342 (Sigma, MO, USA, final concen- tration 1 μg/ml) for 30 min either in normoxia or after 24 h under coverslips. Images were obtained with the fluorescence module of the Nanolive 3D Cell Explorer-Fluo and were analysed in order to determine the pres- ence of nuclear foci. Normalization of the images was performed using Enhance Contrast tool of ImageJ, with 0,35% saturated pixels. Nuclear profiles were segmented with color threshold or ImageJ/Fiji StarDist 2D with the default parameters of the ­plugin47. Afterwards, overlapping or adjacent nuclei were separated with the macro Separate ­Labels48, and on edge nucleus were excluded. Nuclear foci were determined by Find Maxima ImageJ tool, which locates the maxima intensity in an image and, as a result, creates a binary mask of one segmented particle per maximum. Finally, both images were combined in order to measure the number of foci per nucleus. www.nature.com/scientificreports/ were trypsinized and neutralized with culture medium. Then, cells were centrifuged for 5 min at 1200 rpm and the pellet was resuspended in 10 µl of PBS. The cell suspension (20 µl) mixed with 80 µl of 0.75% low melting point agarose (LMP) (Gibco #15,517–014) was extended on 1.0% NMP-agarose (Invitrogen #15,510–019) pre- coated slide. A coverslip was added and the agarose was allowed to set for 10 min at 4 °C. Then, the coverslip was removed and the cells were lysed by immersion of the slide in cold lysis buffer solution (pH 10, 2.5 mol/l NaCl, 100 mmol/l ­Na2EDTA, 10 mmol/l Tris–HCl, 1% Triton X-100) and 10% DMSO for 1 h at 4 °C. Electrophoresis was conducted in alkaline buffer (3 mol/l NaOH, 100 mmol/l EDTA, pH 13) at 25 V for 20 min in a cold unit at 4 °C with a distance between electrodes of 34 cms. Following neutralization, cells were stained with DAPI (6 µg/ml; Invitrogen) for 10 min. Two slides per condition were analyzed in each experiment and 100 cells per slide were scored. Damage was quantified as the damage index, which represents damage in individual cells DNA damage scores vary from 1 to 5, with 1 being the lower damage and 5 the higher damage. The Damage Index is calculated based on 100 cells, with the following equation: 5 1n.α where n is the cell number and α is the damage score. Statistical analysis y Statistical analysis and graphical presentation were performed using GraphPad Prism version 8.0.1 software (GraphPad Software Inc., San Diego, CA, USA). All experiments were performed in triplicate, and data were presented as the mean ± standard deviation (SD), with the exception of the comet assay, in which two experiments were performed induplicate. For all statistical analysis, One-Way unpaired ANOVA statistical test followed by Tukey’s HSD post-hoc test was conducted with the exception of data retrieved from ROS quantification, which was analyzed using Student`s Test in order to compare only two means, and data obtained from protein profile analysis which was analyzed using two-way analysis of variance (ANOVA) test in order to assess statistical sig- nificance among groups along with Dunnett post-hoc test for intra-group comparison. Statistical significance was determined at p < 0.05. Descriptive statistics, Mann–Whitney test and ANOVA was also performed for nuclear foci quantitative image analysis. DNA Damage measurement by comet assay g y y In order to evaluate the DNA damage generated in the hypoxic microenvironment in both HaCaT parental and HaCaTE5/E6/E7-18 cells, we used the alkaline comet assay. 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Phase imaging with computational specificity (PICS) for measuring dry mass changes in sub-cellular compart- ments. Nat. Commun. 11(1), 6256. https://​doi.​org/​10.​1038/​s41467-​020-​20062-x (2020).f p g 3. Baker-Groberg, S. M. et al. Effect of ionizing radiation on the physical biology of head and neck squamous cell carcinoma cells Cell Mol. Bioeng. 8(3), 517–525. https://​doi.​org/​10.​1007/​s12195-​015-​0393-8 (2015). 43. Baker-Groberg, S. M. et al. Effect of ionizing radiation on the physical biology of head and n Cell Mol. Bioeng. 8(3), 517–525. https://​doi.​org/​10.​1007/​s12195-​015-​0393-8 (2015). g ( ) p g ( ) 44. Eldridge, W. J., Steelman, Z. A., Loomis, B. & Wax, A. Optical phase measurements of disorder strength link microstructure to cell stiffness. Biophys. J. 112(4), 692–702. https://​doi.​org/​10.​1016/j.​bpj.​2016.​12.​016 (2017). g p g 44. Eldridge, W. J., Steelman, Z. A., Loomis, B. & Wax, A. Optical phase measurements of disorder strength link microstructure to cell stiffness. Biophys. J. 112(4), 692–702. https://​doi.​org/​10.​1016/j.​bpj.​2016.​12.​016 (2017). f p y J ( ) p g j pj ( ) 45. Pérez, F. et al. Furoxans and tocopherol analogs-furoxan hybrids as anticancer agents. Anticancer Drugs 30(4), 330–338. https://​ doi.​org/​10.​1097/​CAD.​00000​00000​000721 (2019). f p y p g j pj 5. Pérez, F. et al. Furoxans and tocopherol analogs-furoxan hybrids as anticancer agents. Anticancer Drugs 30(4), 330–338. https:// doi.​org/​10.​1097/​CAD.​00000​00000​000721 (2019). g ( ) 6. Phillips, K. G., Jacques, S. L. & McCarty, O. J. Competing interests h p g The authors declare no competing interests. p g The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​44880-3. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​44880-3. Correspondence and requests for materials should be addressed to J.H. or M.A. Correspondence and requests for materials should be addressed to J.H. or M.A. Correspondence and requests for materials should be addressed to J.H. or M.A. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2023 https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 |
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Educação Patrimonial como Base de Preservação.
Diálogos
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Diálogos Volume 05 ISSN em linha 2789-2182 2020 ISSN impresso 2520-5927 dialogosuntl.com https://doi.org/10.53930/27892182.dialogos.5.50 Diálogos Volume 05 ISSN em linha 2789-2182 2020 ISSN impresso 2520-5927 dialogosuntl.com https://doi.org/10.53930/27892182.dialogos.5.50 Diálogos Volume 05 ISSN em linha 2789-2182 2020 ISSN impresso 2520-5927 dialogosuntl.com https://doi.org/10.53930/27892182.dialogos.5.50 Diálogos Volume 05 ISSN em linha 2789-2182 2020 ISSN impresso 2520-5927 dialogosuntl.com https://doi.org/10.53930/27892182.dialogos.5.50 1 Mestre em Ciências da Religião pela Universidade Federal de Sergipe. Advogado e presidente da Comissão de Liberdade Religiosa da OAB/SE. Email: advdouglascosta@gmail.com. https://doi.org/10.53930/27892182.dialogos.5.50 eduCação patrimonial Como base de preservação Douglas Lima da Costa1 Resumo: O presente artigo objetiva conduzir à reflexão sobre o papel que desempe- nha o Patrimônio Histórico Cultural como componente simbólico representativo do poder e da identidade da memória coletiva das comunidades e do local em que constroem sua história. Pontua a contribuição oferecida pela Nova História Cultural como elemento de registro para a identificação e a preservação da memória, sobretudo, das classes populares e dos locais onde constroem sua cultura bem como, analisa a proposta de Educação Patrimonial contida no Guia Básico de Educação Patrimonial organizado pelo IPHAN – Instituto do Patrimônio Histórico e Artístico Nacional. Palavras-chave: Patrimônio Histórico Cultural; Memória; Educação Patrimonial. Douglas Lima da Costa Mestre em Ciências da Religião pela Universidade Federal de Sergipe. Advogado e presidente da Comissão de Liberdade Religiosa da OAB/SE. e-mail: advdouglascosta@gmail.com Submetido: 11 de março de 2020 Aceito: 15 de agosto de 2020 Publicado: 17 de novembro de 2020 Submetido: 11 de março de 2020 Aceito: 15 de agosto de 2020 Publicado: 17 de novembro de 2020 Diálogos | Volume 05 | 2020 heritage eduCation as a preservation base Abstract: This article aims to reflect on the role played by Cultural Historical Heritage as a symbolic component representative of the power and identity of the collective memory of the communities and the place where they build their history. It points out the contribution offered by the New Cultural History as an element of registration for the identification and preservation of memory, above all, of the popular classes and the places where they construct their culture, as well as analyses the proposal of Education Patrimonial contained in the Basic Guide of Patrimonial Education organized by IPHAN – Institute of National Historical and Artistic Heritage. Keywords: Cultural Historical; Heritage Memory; Patrimonial Education. Em seu significado mais primitivo, a palavra patrimônio tem origem atre- lada ao termo grego pater, que significa “pai” ou “paterno”, ou seja, tal qual um pai transmite ao filho seus bens, essa foi a ideia que se relacionou ao patrimônio como legado ao povo. O Patrimônio Histórico e Cultural é o conjunto de bens materiais e imateriais arraigados de valor simbólico para uma dada comunidade, diz respeito a memória que foi valorizada e materializada ao longo do tempo. 1 Mestre em Ciências da Religião pela Universidade Federal de Sergipe. Advogado e presidente da Comissão de Liberdade Religiosa da OAB/SE. Email: advdouglascosta@gmail.com. https://doi.org/10.53930/27892182.dialogos.5.50 Diálogos | Volume 05 | 2020 Diálogos | Volume 05 | 2020 | 129 Douglas Lima da Costa Douglas Lima da Costa Essa noção de patrimônio ganhou força no século XIX, quando a Revolução Francesa ressaltou a necessidade de elencar monumentos que refu- tassem o esquecimento do passado. Nesse período a preservação do passado colocava-se presa a uma noção de “melhoria”, “evolução” e “progresso”, pro- messas da Revolução. As sociedades sempre deixam marcas no lugar onde se estabelecem que identificam a sua história, materializam sua identidade, tradições e seus cos- tumes. Como afirma Susana Gastal (2006, p. 101) nestes ambientes estão “as marcas do local construídas no tempo”. Neste sentido, entende-se que todos os lugares trazem traços peculiares do modo de ver e viver da população que habita ou habitou o local. Para Gastal (2006, p. 101), “o Lugar seria o Locus, no tempo e no espaço, do acúmulo de experiência em forma de história e de tradição, a segurança da identidade”. Figura 1: A liberdade guiando o povo de Eugène Delacroix Fonte: <https://chelios.jusbrasil.com.br/artigos/464544307/os-ide- ais-da-revolucao-francesa-e-o-direito-moderno>. Acessado em: 20 jul. 2018. Figura 1: A liberdade guiando o povo de Eugène Delacroix Fonte: <https://chelios.jusbrasil.com.br/artigos/464544307/os-ide- ais-da-revolucao-francesa-e-o-direito-moderno>. Acessado em: 20 jul. 2018. Fonte: <https://chelios.jusbrasil.com.br/artigos/464544307/os-ide- ais-da-revolucao-francesa-e-o-direito-moderno>. Acessado em: 20 jul. 2018. Diálogos | Volume 05 | 2020 130 | 130 | Educação patrimonial como base de preservação Douglas Lima da Costa Douglas Lima da Costa Portanto, o objeto “fala sempre de um lugar, seja ele qual for, porque está ligado à experiência dos sujeitos com e no mundo, posto que ele representa uma porção significativa da paisagem vivida” (Silveira e Lima Filho, 2005, p. 40). Os autores acrescem que: É nesse sentido que é possível falar numa memória que impregna e restitui “a alma nas coisas”, referida a uma paisagem (inter) subjetiva onde o objeto (re) situa o sujeito no mundo vivido mediante o trabalho da memória, ou ainda, é da força e dinâmica da memória coletiva que o objeto, enquanto expressão da materialidade da cultura de um grupo social remete à elasticidade da memória como forma de fortalecer os vínculos com o lugar, considerando as tensões próprias do esquecimento. (Silveira e Lima Filho, 2005, p. 39). É nesse sentido que é possível falar numa memória que impregna e restitui “a alma nas coisas”, referida a uma paisagem (inter) subjetiva onde o objeto (re) situa o sujeito no mundo vivido mediante o trabalho da memória, ou ainda, é da força e dinâmica da memória coletiva que o objeto, enquanto expressão da materialidade da cultura de um grupo social remete à elasticidade da memória como forma de fortalecer os vínculos com o lugar, considerando as tensões próprias do esquecimento. (Silveira e Lima Filho, 2005, p. 39). É importante ainda tratar, brevemente, sobre o desgaste sofrido pela memória com o passar do tempo. Diehl (2002, p. 118) nomeia este processo de “corrosão temporal” da memória. Para Diehl, quanto mais distante do fato, ou da época, ou do contexto tomado como objeto de investigação, mais desgastada a memória estará. A memória “vai perdendo força, capacidade explicativa, capacidade de informar, tornar-se transparente, sem pontos de referência substantiva para manter suas funções (...)” (p. 118). Dihel (2002, p. 119) aduz então, que a memória seja “refrescada constantemente” e que seja grafada, narrada ou tornada uma fonte histórica ou ainda que seja trans- formada em Patrimônio Histórico Cultural da coletividade. Mas o que deve ser visto como memória a fim de haver a preocu- pação de ser guardada? Chartier (1991) acredita que existem esquemas geradores das classificações e das percepções, que atuam como verdadeiras instituições sociais criadoras do mundo social ou das representações coleti- vas. Para o autor em comento nem sempre a representação é o representado e o signo é o significado. Bourdieu (1989, p. Educação patrimonial como base de preservação Já Bartholo (2005, p. 19) afirma que “o lugar é produzido na relação com o espaço construído socialmente, mediante uma rede de significados e sentidos que são historicamente e culturalmente tecidos”. Astor Antônio Diehl (2002, p. 112) entende a construção da memória dos lugares “como um processo dinâmico da própria rememorização” do passado histórico. Para o autor, essa reconstrução histórica produz um novo sentido e significação da história de modo a possibilitar os grupos perceberem futuro no seu passado. Assim, as comunidades, enquanto oriundas de um processo de materia- lização e reunião de um passado, constituem verdadeiras fontes documentais da história do local. Para Diehl (2002, p. 116), a memória “constitui-se de um saber, formando tradições, caminhos – como canais de comunicação entre di- mensões temporais –, ao invés de rastros e restos como no caso da lembrança”. Em continuidade, o autor afirma que a formalização do passado deva cumprir também o papel de identificação cultural dos indivíduos já que “o ato de rememorar produz sentido e significação através da ressubjetivação do sujeito e a repoetização do passado, produzindo uma nova estética do passado” (p. 114). Assim, a memória torna-se fator de identidade e valorização da cultu- ra dos mesmos. Conforme Andrade (2008, p. 570) estes lugares de memória “são verdadeiros patrimônios culturais, projetados simbolicamente e podem estar atrelados a um passado vivo que ainda marca presença e reforça os traços identitários do lugar". Além de ser arquivos da memória coletiva de um povo, os lugares tam- bém representam a memória coletiva de uma comunidade que pode ser reco- nhecida também em objetos, festas, músicas, danças, práticas alternativas de medicina, técnicas, culinária e diversas outras representações que estão tomadas de significação das diversas formas de viver que constituem as culturas dos povos. Referindo-se sobre este patrimônio, apesar de não ser como afirma Fonseca (2005), feitos de “pedra e cal” também são memórias que podem interligar diálogos entre as gerações. O objeto, afirma Debary (2010, pp. 38-39), “guarda consigo uma história que é retomada com ele” e é um teste- munho que funciona “como um transmissor de história” que é reativado com as lembranças que ele traz ao indivíduo ou sociedade. | 131 Diálogos | Volume 05 | 2020 Educação patrimonial como base de preservação O poder se vê por toda parte e é necessário saber descobri-lo onde ele se deixa ver menos, onde ele é completamente ignorado, portanto, reconhecido: o poder simbólico é, com efeito, esse poder invisível o qual só pode ser exercido com a cumplicidade daqueles que querem saber que lhe estão sujeitos ou mesmo que o exercem. (Bourdieu, 1989, pp. 7-8). O poder se vê por toda parte e é necessário saber descobri-lo onde ele se deixa ver menos, onde ele é completamente ignorado, portanto, reconhecido: o poder simbólico é, com efeito, esse poder invisível o qual só pode ser exercido com a cumplicidade daqueles que querem saber que lhe estão sujeitos ou mesmo que o exercem. (Bourdieu, 1989, pp. 7-8). Para Langer um monumento “não é simplesmente o que restou do tem- po, é o que deve permanecer na História” (1997, p. 121) porque nele a sociedade pode reconhecer-se nele e os poderes nele constituídos, revelados. Aliado ao conceito de patrimônio histórico está o de patrimônio cultural. Segundo o artigo 216.º da Constituição brasileira, o patrimônio cultural repre- senta os bens: “(...) de natureza material e imaterial, tomados individualmente ou em conjunto, portadores de referência à identidade, à ação, à memória dos diferentes grupos formadores da sociedade brasileira”. Canani (2005, p. 3) afirma que o termo patrimônio “pode ser entendido como um conjunto de bens, materiais ou não, direitos, ações, posse e tudo o mais que pertença a uma pessoa e seja suscetível de apreciação econômica”. Desse modo, o termo patrimônio cultural está relacionado a um bem paternal, de tamanha valia que justifica sua herança. O roteiro histórico da construção do conceito Patrimônio Cultural no Brasil está vinculado ao pertencimento do patrimônio como um bem. O decreto-lei que Getúlio Vargas assinou em 1937 utiliza este vocábulo. O decreto-lei nº 25, de 30 de novembro de 1937, além de criar o Serviço do Patrimônio Histórico e Artístico Nacional (SPHAN), definiu que patrimônio é o “conjunto de bens móveis e imóveis de interesse público” que possuam “excepcional valor arqueo- lógico ou etnográfico, bibliográfico ou artístico”. No Brasil, este patrimônio histórico é gerido pelo Instituto do Patrimônio Histórico e Artístico Nacional (Iphan), o qual está atualmente vinculado ao Ministério do Turismo, sendo anteriormente vinculado ao extinto Ministério da Cultura que fora transformado em Secretaria da Cultura pela Lei 8.028 de 12 de abril de 19902. 2 Em 2019, a estrutura da Cultura foi incluída no Ministério da Cidadania por meio do Decreto Nº 9.674, de 2 de janeiro. O Ministério da Cidadania ainda incorporou as pastas de Desenvolvimento Social, Esporte, e parte da Secretaria Nacional de Políticas sobre Drogas. Disponível em http://cultura.gov.br/acesso-a-informacao/institucional/historico/ Acesso 19 maio. 2020. Douglas Lima da Costa 12) aplica a expressão poder simbólico como sen- do aquele que é capaz de construir uma realidade. O autor acredita que “o campo da produção simbólica é um microcosmos da luta simbólica entre as classes” e que ele é uma forma transformada, irreconhecível, transfigurada e legitimada, das outras formas de poder. E mais: Diálogos | Volume 05 | 2020 132 | Educação patrimonial como base de preservação Ele foi criado em 1937 pela Lei n.º 378 no governo de 2 Em 2019, a estrutura da Cultura foi incluída no Ministério da Cidadania por meio do Decreto Nº 9.674, de 2 de janeiro. O Ministério da Cidadania ainda incorporou as pastas de Desenvolvimento Social, Esporte, e parte da Secretaria Nacional de Políticas sobre Drogas. Disponível em http://cultura.gov.br/acesso-a-informacao/institucional/historico/ Acesso 19 maio. 2020. Diálogos | Volume 05 | 2020 | 133 Douglas Lima da Costa Douglas Lima da Costa Getúlio Vargas. Ribeiro (2005, p. 52) afirma que, no Brasil “a atribuição de valores aos bens segue a tradição européia, em que os patrimônios nacionais são constituídos a partir das categorias de história da arte”. E acrescenta: “na prática, as ações são direcionadas para a proteção da cultura da elite”. Getúlio Vargas. Ribeiro (2005, p. 52) afirma que, no Brasil “a atribuição de valores aos bens segue a tradição européia, em que os patrimônios nacionais são constituídos a partir das categorias de história da arte”. E acrescenta: “na prática, as ações são direcionadas para a proteção da cultura da elite”. Nesse sentido, com relação à preservação, quando os bens são tomba- dos pelo órgão responsável, significa que possuem estimado valor histórico e cultural. Essa intervenção tem como objetivo preservar o patrimônio, uma vez que depois do tombamento eles não podem ser demolidos ou reformados. Abaixo, segue imagem da Praça São Francisco que é uma praça localizada no centro histórico de São Cristóvão, no estado de Sergipe. A Praça foi fundada junto com a cidade em 1607, e seu entorno possui edificações construídas entre os séculos XVII e XIX. Foi protegida em nível estadual e nacional e designada Patrimônio da Humanidade em 1º de agosto de 2010 pela Organização das Nações Unidas para a Educação, a Ciência e a Cultura – UNESCO. Todo mês de agosto são feitas manifestações para que a população possa se sentir inserida nesse complexo patrimonial e sinta o interesse de protegê-lo como algo de sua identidade. Normalmente a praça é palco de várias manifestações artísticas e culturais como uma forma de apropriação do local. Figura 2: Praça São Francisco, São Cristovão - Sergipe Fonte: < ht t p://e x pre s s aoserg ipa n a .c om.br/10 -ben s- de-serg i- pe-que-orgulham-o-povo-sergipano/>. Acessado em: 20 jul. 2018. Figura 2: Praça São Francisco, São Cristovão - Sergipe Fonte: < ht t p://e x pre s s aoserg ipa n a .c om.br/10 -ben s- de-serg i- pe-que-orgulham-o-povo-sergipano/>. Acessado em: 20 jul. 2018. Diálogos | Volume 05 | 2020 134 | 134 | Educação patrimonial como base de preservação Educação patrimonial como base de preservação Não obstante, o compromisso de preservação não diz respeito apenas ao ente público, Lemos (1981, p. 29) em seu livro sobre “O que é Patrimônio Histórico” sugere que preservemos para “garantir a compreensão de nossa memória social preservando o que for significativo dentro de nosso vasto repertório de elementos componentes do Patrimônio Cultural”. E acrescenta Assim, preservar não é só guardar uma coisa, um objeto, uma construção, um miolo histórico de uma grande cidade velha. Preservar também é gravar depoimentos, sons, músicas populares e eruditas. Preservar é manter vivos, mesmo que alterados, usos e costumes populares. È fazer, também, levantamentos, levantamentos de qualquer natureza, de sítios variados, de cidades, de bairros, de quarteirões significativos dentro do contexto urbano. É fazer levantamentos de construções, especialmente aquelas sabidamente da especulação imobiliária. (Lemos, 1981, p. 29). Portanto, uma vez ampliada à concepção de patrimônio através das possibilidades de preservação a partir da nova Constituição de 1988 resta o desafio de estabelecer uma nova política e consciência de como preservar, um caminho apontado é a participação da comunidade na tomada de decisões com relação à preservação do patrimônio cultural, sensibilizando-as inicialmente a fim do inventário participativo, conforme orientado pela cartilha do IPHAN, esse trabalho possibilita uma identificação, reconhecimento e autovalorização dos cidadãos como artífices da identidade social. Assim, a construção de uma identidade, ligada ao patrimônio cultural necessita de um importante cuidado “ao modo como esse objeto tem sido construído e ideologicamente elaborado por determinados sujeitos sociais, que têm tido, no Brasil, o monopólio dessa construção” (Fonseca, 2005, p. 28). Fonseca ainda considera o valor do patrimônio nacional, mas tem se tornado pesado e mudo: Pesado, não só por sua monumentalidade, pela solidez dos materiais e pelo lugar que ocupa no espaço público. Pesado porque mudo, na medida em que, ao funcionar apenas como símbolo abstrato e distante da nacionalidade, em que um grupo muito reduzido se reconhece, e referido a valores estranhos ao imaginário da grande maioria da população brasileira, o ônus de sua proteção e conservação acaba sendo considerado como um fardo por mentes mais pragmáticas. (Fonseca, 2005, pp. 26-27). Pesado, não só por sua monumentalidade, pela solidez dos materiais e pelo lugar que ocupa no espaço público. Educação patrimonial como base de preservação Pesado porque mudo, na medida em que, ao funcionar apenas como símbolo abstrato e distante da nacionalidade, em que um grupo muito reduzido se reconhece, e referido a valores estranhos ao imaginário da grande maioria da população brasileira, o ônus de sua proteção e conservação acaba sendo considerado como um fardo por mentes mais pragmáticas. (Fonseca, 2005, pp. 26-27). | 135 Diálogos | Volume 05 | 2020 Diálogos | Volume 05 | 2020 Douglas Lima da Costa Douglas Lima da Costa Neste contexto surge a Educação Patrimonial como um instrumento pedagógico eficaz e capaz de efetivar a identificação, a valorização e a preser- vação do Patrimônio Histórico e Cultural dos lugares de memória. Assunção (2003, p. 55) entende que “o patrimônio cultural constitui uma herança histórica, deixada pelas gerações anteriores, que cabe a todos preservar para que seja transmitida às gerações vindouras”. A autora acredita que por aproximar o indivíduo com o patrimônio, a Educação Patrimonial Contribui para a formação de um cidadão consciente dos seus direitos e deveres, que compreenderá a importância da preservação dos bens culturais para a preservação da memória e da identidade de um povo ou nação e da necessidade da ação de proteger e escolher seus bens patrimoniais. (Assunção, 2003, p. 51). Segundo Horta (1999) a proposta de Educação Patrimonial surgiu efe- tivamente e foi organizada em termos conceituais e práticos no Brasil, a partir de 1983, quando no Museu Imperial de Petrópolis, RJ, ocorreu o I Seminário de Educação Patrimonial. Para a autora, a Educação Patrimonial, além de ser uma meta da educação contemporânea e dos órgãos responsáveis pela preser- vação patrimonial, torna-se, [...] um instrumento de “alfabetização cultural” que possibilita ao indivíduo fazer a leitura do mundo que o rodeia, levando-o a compreensão do universo sóciocultural e da trajetória histórico- temporal em que está inserido. Este processo leva ao reforço da auto-estima dos indivíduos e comunidades e à valorização da cultura brasileira, compreendida como múltipla e plural. (Horta, 1999, p. 6). Neste viés, o IPHAN elaborou um Guia Básico de Educação Patrimonial, que auxilia todas as unidades do país no desenvolvimento de atividades que contribuem com o trabalho educacional em museus, monumentos e sítios históricos, trabalhando com conceitos ligados ao patrimônio além de sugerir metodologias para o desenvolvimento das mesmas. As bases conceituais e práticas da metodologia da Educação Patrimonial objetiva orientar, em especial os professores e profissionais responsáveis pela preservação da cultura, em como preservar os bens cul- turais e do meio ambiente histórico em que estão inseridos considerando a Educação Patrimonial como “um processo permanente e sistemático de 136 | Diálogos | Volume 05 | 2020 Educação patrimonial como base de preservação Educação patrimonial como base de preservação trabalho educacional” (Horta, 1999, p. 6), a partir da observação direta dos bens culturais. Este é o ponto que dá centralidade a todo o trabalho de Educação Patrimonial sugerido pelo IPHAN. Figura 3: Slogan IPHAN – principal autarq sobre patrimônio no Brasil. Fonte: <http://infon com.br/noticias/livrarias-po rao-vender-livros-do-iphan Acessado em: 20 jul. 20 Figura 3: Slogan do IPHAN – principal autarquia sobre patrimônio no Brasil. Figura 3: Slogan do IPHAN – principal autarquia sobre patrimônio no Brasil. Fonte: <http://infonet. com.br/noticias/livrarias-pode- rao-vender-livros-do-iphan/>. Acessado em: 20 jul. 2018. Fonte: <http://infonet. com.br/noticias/livrarias-pode- rao-vender-livros-do-iphan/>. Acessado em: 20 jul. 2018. A partir da concepção de que todo e qualquer objeto cultural é uma fonte primária de conhecimento, o IPHAN estruturou uma metodologia específica que possibilita a percepção e a compreensão dos fatos e dos fenômenos sociais estudados para que os professores se apropriem dos objetos culturais “na sala de aula ou nos próprios locais onde são encontrados, como peças chave no desenvolvimento dos currículos e não simplesmente como mera ilustração das aulas” (Horta, 1999, p. 9). Assim, o Patrimônio Cultural da comunidade ganha vida através da valorização da história do seu espaço além de provocar nos alunos sentimentos de curiosidade essencial para que adquiram habilidades e conceitos relaciona- dos ao Patrimônio Cultural. Essa aproximação, segundo Assunção (2003, p. 51) desperta quatro atitudes: a observação e a reflexão sobre o bem cultural; a manifestação das impressões sobre o mesmo; a capacidade de pesquisa e discussão sobre os resultados e a apropriação de um novo significado do bem para cada um que participa da proposta. Assunção (2003, p. 51) entende que os projetos de Educação Patrimonial desenvolvidos nas comunidades devam procurar “envolver o indivíduo e a comunidade como agentes ativos, na preservação sustentável e na gestão do patrimônio cultural”. Assim, é indispensável que a comunidade participe de todas as etapas do processo de seu amadurecimento cultural na área da preser- vação de seus bens patrimoniais. Diálogos | Volume 05 | 2020 | 137 Educação patrimonial como base de preservação Portanto, o Patrimônio Cultural deve ser visto como um conjunto de coisas da invenção do homem para o próprio homem sejam elas para facilitar a vivência em grupo e presença nos espaços. É ainda, resultante de construções da coletividade através dos tempos e um determinado espaço, esse vínculo direto com a memória e a construção de uma identidade deve ser tratada como material a ser preservado. Para isso, a educação patrimonial cumpre o papel aproximador e sensibilizador. Em um movimento de continuidade, a preservação e a conservação dependem da participação de todas as pessoas, independente do grupo social e do papel que exerçam. Assim sendo, enxergar-se nos elementos compostos do passado e no passado, gerará no indivíduo o senso de comprometimento em olhar o preté- rito como elo de compreeensão do presente e respeito para com a história e o povo que a constrói. Douglas Lima da Costa Douglas Lima da Costa A utilização da metodologia do Guia Básico de Educação Patrimonial sugerida pelo IPHAN nos programas curriculares do sistema educacional contribue para o diálogo e enriquecimento de dois segmentos que quase nun- ca estão conectados: os órgãos responsáveis pela preservação do patrimônio histórico e cultural e as comunidades que o produziram. O patrimônio cultural tem que ser considerado um instrumento valioso das experiências humanas, mas a participação popular não pode mais ser ignorada pelo poder público. Aos indivíduos que são excluídos desse processo, a educação patrimonial deve ser uma prática de participação social. E essas práticas políticas que envolvem a experiência da sociabilidade, exigem de todos os envolvidos uma compreensão do uso social do espaço de convívio entre os indivíduos que compõem àquela comunidade. (Certeau, 1996, p. 233). Figura 4: Cartilha de Educação Patrimonial Desse modo, seria relevante que a educação patrimonial estivesse presente nos currículos escolares, nas ações edu- cacionais, nos mais variados espaços e de outras instituições e atividades de educação informal, de forma interdis- ciplinar. Fonte: < https://issuu.com/letiz/ docs/educa_o_web>. Acessado em: 20 jul. 2018. Assim, é necessário entender o pa- pel que o Patrimônio Histórico Cultural desempenha no resgate e valorização da identidade de cada indivíduo e da coleti- vidade que constrói num mesmo local a sua história. Este é também um desafio que a escola deveria imprimir. Dessa forma, os bens culturais adquiririam a conotação de bem públi- co, ou seja, que pertence ao povo, e não ao Estado ou a uma restrita parcela da sociedade, e a preservação ganharia um novo olhar da comunidade. Fonte: < https://issuu.com/letiz/ docs/educa_o_web>. Acessado em: 20 jul. 2018. Diálogos | Volume 05 | 2020 138 | Educação patrimonial como base de preservação reFerênciAS Andrade, C. (2008). Lugar de memória... memórias de um lugar: patrimô- nio imaterial de Iguatu, Andaraí, BA. Passos Revista de Turismo y Patrimonio Cultural, v. 6, pp. 569-590. https://doi.org/10.25145/j.pasos.2008.06.042 Assunção, P. (2003). O Patrimônio. Edições Loyola. Bartholo, R. (2005). Turismo e Sustentabilidade no Estado do Rio de Janeiro. Rio de Janeiro: Garamond. Bourdieu, P. (1989). O Poder Simbólico. Editora Bertrand Brasil S.A. Brasil. (1937). Decreto-Lei nº 25, de 30 de novembro de 1937. Organiza a proteção do patrimônio histórico e artístico nacional. Disponível em: http://www.planalto.gov.br/ccivil_03/Decreto-Lei/Del0025.htm. Acesso em: 23 jul. 2018. Brasil. (1988). Constituição da República Federativa do Brasil de 1988. Disponível em: http://www.planalto.gov.br/ccivil_03/Constituicao/Constituicao.htm Acesso em: 23 jul. 2018. Brasil. (1990). Lei nº 8.028 de 12 de abril de 1990. Dispõe sobre a organiza- Diálogos | Volume 05 | 2020 | 139 Douglas Lima da Costa ção da Presidência da República e dos Ministérios, e dá outras providências. Disponível: http://www.planalto.gov.br/ccivil_03/leis/l8028.htm Acesso em: 19 maio. 2020. ção da Presidência da República e dos Ministérios, e dá outras providências. Disponível: http://www.planalto.gov.br/ccivil_03/leis/l8028.htm Acesso em: 19 maio. 2020. Brasil. (2019). Decreto nº 9.674, de  2 de janeiro de 2019. Disponível: http://www.planalto.gov.br/ccivil_03/_ato2019-2022/2019/decreto/D9674.htm Acesso em: 17 jun. 2020. Canani, A. S. K. B. (2005). Herança, sacralidade e poder: sobre as diferentes categorias do patrimônio histórico e cultural no Brasil. Horiz. antropol. [online], vol. 11, n. 23, pp. 163-175. https://doi.org/10.1590/S0104-71832005000100009 Acesso em: 26 jul. 2011. Certeau. M. (1996). A invenção do Cotidiano 2 (morar, cozinhar). Rio de Janeiro: Editora Vozes. Chartier, R. (1991). O mundo como representação. Estudos Avançados [on- line] vol. 5, n. 11, pp. 173-191. Disponível em: https://doi.org/10.1590/S0103-40141991000100010 Acesso em: 30 jul. 2011. Debary, O. (2010). Segunda mão e segunda vida: objetos, lembranças e foto- grafias. Revista Memória em Rede, Pelotas, v. 2, n. 3, ago.-nov. 2010. Disponível em: www. ufpel.edu.br Acesso em: 21 jul. 2011. Diehl, A. (2002). Teorias da História. Cultura historiográfica (memória, iden- tidade e representação). Bauru: EDUSC. Fonseca, M. C. L. (2005). O patrimônio em processo. Rio de Janeiro: UFRJ / Minc - IPHAN. Gastal, S. (2006). Alegorias urbanas: o passado como subterfúgio tempo, espaço e visualidade na pós-modernidade. Papirus Editora. Disponível em: http://books. google.es Acesso em: 03 jun. 2011. Horta, M. L. P. (1999). Guia Básico de Educação Patrimonial. – Brasília: Instituto do Patrimônio Histórico e Artístico Nacional, Museu Imperial. Langer, J. (1997). Mitos arqueológicos e poder. Clio – Série Arqueológica (UFPE), Recife, v. 1, n. 12, pp. 109-125. Lemos, C. Ribeiro, S. B. (2005). Memória, cidadania e gestão do patrimônio cultural. São Paulo: Annablume. Silveira, F. L. A., & Lima Filho M. F. (2005). Por uma antropologia do objeto documental: entre a “alma nas coisas” e a coisificação do objeto. Horizontes Antropológicos. Porto Alegre, ano 11, n. 23, jan/jun, pp. 37-50. https://doi.org/10.1590/S0104-71832005000100003 reFerênciAS A. C. (1981). O que é patrimônio histórico. São Paulo: Editora Brasiliense S.A. Diálogos | Volume 05 | 2020 140 | Educação patrimonial como base de preservação Diálogos | Volume 05 | 2020 | 141 | 141 Direitos Autorais (c) 2020 Douglas Lima da Costa Resumodalicença Texto completo dalicença Este texto está protegido por uma licença Creative Commons Você tem o direito de Compartilhar - copiar e redistribuir o material em qualquer suporte ou formato - e Adaptar o documento - remixar, transformar, e criar a partir do material - para qualquer fim, mesmo que comercial, desde que cumpra a condição de: Atribuição: Você deve atribuir o devido crédito, fornecer um link para a licença, e indicar se foram feitas alterações. Você pode fazê-lo de qualquer forma razoável, mas não de uma forma que sugira que o licenciante o apoia ou aprova o seu uso. Resumodalicença Texto completo dalicença
https://openalex.org/W4236472041
https://revistadaanpoll.emnuvens.com.br/revista/article/download/610/621
Portuguese
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DADOS TÉCNICOS E EDITORIAIS
Revista da ANPOLL
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cc-by
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n. 32, p. 1-208, Brasília-DF, Jan./ Jul. 2012 ISSN 1414-7564 Revista da 32 Linguística: linguagem, história e acontecimento Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística Associaçao Nacional de Pós-Graduação e Pesquisa em Letras e Linguística n. 32, p. 1-208, Brasília-DF, Jan./ Jul. 2012 ISSN 1414-7564 Revista da Reitor: Roberto de Souza Salles Vice-Reitor: Sidney Luiz de Matos Mello Universidade Federal Fluminense Reitor: Roberto de Souza Salles Vice-Reitor: Sidney Luiz de Matos Mello Comissão Editorial Ida Maria Santos Ferreira Alves (UFF - 2010/12) Mariangela Rios de Oliveira (UFF - 2010/12) Maria Antonieta Jordão de Oliveira Borba (UERJ - 2010/12) Lucia Teixeira (UFF - 2010/12) Audemaro Goulart (PUC/MG - 2008/10) Fábio Alves (UFMG - 2008/10) André Luís Gomes (UnB - 2006/08) Maria Cristina Faria Dalacorte (UFG - 2006/08) Conselho Editorial Almuth Grésillon (ITEM - França) Aryon Dall’ Igna Rodrigues (UnB) Carlos Reis (Univ. de Coimbra - Portugal) Diana Luz Pessoa de Barros (MACKENZIE - USP) Edna Maria Fernandes do Nascimento (UNIFRAN) Eduardo Guimarães (UNICAMP) Eni Pulcinelli Orlandi (UNICAMP - Univ. do Vale do Sapucaí) Fernando Cristóvão (Univ. de Lisboa - Portugal) Freda Indursky (UFRGS) Francisco da Silva Borba (UNESP - CNPq) Francisco Gomes de Matos (UFPE) Jacqueline-Authier-Revuz (Univ. Paris II/CNRS - França) John Gledson (Univ. de Liverpool - Inglaterra) Laura Padilha (UFF) Leci Barbisan (PURS) Margarida T. Petter (USP) Maria Célia Leonel (UNESP - Araraquara) Milton Azevedo (Univ. de Berkeley - EUA) Philippe Léon M. G. Willemar (NAP/USP - SP) Pierre Rivas (Univ. de Paris 10 - França) Sylvain Aurox (CNRS - França) Teresa Cristina Cerdeira da Silva (UFRJ) Conselho da ANPOLL Estudos Literários: Titulares: José Luiz Jobim de Salles Fonseca (UERJ - 2010/14), Eurídice Figueiredo (UFF - 2010/14), Rogério da Silva Lima (UnB - 2008/12), Ivete Lara Camargo Walty (PUC/MG - 2008/12) Suplentes: Luiz Roberto Velloso Cairo (UFRJ - 2010/14), Antônia Torreão Herrero (UFBA - 2010/14), Maria Zaira Turchi (UFG - 2008/12), Lúcia Rebello (UFRGS - 2008/12) Endereço Editorial: Universidade Federal Fluminense; Instituto de Letras - Bloco C; Campus Gragoatá - São Domingos Niterói - RJ; CEP 24.210-200. Proibida a reprodução parcial ou integral desta obra, por qualquer meio eletrônico ou mecânico, inclusive por processo xerográfico, ou sem permissão do editor (Lei n. 9.610 de 19/2/1998). Os conceitos emitidos em artigos assinados são de absoluta e exclusiva responsabilidade de seus autores. Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística CIP-BRASIL. CATALOGAÇÃO-NA-FONTE SINDICATO NACIONAL DOS EDITORES DE LIVROS, RJ CIP-BRASIL. CATALOGAÇÃO-NA-FONTE SINDICATO NACIONAL DOS EDITORES DE LIVROS, RJ R349 Revista da ANPOLL / Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística. - 1(1994)- . - - Brasília, DF : ANPOLL, 1994-. v. : Semestral Descrição baseada no: v. 18, N.32 (2012) Tema especial: Linguística: linguagem, história e acontecimento Organizador: André Luís Gomes ISSN 1414-7564 1. Linguística. 2. Linguagem e línguas. 3. Linguística histórica. 4. Linguística - Periódicos. I. Gomes, André Luís, 1963-. II. Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística (Brasil). III. Título: Linguística: linguagem, história e acontecimento. 12-3743. CDD: 410 CDU: 81’1 06.06.12 15.06.12 036125 R349 Revista da ANPOLL / Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística. - 1(1994)- . -. - Brasília, DF : ANPOLL, 1994-. v. : Semestral Descrição baseada no: v. 18, N.32 (2012) Tema especial: Linguística: linguagem, história e acontecimento Organizador: André Luís Gomes ISSN 1414-7564 Descrição baseada no: v. 18, N.32 (2012) Tema especial: Linguística: linguagem, história e acontecimento Organizador: André Luís Gomes ISSN 1414-7564 1. Linguística. 2. Linguagem e línguas. 3. Linguística histórica. 4. Linguística - Periódicos. I. Gomes, André Luís, 1963-. II. Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística (Brasil). III. Título: Linguística: linguagem, história e acontecimento. 12-3743. CDD: 410 CDU: 81’1 06.06.12 15.06.12 036125 Organização deste número: Mariangela Rios de Oliveira e André Luís Gomes Pareceristas deste número: Beatriz Feres dos Santos: beatrizferes@yahoo.com.br, Bethania Sampaio Mariani: bmariani@uol.com.br, Décio Rocha: rochadm@uol.com.br, Edila Vianna: edila@openlink.com.br, Eduardo Kenedy: edu.kenedy@gmail.com, Fernando Afonso de Almeida: fealfal@gmail.com, Fernando Muniz: fernandomuniz@id.uff.br, Jussara Abraçado: almeidajussara@hotmail.com, Lívia Lindóia Paes Barreto: liviapaesbarreto@yahoo.com.br, Lygia Gonçalves Trouche: lymt@terra.com.br, Maria Del Carmen Daher: deldaher@terra.com.br, Maria Elizabeth Chaves de Mello: bethcmello@gmail.com, Maurício Beck: pardalbeck@gmail.com, Mônica Savedra: msavedra@faperj.br, Patrícia Ferreira Ribeiro: patleitura@gmail.com, Paulo Antonio Pinheiro Correa: papicorrea@gmail.com, Renata Mancini: renata.mancini@gmail.com, Ricardo Stavola Cavaliere: cavaliere@oi.com.br, Rosane Santos Mauro Monnerat: rosanemmonnerat@gmail.com, Silmara Cristina Dela da Silva: silmaradela@gmail.com, Silvia Maria de Sousa: smsousa2002@yahoo.com.br, Solange Coelho Vereza: svereza@uol.com.br, Vanda Maria Cardozo de Menezes: vcmenezes@terra.com.br, Victoria Wilson: victoriawilson@superig.com.br, Xoán Lagares Diez: xlagares@gmail.com Projeto gráfico e capa: Grupodesign Revisão: Ana Cristina Vilela Produção gráfica: Sermograf Todos os direitos reservados Universidade Federal Fluminense Instituto de Letras Campus Gragoatá - São Domingos - Niterói - RJ CEP: 24.210-200 Fone: +55 (21) 2629-2601 egl@vm.uff.br Organização deste número: Mariangela Rios de Oliveira e André Luís Gomes Pareceristas deste número: Beatriz Feres dos Santos: beatrizferes@yahoo.com.br, Bethania Sampaio Mariani: bmariani@uol.com.br, Décio Rocha: rochadm@uol.com.br, Edila Vianna: edila@openlink.com.br, Eduardo Kenedy: edu.kenedy@gmail.com, Fernando Afonso de Almeida: fealfal@gmail.com, Fernando Muniz: fernandomuniz@id.uff.br, Jussara Abraçado: almeidajussara@hotmail.com, Lívia Lindóia Paes Barreto: liviapaesbarreto@yahoo.com.br, Lygia Gonçalves Trouche: lymt@terra.com.br, Maria Del Carmen Daher: deldaher@terra.com.br, Maria Elizabeth Chaves de Mello: bethcmello@gmail.com, Maurício Beck: pardalbeck@gmail.com, Mônica Savedra: msavedra@faperj.br, Patrícia Ferreira Ribeiro: patleitura@gmail.com, Paulo Antonio Pinheiro Correa: papicorrea@gmail.com, Renata Mancini: renata.mancini@gmail.com, Ricardo Stavola Cavaliere: cavaliere@oi.com.br, Rosane Santos Mauro Monnerat: rosanemmonnerat@gmail.com, Silmara Cristina Dela da Silva: silmaradela@gmail.com, Silvia Maria de Sousa: smsousa2002@yahoo.com.br, Solange Coelho Vereza: svereza@uol.com.br, Vanda Maria Cardozo de Menezes: vcmenezes@terra.com.br, Victoria Wilson: victoriawilson@superig.com.br, Xoán Lagares Diez: xlagares@gmail.com Projeto gráfico e capa: Grupodesign Revisão: Ana Cristina Vilela Produção gráfica: Sermograf Todos os direitos reservados Universidade Federal Fluminense Instituto de Letras Campus Gragoatá - São Domingos - Niterói - RJ CEP: 24.210-200 Fone: +55 (21) 2629-2601 egl@vm.uff.br
https://openalex.org/W3129646928
https://link.springer.com/content/pdf/10.1007/s00265-021-02996-4.pdf
English
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Individual behavioural traits not social context affects learning about novel objects in archerfish
Behavioral ecology and sociobiology
2,021
cc-by
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https://doi.org/10.1007/s00265-021-02996-4 Behavioral Ecology and Sociobiology (2021) 75: 58 https://doi.org/10.1007/s00265-021-02996-4 Behavioral Ecology and Sociobiology (2021) 75: 58 ORIGINAL ARTICLE Abstract Learning can enable rapid behavioural responses to changing conditions but can depend on the social context and behavioural phenotype of the individual. Learning rates have been linked to consistent individual differences in behavioural traits, especially in situations which require engaging with novelty, but the social environment can also play an important role. The presence of others can modulate the effects of individual behavioural traits and afford access to social information that can reduce the need for ‘risky’ asocial learning. Most studies of social effects on learning are focused on more social species; however, such factors can be important even for less-social animals, including non-grouping or facultatively social species which may still derive benefit from social conditions. Using archerfish, Toxotes chatareus, which exhibit high levels of intra-specific competition and do not show a strong preference for grouping, we explored the effect of social contexts on learning. Individually housed fish were assayed in an ‘open-field’ test and then trained to criterion in a task where fish learnt to shoot a novel cue for a food reward—with a conspecific neighbour visible either during training, outside of training or never (full, partial or no visible presence). Time to learn to shoot the novel cue differed across individuals but not across social context. This suggests that social context does not have a strong effect on learning in this non-obligatory social species; instead, it further highlights the importance that inter- individual variation in behavioural traits can have on learning. Significance statement Some individuals learn faster than others. Many factors can affect an animal’s learning rate—for example, its behav- ioural phenotype may make it more or less likely to engage with novel objects. The social environment can play a big role too—affecting learning directly and modifying the effects of an individual’s traits. Effects of social context on learning mostly come from highly social species, but recent research has focused on less-social animals. Archerfish display high intra-specific competition, and our study suggests that social context has no strong effect on their learning to shoot novel objects for rewards. Our results may have some relevance for social enrichment and welfare of this increasingly studied species, suggesting there are no negative effects of short- to medium-term isolation of this species— at least with regards to behavioural performance and learning tasks. Keywords Social context . Inter-individual differences . Social learning . Toxotes . Exploratory tendency . Learning ndividual behavioural traits not social context affects learning about novel objects in archerfish Nick A. R. Jones1 & Helen C. Spence-Jones1 & Mike Webster1 & Luke Rendell1 Received: 25 November 2020 /Revised: 9 February 2021 /Accepted: 12 February 2021 # The Author(s) 2021 / Published online: 22 February 20 1 Centre for Social Learning and Cognitive Evolution, School of Biology, University of St Andrews, Sir Harold Mitchell Building, St Andrews, Fife KY16 9TH, UK * Nick A. R. Jones narj@st-andrews.ac.uk Introduction 2016; Pearish et al. 2019; Tucker and Suski 2019); however, the importance of social context may depend on the species. Studies exploring the effects of social context on learning have focused on relatively few highly social species, and there is current debate about the evolution of social learning on social and non-social species (Heyes 2012; Reader 2016). We were interested in exploring whether social context would facilitate learning in a species not generally considered social but capable of receiving social information, and whether this could overcome pre-existing variation in relevant traits such as exploratory tendency. Specifically, we tested the effect of different social context on the rate of learning an association between shooting novel objects and obtaining a food reward in archerfish, Toxotes chatareus. Famous for their ability to ‘shoot’ down prey (Bekoff and Dorr 1976; Dill 1977; Schuster 2007), archerfish exhibit low sociality with no grouping pref- erence (Timmermans and Maris 2000), possibly linked to their highly competitive foraging behaviour (Rischawy et al. 2015) and tendency for intra-specific kleptoparasitism (Davis and Dill 2012). Previous work has suggested that they use social information (Schuster et al. 2006) and alter their shooting behaviour in response to a conspecific in a neighbouring tank (Jones et al. 2018). However, little else is known about their social behaviour and social effects on learning. Most research has focused on understanding the mechanisms underlying their shooting abilities (Timmermans and Vossen 2000; Gerullis and Schuster 2014; Reinel and Schuster 2018) and related abilities (Ben-Tov et al. 2018; Newport and Schuster 2020) including image search (Temple et al. 2010; Rischawy and Schuster 2013; Ben-Tov et al. 2015) and associated neu- robiology (Schlegel and Schuster 2008; Ben-Simon et al. 2012; Machnik et al. 2018a, b). Archerfish have been shown to exhibit consistent individual differences in latency to shoot—with consequences for their performance in learning and discrimination trials (Jones et al. 2020): fish with low latencies were quicker to learn to shoot targets for reward. The effects of these individual differences have only been explored in fish in solitary conditions, and no social element has been tested. Our main question in this study was whether archerfish learn to shoot a novel object faster when housed in solitary conditions or with a visible conspecific. We included a variation of the open-field assay to determine whether indi- vidual variation in learning rates was also associated with individual differences in exploratory tendency. Introduction Communicated by I. Hamilton Learning enables rapid responses to change (Shettleworth 1972, 2009), but in order to learn about a novel object, an animal must first engage with it. Exploring a potential new food source can provide obvious foraging benefits but may expose an individual to potential risk from predation or nox- ious substances (Lima and Dill 1990). The social environment * Nick A. R. Jones narj@st-andrews.ac.uk 58 Page 2 of 11 Behav Ecol Sociobiol (2021) 75: 58 can play a large role in an individual’s motivation to engage with novelty and subsequent learning opportunities (Boogert et al. 2006; Mirville et al. 2016; Langley et al. 2018; Kent et al. 2019; Hansen et al. 2020). Individuals can be more likely to approach novel foods with conspecifics present (Gómez- Laplaza and Morgan 1986; Sherwin 2003). For example, ca- puchin monkeys (Cebus apella) in social conditions had higher levels of engagement with and consumption of novel foods than solitary individuals (Visalberghi and Fragaszy 1995). Being in a group also affords learning opportunities through social learning mechanisms (Coussi-Korbel and Fragaszy 1995; Hoppitt and Laland 2013; Ward and Webster 2016) such as social facilitation (Zentall and Hogan 1976; Dindo et al. 2009). The social environment can also enhance learning effects through the ‘social buffering of stress’ (Smith et al. 1998; DeVries et al. 2003; Allen et al. 2009; Crane et al. 2018; Culbert et al. 2019), for example cichlids (Cichlasoma paranaense) in groups showed lower stress and faster learning rates than isolated individuals (Brandão et al. 2015). These social benefits are not limited to obligate social species. The solitary octopus (Octopus vulgaris) (Fiorito and Scotto 1992) and several non- grouping species of fish (Webster and Laland 2017), includ- ing solitary sharks (Vila Pouca and Brown 2019; Vila Pouca et al. 2020), use social learning. However, the effects of the social environment on learning can be complex. Individuals exhibiting specific ‘coping styles’ (Koolhaas et al. 1999) can perform differently in cognition studies (Zidar et al. 2017; Mazza et al. 2019). Several studies have shown that ‘proactive’ individuals, with low cortisol levels and high levels of exploratory activity, learn faster than ‘reactive’ individuals which tend to take longer to approach novel objects (Mesquita et al. 2015; Bensky et al. 2017; Raoult et al. 2017). These individual differences in behavioural traits can be moderated by social environment (Webster and Ward 2011; Jolles et al. Subjects We tested 19 archerfish, T. chatareus, 12 ± 0.5 cm (total length), from animals housed at the University of St. Andrews. All fish were wild-caught as juveniles from a fresh- water population, sourced from an accredited ornamental fish supplier. Fish were of unknown sex as archerfish are sexually monomorphic. The fish had been kept in their stock tanks for a minimum of 6 months prior to use in the study and had not been trained to shoot or exposed to experimental tanks in that time. Stock tanks (180 × 45 cm and 35 cm deep) held at temperatures between 25 and 26°C under a 12:12 h light:dark cycle, had environmental enrichment in the form of 3-cm- deep gravel substrate and several plastic plants. Water quality parameters (pH, nitrite, ammonia and nitrate concentrations) were measured weekly, and levels were kept within a range appropriate for archerfish (Newport et al. 2013; Jones et al. 2018), using external canister filters and regular water changes. Each of four stock tanks held between 5 and 8 fish, with a total of 27 fish. We aimed to test groups of familiar fish so we selected 21 fish for the experiment to ensure all groups of fish came from the same stock tank. At the time of the experiment, the fish were estimated to be 18–24 months old, based on their size and date of arrival from the retailer; however, this is an estimate as the correlation between age and growth rate in captivity is not known for this species. The fish were fed daily with an alternating mixture of commercial fish food (Tetra Cichlid Sticks) and freeze-dried bloodworms. When moving fish into the experimental setup, fish were always captured from the same tank. Fish were measured as they were captured from the stock tank—using a ruler taped to the side of the tank, netted fish were gently pressed against the side of the tank to record their length before being moved to the experimental tank. All the fish that were tested were used only once in this experiment. General procedure Fish were tested in groups of three; each fish in a tested trio came from the same stock tank and had been kept together for at least 6 months prior to the experiment to ensure they were familiar with each other. Each fish was moved from the stock tank and introduced into one of three experimental tanks which had been randomly assigned to one of the social treat- ments. Each fish was then given a full day to acclimate to the experimental tank. After 24 h, the video recordings were taken for the behavioural assay. At the time of introduction and until after the behavioural assay, all barriers were kept in place such that all fish were in the same solitary conditions with no other fish visible to them. Introduction Learning performance can depend on the social dynamics (Nicol and Pope 1999; Soma and Hasegawa 2004; Boogert et al. 2006; Grampp et al. 2019), behavioural traits (An et al. 2011; González-Bernal et al. 2014; Krueger et al. 2014), sex (Snijders et al. 2019) and level of familiarity (Guillette et al. 2016) of the individuals involved. In some species, individ- uals show no benefit from the presence of others when learn- ing, for example the facultatively social tree skinks, Egernia striolata, (Riley et al. 2017, 2018). The presence of conspe- cifics can even worsen learning performance (Zajonc et al. 1969; Webster and Laland 2012; Fei et al. 2019). Notwithstanding social factors, individual variation can also have significant effects on learning. Many of the commonly measured behavioural traits and syndromes relate to explorato- ry tendency and associated neophobia (aversion to novelty (Greenberg and Mettke-Hofmann 2001; Mettke-Hofmann 2017)), and these traits can affect learning, as well as cognitive performance more generally (Sih and Del Giudice 2012; Greggor et al. 2015; Morand-Ferron et al. 2016; Boogert et al. 2018; Dougherty and Guillette 2018). Several studies have shown that individuals which exhibit consistently high levels of exploratory tendency tend to show lower levels of neophobia and engage with/learn-to-associate cues with food rewards more quickly than individuals with low activity levels (An et al. 2011; Guenther et al. 2014; Guillette et al. 2015; Reader 2015; Mazza et al. 2018). Differences in response to stress and physiology may drive variation in learning. Behav Ecol Sociobiol (2021) 75: 58 Page 3 of 11 58 Page 3 of 11 58 Experimental setup Experimental tanks (55 × 55 cm and 45 cm deep) were each set up with an immersion heater (kept at 24.5 ± 0.5°C), a small internal filter (Eheim Biopower 200 Internal Filter), a 1-cm- deep gravel bottom, and plastic plants (positioned to provide structure and refuge but allow a clear view of neighbouring tanks). We used two replicate setups of three tanks each to run up two groups of fish at a time. In each setup, three tanks were set up side by side with a 0.5-cm gap between them, using the same setup as in Jones et al. (2018) (see Fig. 1). A 3-mm-thick black opaque plastic barrier could be easily inserted between each tank to block or allow vision between tanks. The barriers were used to create different social conditions detailed below, by controlling the visibility of the side tanks. Methods Between 46 and 48 h after being moved into experimental tanks, the barriers were re-positioned in accordance with the social treatment assigned to each tank. Fish were then given another period of ~24 h acclimation to the conditions of their respective social treatment before training began. During the training period, the fish were trained to learn to shoot at a novel target to obtain a food reward. Archerfish will readily shoot novel targets and can learn to associate shooting a target with receiving food rewards (Newport et al. 2013; Karoubi et al. 2017). Each fish was given a single training session per day until it reached the training criterion: 10 on-target shots a day for two consecutive days. In each session, an initially novel target (a five-armed star-shaped cut from black plastic, max diameter 2cm) was placed on the glass 15 cm above the water level of the tank. A training session lasted 45 min, or until a fish had shot at the target 10 times. Shots were easily visible as they left water droplets on the underside of the transparent platform on which the target was placed, signalling where the shot had impacted. A shot was consid- ered on-target if it was within 2 cm of the target; successful shots were rewarded with food, and the target was removed and repositioned on the glass for a subsequent trial. We con- tinued training for all fish in each set until all three of the fish had reached the criterion or until the 50th session. Fish were given a maximum of 50 sessions to reach the criterion. Behavioural assay We scored fish in their experimental tanks using a simple ‘open-field’ assay. The ‘open-field test’ exposes animals to a novel area and allows measurements such as amount of activ- ity and time spent in the open as a measure of exploratory behaviour, where animals that spend more time in the open are considered to have greater exploratory behaviour (Burns 2008). This assay is widely used in fishes (Webster et al. 2007; Chapman et al. 2011; Conrad et al. 2011; Lucon-Xiccato et al. 2020). For this assay, each fish was recorded for 10 min, and 58 Page 4 of 11 Behav Ecol Sociobiol (2021) 75: 58 Fig. 1 Experimental set-up, from a top-down view as recorded by the camera above the tanks. The different experimental treatments and level of social context was manipulated by use of the opaque barriers. Solo treatments were always set up in one of the two end tanks Fig. 1 Experimental set-up, from a top-down view as recorded by the camera above the tanks. The different experimental treatments and level of social context was manipulated by use of the opaque barriers. Solo treatments were always set up in one of the two end tanks ‘partial’ treatment, fish had partial social exposure: they were able to see a neighbouring fish when given opportunities to shoot the presented target. However, they could not observe when the neighbour was being trained. Fish in the partial treatment were therefore not exposed to social information about the presented target and associated food reward by other fish, but they may have been impacted by the presence of another fish. This presence could have had either positive impacts on learning rates through social buffering of stress, or, more likely, negative impacts due to the presence of po- tential competitors per (Simon et al. 2011; Jones et al. 2018). Fish in the ‘social’ treatment were exposed to full social con- ditions and had visible contact with a neighbouring fish at all times (including during the neighbour’s training sessions), except when presented with the target itself. A visible neigh- bour fish may act as a potential distraction when shooting, as shown by Jones et al. (2018), so the social condition was designed to afford social information about the target while reducing the chance of distraction. The treatments were pseudo-randomly assigned to each tank prior to transferring fish into them. Behavioural assay For each trio of fish tested, the solo treatment was randomly assigned to one of the two end tanks: the social and partial treatments were then randomly allocated a tank from the two remaining. the proportion of time spent in the open, where the fish was more than 1 body length away from the plant shelter or corners of the tank, was recorded. We used a 24-h acclimation period prior to conducting the assay, informed by our previous work with this species in a similar setup (Jones et al. 2018); while a single measurement per individual is not ideal for a labile trait (Biro and Stamps 2010), suitable acclimation periods can pro- vide more robust estimates of individual behavioural traits (Biro 2012), as demonstrated by high estimates of repeatabil- ity in guppy activity levels following acclimation (O’Neill et al. 2018). To minimize observer bias, blinded methods were used; specifically, a hypothesis naïve scorer was used to score videos of the behavioural assays. We initially planned on testing 21 fish, but the social con- text of two fish (in a solo and partial treatment) may have been compromised as there was a gap in the barrier between the two tanks for at least one session. We noticed this on day 4 of testing this group. These two fish were excluded from the data and were not tested further except to provide a social context for the fish in the social treatment. Of the remaining 19 fish tested, one—in the social treatment—was unable to hit the target or shoot within 2 cm of it. This fish showed no obvious injury or morphological difference from other fish but was unable to reach training criterion for this reason. All other fish were able to shoot and hit the target. Experimental treatments (social context) All analysis was conducted using R base package (R Core Team 2019) and specific packages as detailed below. The opaque barriers were employed to block visible contact between fish in neighbouring tanks. Fish in the ‘solo’ treat- ment were left in isolation, with no visible neighbour. In the In order to determine whether the different treatments affect- ed learning rates, we conducted a survival analysis using a Cox Behav Ecol Sociobiol (2021) 75: 58 Page 5 of 11 58 were more exploratory) were faster learners, taking fewer ses- sions on average to reach the criterion. proportional hazards regression model, as per similar studies exploring time to reach the criterion in learning tasks (Bensky and Bell 2018). We used this approach as six of the 19 fish failed to reach criterion in the given 50 sessions. The numbers of sessions required to reach training criterion were used as the dependent variable. The social treatment (solo, partial, or so- cial) and score from the behavioural assay (the proportion of time spent in the open) were the two independent variables included in the models. We fitted a model using the coxph function in the ‘survival’ package in R (Therneau 2019), with the variables detailed above, where survival equated to failing to reach the learning criterion. We included an interaction be- tween the two independent variables, as we expected that the effect of social treatment may depend on the behavioural traits of individual fish. This analysis allowed us to explore the ef- fects of the independent variables of interest on the probability of reaching learning criterion using all the available data. The link between individual behavioural traits and learning rates is apparent in an increasing number of species (Dougherty and Guillette 2018; Smit and van Oers 2019); our results com- plement earlier findings showing that inter-individual differ- ences in latency to shoot relate to differences in learning in archerfish (Jones et al. 2020). Our inability to detect any effect of social context on learning rates is perhaps more interesting given that archerfish were affected by the presence of others in a previous experiment with similar conditions and setup: visible conspecifics in a neighbouring tank increased the latency to shoot and behaviour of shooting fish (Jones et al. 2018). Results There was no effect of social context on the probability of reaching the learning criterion (Table 1; Fig. 2); however, the probability of an individual reaching criterion did depend on the proportion of time that the individual spent in the open in the open-field assay. Time spent in the open during the initial behavioural assay is significantly associated with time to learn: greater time spent in the open, our measure of explor- atory tendency, is associated with increased learning rates (Table 1; Fig. 3). Experimental treatments (social context) It certainly raises more questions: is there no effect of social con- text on learning, or are these effects more subtle and therefore masked by the individual differences? Our limited sample size prevents us from being able to address this issue. Effects of social environment on learning can vary with behavioural phe- notype where more exploratory individuals tend (relative to less exploratory individuals) to be less sensitive to external stimuli (Guillette et al. 2011; Kelly et al. 2020) and derive less benefit from social environment (Barou Dagues et al. 2020). The lack of significant effect in our study may therefore be a result of exploratory fish that are more likely to engage in risk-taking behaviour but less likely to use social information. Five fish that did not reach training criterion in the allotted time (but could shoot), and it is possible that these individuals may have been more affected by different social contexts. Given the large var- iation between individuals we found, future studies with a great- er sample size will be needed to detect any social effects in less exploratory, potentially more socially sensitive fish, or at least be more confident that there is no social effect on learning to shoot a novel target in this species. Our results, however, do correspond with other studies, across several different species, which have shown that social context does not always have a strong effect on learning performance, at least not when there are strong inter-individual differences in behavioural traits (Seferta et al. 2001; Krueger et al. 2014; Riley et al. 2017, 2018). Discussion Black lines indicate median number of sessions to reach criterion for each treatment; coloured dots represent data points for individual fish. White points indicate fish which failed to reach criterion within 50 sessions had access to social information regarding the novel objects and associated food rewards. Black lines indicate median number of sessions to reach criterion for each treatment; coloured dots represent data points for individual fish. White points indicate fish which failed to reach criterion within 50 sessions Fig. 2 Effect of different social contexts on time taken to reach training criterion. The treatments relate to level of exposure to a visible conspecific: solo (no social exposure); partial (visual social contact except during training of neighbours); social (full visual social contact including during training of neighbours). Only fish with ‘social’ exposure constrain learning (Shettleworth 1972). In addition to showing no preference for shoaling with conspecifics as juveniles (Timmermans and Maris 2000), archerfish are highly compet- itive predators (Rischawy et al. 2015). Intra-specific competi- tion can be a major driver of individual differences in behav- iour, with stronger competition potentially leading to more defined inter-individual differences (Bolnick et al. 2003; Svanbäck and Bolnick 2007; Laskowski and Bell 2013) and archerfish like other predators show strong inter-individual variation in latency to respond to and attack prey (Szopa- Comley et al. 2020a, b). These individual differences may override effects of the social environment, particularly in so- cial species—as shown in threespine sticklebacks (Gasterosteus aculeatus) where, individual differences were suppressed in certain social contexts, but bolder individuals were more likely to feed first in social conditions (McDonald et al. 2016). Our experimental design limited our ability to detect more subtle effects or interactions of this nature. Archerfish do respond to the presence of other fish by increas- ing their latency to shoot (Jones et al. 2018) and exhibit lower shooting accuracy in the presence of others (Simon et al. 2011). This may have impacted training results of fish in the partial social condition, the only condition in which subject fish were exposed to a visible conspecific when given oppor- tunities to shoot. The presence of other fish may have inhibited learning through distraction by the conspecific (as demonstrated in zebrafish Dario rerio; Ayoub et al. 2019) or inhibition from potential conspecific competition. Fig. 3 Relationship between the proportion of time spent in the open and number of sessions taken to reach training criterion. Discussion The social environment did not influence the associative learning rates of the archerfish in this experiment. Instead, learning rates were related to behavioural traits linked to ex- ploratory tendency—specifically, time spent in the open in an open-field test. Fish which spent more time in the open (i.e., Despite the potential benefits of social learning, public in- formation use appears to be species-specific (Webster et al. 2019), and individual biases can have a greater effect on learn- ing than social information even when it is employed (Szabo et al. 2017). Even in species known to use social information, social learning may be dependent upon an individual having some experience with novel foraging opportunities, as recent- ly shown in American crows (Corvus brachyrhynchos) (Pendergraft et al. 2020) and stickleback species (Webster and Laland 2018). While it is likely that archerfish do use social information, given previous findings suggesting social learning in this species (Schuster et al. 2006), many factors may impact the use of social leaning or our ability to detect it. Especially, as both social and asocial learning may depend on the same basic mechanisms (Heyes 2012) and factors that Table 1 Summary output of survival model Coef (β) SE (coef) z Pr(>|z|) Social treatment 2.411 1.735 1.389 0.1648 Solo treatment 1.181 1.422 0.83 0.4063 Open-field score 11.14 5.154 2.162 0.0307 Social: open-field score −10.77 6.292 −1.711 0.0871 Social: open-field score 2.911 4.466 0.652 0.5146 Positive coefficients are associated with shorter time to reach learning criterion. Significant coefficients (p < 0.05) are highlighted in bold. Likelihood ratio test = 16.61 on 5 df, p = 0.005, concordance = 0.724 Table 1 Summary output of survival model Positive coefficients are associated with shorter time to reach learning criterion. Significant coefficients (p < 0.05) are highlighted in bold. Likelihood ratio test = 16.61 on 5 df, p = 0.005, concordance = 0.724 58 Page 6 of 11 Behav Ecol Sociobiol (2021) 75: 58 Fig. 2 Effect of different social contexts on time taken to reach training criterion. The treatments relate to level of exposure to a visible conspecific: solo (no social exposure); partial (visual social contact except during training of neighbours); social (full visual social contact including during training of neighbours). Only fish with ‘social’ exposure had access to social information regarding the novel objects and associated food rewards. Declarations Ethics approval This research was approved by the University of St Andrews Animal Welfare and Ethics Committee (AWEC). No procedures required U.K. Home Office licensing. All tanks were enriched with gravel and plastic plants for cover. All fish were retained in the laboratory after the study period to be used in future projects. Handling was kept to a mini- mum, and when fish had to be moved between tanks, they were caught using two large hand nets to reduce the likelihood of extended capture periods. In the experimental tanks, fish were kept singly in a volume of at least 125 l. Archerfish are not considered a social species. There are no published studies on the effects or preference for any social context and or isolation in this species, but several previous studies have maintained ar- cherfish in isolation with no reported ill effects or perceived likelihood of stress (Gabay et al. 2013; Gerullis and Schuster 2014; Newport et al. 2014, 2018). During our study, we closely monitored each fish, specifically for signs of reduced feeding rate, responsiveness, stereotypic behaviour and colour changes. We observed few instances of these signs: all of the in- stances of short-term colour change occurred immediately post-transfer between tanks and reduced feeding rate for the first 12 h post-transfer. Consent for publication All authors consent to publication of this article. Conflict of interest The authors declare no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 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, pro- vide 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/. Archerfish are not a highly social species but do modify their shooting behaviour in response to the presence of con- specifics. Declarations The lack of variation in learning rates with social context in this study suggests that learning about what to shoot and the decision to take a shot may not be affected equally by social factors. Other non-grouping species may similarly be less likely to show strong effects of social context on learning behaviour, even when their behaviour can be affected by so- cial context in other situations. Discussion Each point represents a single fish that reached criterion (n = 13) and is coloured according to the social treatment they were exposed to. The predicted response and estimated standard error from a linear model are given by the black line and grey shading, respectively Behav Ecol Sociobiol (2021) 75: 58 Page 7 of 11 58 One area where our results may be useful is in informing future studies, particularly with regard to acclimation, train- ing and related ethical and welfare concerns. Archerfish, like other animals perhaps especially other wild caught animals—which may be slower or less likely to engage with novelobjectsthancaptive-bredorurbanindividuals(Gajdon et al. 2004; Jarjour et al. 2020)—can require extended pe- riods to acclimate and train (Archard and Braithwaite 2010; Patton and Braithwaite 2015). Acclimation can play an im- portant role in the expression of behaviours and performance in behavioural assays (O’Neill et al. 2018), and sufficient acclimationmayhelpreducetheissuesinherentinmeasuring cognition in the laboratory (Webster and Rutz 2020). Cognition studies may be particularly susceptible to factors affecting stress and environmental conditions which can af- fect measures (Pritchard et al. 2016), and this is especially important when exploring consistent individual variation (Strand et al. 2010; Rowe and Healy 2014; Griffin et al. 2015; White et al. 2017; Boogert et al. 2018; da Silva et al. 2020). There have been no studies of archerfish welfare, and further work into social and environmental enrichment is required, but our study suggests that keeping archerfish alone may not be detrimental to their welfare in the short term. 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Genetic risk, incident stroke, and the benefits of adhering to a healthy lifestyle: cohort study of 306 473 UK Biobank participants
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7,932
ABSTRACT Objective For numbered affiliations see end of article. In this cohort study, genetic and lifestyle factors were independently associated with incident stroke. These results emphasise the benefit of entire populations adhering to a healthy lifestyle, independent of genetic risk. To evaluate the associations of a polygenic risk score and healthy lifestyle with incident stroke. Introduction Stroke is one of the leading reasons for disability and death worldwide.1 It is a complex disease, caused by both genetic and environmental factors, including diet and lifestyle.2 Results on 3 December 2018 by guest. Protected by copyright. ttp://www.bmj.com/ During a median follow-up of 7.1 years (2 138 443 person years), 2077 incident strokes (1541 ischaemic stroke, 287 intracerebral haemorrhage, and 249 subarachnoid haemorrhage) were ascertained. The risk of incident stroke was 35% higher among those at high genetic risk (top third of polygenic score) compared with those at low genetic risk (bottom third): hazard ratio 1.35 (95% confidence interval 1.21 to 1.50), P=3.9×10−8. Unfavourable lifestyle (0 or 1 healthy lifestyle factors) was associated with a 66% increased risk of stroke compared with a favourable lifestyle (3 or 4 healthy lifestyle factors): 1.66 (1.45 to 1.89), P=1.19×10−13. The association with lifestyle was independent of genetic risk stratums. on 3 December 2018 by guest. Protected by copyright. m/ We investigated whether a weighted genetic risk score based on the genome wide association results for stroke in MEGASTROKE is associated with incident stroke in a large population based cohort (UK Biobank). We also investigated whether adherence to a healthy lifestyle influences this association. What is already known on this topic Stroke is a complex disease caused by both genetic and environmental factors, including diet and lifestyle Whether adhering to a healthy lifestyle could attenuate the effect of genetic background on risk of incident stroke is currently unknown What this study adds Genetic and lifestyle factors were independently associated with risk of incident stroke An unfavourable lifestyle profile was associated with increased risk of stroke across all genetic risk stratums These findings highlight the potential of lifestyle interventions to reduce risk of stroke across entire populations, even in those at high genetic risk of stroke uest. Protected by copyright. RESEARCH RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Genetic risk, incident stroke, and the benefits of adhering to a healthy lifestyle: cohort study of 306 473 UK Biobank ­participants Loes CA Rutten-Jacobs,1,2 Susanna C Larsson,3 Rainer Malik,4 Kristiina Rannikmäe,5,6 Cathie L Sudlow,5,6,7 Martin Dichgans,4,8,9 Hugh S Markus,2 Matthew Traylor2 Conclusion Main outcome measurei Early evidence supporting a role for genetics in risk of stroke came from twin studies and family history studies.3 4 Further evidence has emerged from genome wide association studies. MEGASTROKE, a large meta- analysis of genome wide association studies tripled the number of loci robustly associated with stroke risk.5i Hazard ratios for a first stroke, estimated using Cox regression. A polygenic risk score of 90 single nucleotide polymorphisms previously associated with stroke was constructed at P<1×10−5 to test for an association with incident stroke. Adherence to a healthy lifestyle was determined on the basis of four factors: non-smoker, healthy diet, body mass index <30 kg/m2, and regular physical exercise. Lifestyle is an important modifiable risk factor for stroke. Clear evidence shows that adhering to a healthy lifestyle, including not smoking, reducing the risk of diabetes, regular physical activity, and a healthy diet, decreases the risk of stroke substantially.6  7 It might be hypothesised that adhering to a healthy lifestyle could attenuate the effect of genetics on stroke risk. A previous study in coronary artery disease—a condition closely related to ischaemic stroke, found a statistically significant interplay between genetic and lifestyle risk factors in the risk of coronary artery disease.8 Study population Stroke is a complex disease caused by both genetic and environmental factors, including diet and lifestyle UK Biobank is a prospective study that recruited 500 000 community dwelling participants, aged 40- 69 years, from across the United Kingdom between 2006 and 2010 (www.ukbiobank.ac.uk).9 Participants attended one of 22 assessment centres across England, Scotland, and Wales. The study collects extensive data from questionnaires, interviews, health records, physical measures, biological samples, and imaging. Main outcomes and exposures of interest in the current study include imputed genetic data, incident stroke, and lifestyle (smoking, diet, body mass index, and physical activity). These findings highlight the potential of lifestyle interventions to reduce risk of stroke across entire populations, even in those at high genetic risk of stroke the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 1 RESEARCH RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from In the present study we included all people who were classified as white British (all who self identified as white British, followed by the exclusion of ethnic outliers identified by principal components analysis on the genotype data), without a history of stroke or myocardial infarction on the basis of self report or medical records, or both, and with complete data on lifestyle. people) and the UK Biobank Axiom array, to genotype about 805 426 markers with good genome wide coverage. Phasing was performed using SHAPEIT3 and imputation using IMPUTE4.12  15 Two reference panels were used for imputation; the Haplotype Reference Consortium reference panel (39 131 578 autosomal single nucleotide polymorphisms, SNPs) and a merged UK10K and 1000 Genomes Phase 3 panel.14 Imputed genotypes were available for 488 369 participants in this study.12 From the resulting dataset, we excluded those who self reported ancestry other than white British, related people (second degree or greater: kinship coefficient ≥0.884), people with high levels of heterozygosity and missingness (>5%), and people whose reported sex was inconsistent with sex inferred from the genetic data. The UK Biobank core team centrally performed a check for excessive heterozygosity.13 Extreme heterozygosity or high rates of missingness, or both, can be indicators of poor sample quality due to, for example, DNA contamination. UK Biobank provided a list of samples with unusually high heterozygosity and we excluded those samples according to its recommendations. Study population To evaluate a mismatch in sex self reported sex was compared with sex inferred from the genetic data (based on relative intensity of markers on the Y and X chromosomes). This sex mismatch evaluation was centrally performed by the UK Biobank core team and is described in detail elsewhere.12 This evaluation can be used as a way to detect sample mishandling or other kinds of clerical error. However, in a dataset of this size, some such mismatches would be expected owing to transgender people or instances of real (but rare) genetic variation, such as aneuploidies in sex chromosomes. on 3 December 2018 by guest. P http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Polygenic risk score derived from MEGASTROKE 2 We derived three sets of independent (r2<0.05 or 1000 Kb apart) SNPs associated with any stroke in people of European ancestry in MEGASTROKE (see supplemental text S2) at P<5×10−8, P<1×10−6, and P<1×10−5 using an LD clumping procedure employed using plink v1.90b3.45.5  16 For each individual in the UK Biobank sample we calculated quantitative aggregate risk scores, defined as the sum of the number of risk alleles present at each locus weighted by the log of the odds ratio for that locus estimated from the MEGASTROKE sample using the plink “–score” command. We took into account the occurrence of myocardial infarction during the study as this could potentially result in lifestyle changes during follow-up that affect the risk of stroke. The occurrence of myocardial infarction was defined according to the UK Biobank algorithmic definition (see supplemental table S2). Details of the myocardial infarction algorithm have been described previously and are available on the UK Biobank website (www.ukbiobank.ac.uk).11 We excluded people from the analysis who self reported stroke or myocardial infarction. The three polygenic scores were tested for an association with incident any stroke, and for further analyses we used the polygenic risk score most statistically significantly associated with incident stroke. Supplemental table S3 lists the SNPs included in the MEGASTROKE risk score (all stroke, P<1×10−5). We repeated the previous steps while restricting to ischaemic stroke in those of European ancestry to create a genetic risk score for ischaemic stroke (P<1×10−5). The three polygenic scores were tested for an association with incident any stroke, and for further analyses we used the polygenic risk score most statistically significantly associated with incident stroke. Supplemental table S3 lists the SNPs included in the MEGASTROKE risk score (all stroke, P<1×10−5). on 3 December 2018 by guest. http://www.bmj.com/ 168 on 24 October 2018. Downloaded from The UK Biobank participants completed a questionnaire on their usual diet pattern. In this analysis, a healthy diet was determined according to the increased consumption of fruit, vegetables, and fish and the decreased consumption of processed meats and red meats. We defined a healthy diet as adherence to at least two of the healthy food items. Supplemental text S1 and table S1 provide additional details on the specific questions asked and the construction of a healthy diet score. Moderate physical activity was defined as at least 150 minutes of moderate intensity activity weekly or 75 minutes of vigorous activity weekly. Incident stroke Incident stroke in UK Biobank was based on medical history and linkage to data on hospital admissions and mortality. We used the stroke variables provided by UK Biobank, which were created by combining information from these different data sources (see supplemental table S2). Details of the algorithms used to combine the data from different sources to identify stroke have been described previously and are available on the UK Biobank website (www.ukbiobank.ac.uk). We subtyped stroke as ischaemic stroke, intracerebral haemorrhage, or subarachnoid haemorrhage. In this analysis we only included SNPs imputed from the Haplotype Reference Consortium panel. Healthy lifestyle factorsi We defined four healthy lifestyle factors on the basis of the American Heart Association guidelines: no current smoking, healthy diet, body mass index <30 kg/m2, and moderate physical activity two or more times weekly.10 Statistical analysisi fatal or non-fatal strokes were reported as first incident vascular event or death, of which 1541 were ischaemic stroke, 287 intracerebral haemorrhage, and 249 subarachnoid haemorrhage. Furthermore, 3436 cases of fatal or non-fatal myocardial infarction and 6646 deaths due to other causes than stroke or myocardial infarction were reported as first incident vascular event or death. We defined genetic risk in thirds: “low risk” (lowest third of genetic risk score), “intermediate risk” (second third), “high risk” (highest third). Lifestyle was recorded as “favourable” (three or four healthy lifestyle factors), “intermediate” (two healthy lifestyle factors), “unfavourable” (no or one healthy lifestyle factor). To test the association of genetic and lifestyle factors with incident stroke we used Cox proportional hazards models. The duration of follow-up was calculated as time between the baseline assessment and the first event of either stroke, myocardial infarction, death, or 1 March 2016, which was the end of follow-up for the current data release. Participants who had a myocardial infarction or died before a stroke occurred were censored at the time of the respective event. Polygenic risk scores containing independent SNPs (on basis of linkage disequilibrium patterns) derived from MEGASTROKE at three different significance thresholds were tested for association with incident stroke in UK Biobank (see supplemental figure S1). The three polygenic risk scores were associated with risk of incident stroke, but the genetic risk score including all SNPs associated with stroke in MEGASTROKE at P<1×10−5 (90 SNPs, see supplemental table S3) showed the strongest association and was therefore selected for subsequent analyses. The polygenic risk approximated a normal distribution (see supplemental figure S2). We repeated the previous steps for the ischaemic stroke genetic risk score and assessed the association between this score and incident ischaemic stroke. In this analysis we censored participants with a diagnosis of intracerebral haemorrhage, subarachnoid haemorrhage, or myocardial infarction or who died before an ischaemic stroke occurred at the time of the respective event. In Cox proportional hazards analysis, the risk of incident stroke was higher for those with intermediate (hazard ratio 1.20, 95% confidence interval 1.08 to 1.34) and high genetic risk scores (1.35, 1.21 to 1.50) compared with those with a low genetic risk score (fig 2). We tested available cardiometabolic risk factors for an association with the genetic risk score, adjusting for the first 10 principal components of ancestry, genotyping batch, age, and sex. Statistical analysisi The genetic risk score was significantly associated with systolic blood pressure (P=1.5×10−15), diastolic blood pressure (P=1.1×10−7), use of lipid lowering drugs (P=7.5×10−13), and diabetes (7.6×10−4), but not with body mass index (P=0.18). Cox proportional hazards models included adjustment for age and sex for the lifestyle score models. For the models including the genetic score we additionally adjusted for the first 10 principal components of ancestry and genotyping batch. Model discrimination was evaluated with the concordance (c) statistic. We included an interaction term in the regression model to test for statistical interaction between the lifestyle and genetic risk score. To obtain cumulative incidence for lifestyle and genetic risk stratums we used competing risk regression; the cumulative incidence function. We compared the hazard ratios for the genetic and lifestyle score in the risk of stroke derived from Cox proportional hazards models with the subdistribution hazard ratios, calculated using proportional subdistribution hazards regression models.17 Total UK Biobank participants Excluded Missingness >5% Sex mismatch Excessive heterozygosity 199 298 528 1025 Excluded History of stroke History of myocardial infarction Self report only of incident myocardial infarction or stroke Any missing lifestyle information 5821 8469 60 56 217 70 567 502 619 Genetic data 488 369 Unrelated White British 378 065 Passed genetic quality control 377 040 Total included in analysis 306 473 Fig 1 | Flow of participants through study R software version 3.4.2 was used for the Cox proportional hazards regression (package “survival”) and proportional subdistribution hazards regression (package “cmprsk”). Patient and public involvement The development of the research question or outcome measures was not informed by patients’ priorities, experience, or preferences. No patients were involved in the design and conduct of the present study. There are no plans to disseminate the results to study participants. Genetic data We used the June 2017 release of the imputed genetic data from UK Biobank (downloaded 3 June 2017). Details of the design of the arrays, sample processing, and stringent quality control have been described in detail elsewhere12 and summarised previously.13 Briefly, we used two closely related arrays from Affymetrix, the UK BiLEVE Axiom array (9.9% of We repeated the previous steps while restricting to ischaemic stroke in those of European ancestry to create a genetic risk score for ischaemic stroke (P<1×10−5). doi: 10.1136/bmj.k4168 | BMJ 2018;363:k4168 | the bmj RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Statistical analysisi on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ MJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from genetic risk score (c statistic 0.69 (SE) 0.01), lifestyle score (0.69 (SE 0.01)), and the combined genetic and lifestyle score (0.70 (SE 0.01)). The cumulative risk of stroke increased with age. Blood pressure, diabetes, and the use of lipid lowering drugs did not seem to influence the association between genetic risk of stroke and incident stroke (see supplemental table S4). Similarly, the risk of stroke was increased in those with an unfavourable (hazard ratio 1.66, 95% confidence interval 1.45 to 1.89) and intermediate (1.27, 1.16 to 1.40) lifestyle compared with those with a favourable lifestyle (fig 2).i We repeated the analysis to test the associations of the genetic and lifestyle scores with incident stroke while restricting only to ischaemic stroke for both genetic risk score and outcome. In addition, we compared the results derived from the Cox proportional hazards model with those derived from the competing risk proportional subdistribution hazards model. Results did not change substantially when restricting to ischaemic stroke or when using the subdistribution hazards model compared with the original analyses (see supplementary tables S7-S9). Supplemental figure S3 shows the distribution of thirds of genetic risk and lifestyle scores. The genetic risk score was not associated with any of the single healthy lifestyle factors: odds ratio 0.98 (95% confidence interval 0.93 to 1.04) for body mass index score, 1.04 (0.99 to 1.09) for diet score, 0.97 (0.88 to 1.07) for smoking score, and 0.98 (0.94 to 1.03) for exercise score. Furthermore, the association of genetic risk with incident stroke was unchanged after adjustment for lifestyle (see supplemental table S5). Likewise, the association of lifestyle with incident stroke was essentially unchanged after adjustment for the genetic risk score (see supplemental table S5). Among individual components of the lifestyle score, smoking and body mass index ≥30 kg/m2 contributed most to the risk of incident stroke (table 3). For all lifestyle factors, the effects were similar across genetic risk stratums.f As the effect of smoking was about twice as strong as the other individual lifestyle scores (table 3). We repeated the analysis of the risk of incident stroke for combined genetic risk and lifestyle profile in which smoking was counted twice, and this resulted in slightly increased point estimates in the unfavourable versus favourable lifestyle categories (see supplementary table S10). Results Complete data for the present analysis were available for 306 473 participants in the UK Biobank Study (fig 1). Table 1 shows the baseline characteristics of the study population. During a total of 2 138 443 person years (median follow-up 7.1 years), 2077 incident Fig 1 | Flow of participants through study the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 3 RESEARCH Table 1 | Characteristics of participants at baseline. Values are numbers (participants) unless stated otherwise Characteristic All (n=306 473) Incident stroke (n=2077) No stroke (n=304 396) Mean (SD) age (years) 56.7 (7.9) 61.2 (6.8) 56.6 (7.9) Men 136 654 (44.6) 1184 (57.0) 135 470 (44.5) Mean (SD) systolic blood pressure (mm Hg) 138 (18) 146 (21) 138 (19) Mean (SD) diastolic blood pressure (mm Hg) 82 (10) 85 (11) 82 (10) Mean (SD) body mass index 27.2 (4.7) 28.0 (4.8) 27.2 (4.7) Diabetes 12 927 (4.2) 209 (10.1) 1865 (4.2) Use of lipid lowering drugs 44 785 (14.7) 497 (24.1) 44 288 (14.6) Use of antihypertensives 61 218 (20.0) 697 (33.6) 60 521 (19.9) Healthy lifestyle factors:   No current smoking 286 352 (93.4) 1822 (87.7) 284 530 (93.5)   Body mass index <30 236 326 (77.1) 1489 (71.7) 588 (77.1)   Regular moderate physical activity 181 234 (59.1) 1203 (57.9) 180 031 (59.1)   Healthy diet 139 328 (45.5) 884 (42.6) 138 444 (45.5) Healthy lifestyle score:   Favourable (3 or 4 healthy lifestyle factors) 191 003 (62.3) 1157 (55.7) 189 846 (62.4)   Intermediate (2 healthy lifestyle factors) 86 710 (28.3) 652 (31.4) 86 058 (28.3)   Unfavourable (0 or 1 healthy lifestyle factor) 28 760 (9.4) 268 (12.9) 28 492 (9.4) Genetic risk category:   Low 101 977 (33.3) 589 (28.4) 101 388 (33.3)   Intermediate 102 300 (33.4) 703 (33.8) 101 597 (33.4)   High 102 196 (33.3) 785 (37.8) 101 411 (33.3) Discussion We investigated the association between genetic risk of stroke, lifestyle, and incident risk of stroke in 306 473 people within the population based UK Biobank study. Risk of incident stroke was 35% higher among those at high genetic risk compared with those at low genetic risk, and these associations were independent of lifestyle profile. Furthermore, an unfavourable lifestyle was associated with a 66% increased risk of incident stroke compared with a favourable lifestyle, and this increased risk was present within any genetic risk category. A high genetic risk combined with an unfavourable lifestyle profile was associated with a more than twofold increased risk of stroke compared with a low genetic risk and a favourable lifestyle. The present study provides further support that common genetic variants are implicated in the development of stroke. Our findings showing that a polygenic risk score is associated with incident stroke is in line with both clinical and population based studies.18-21 Cumulative risk of stroke (%) The genetic risk score was also associated with blood pressure and use of lipid lowering drugs, which suggest that the effect of the genetic variants on risk of incident stroke might at least in part be mediated by vascular risk factors. However, adjusting for those factors did not change the effect size of association between genetic risk and incident stroke, which emphasises that other mechanisms than those that involve the traditional cardiovascular risk factors are likely important. In the MEGASTROKE genome wide association analysis of stroke, only about half of the identified loci shared genetic variation with related vascular traits, including blood pressure and lipid levels, which support that the genetic risk might act through additional mechanisms.5 on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ Fig 2 | Standardised risk of incident stroke according to genetic risk and lifestyle profile. on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Table 2 shows the risk of incident stroke for combined genetic risk and lifestyle profiles. An additive effect was found for genetic risk and lifestyle on risk of incident stroke. Within each genetic risk stratum there was an increase in strength of association with decreasing number of favourable life style factors (see supplemental table S6). The highest risk of incident stroke was observed in participants with a high genetic risk and an unfavourable lifestyle: hazard ratio 2.30 (95% confidence interval 1.84 to 2.87); see supplemental figure S4. The test for statistical interaction between lifestyle score and genetic risk score in relation to incident stroke was not significant (P=0.57) compared with participants with low genetic risk and favourable lifestyle. guest. Protected by copyright. The associations of genetic risk score with incident stroke were consistent in men and women (interaction P=0.70, supplemental figure S5). However, across all genetic risk stratums the absolute risk of incident stroke was lower in women than in men.i A statistically significant interaction on the multiplicative scale was found between sex and lifestyle profile in the risk of incident stroke (interaction P=0.01, supplemental figure S6). For men there was an increase in association with decreasing number of healthy lifestyle factors: hazard ratio 1.20 (95% confidence interval 1.05 to 1.36) and 1.82 (1.55 Model discrimination was similar between the main Cox proportional hazards models including the doi: 10.1136/bmj.k4168 | BMJ 2018;363:k4168 | the bmj RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Downloaded from Genetic risk No at risk High Intermediate Low Age (years) Cumulative risk of stroke (%) 0 2 3 5 4 1 50 55 60 65 70 75 16 693 16 718 16 628 19 885 20 106 19 895 23 927 24 020 23 608 31 663 31 569 31 823 24 740 25 312 25 044 4906 4957 5185 High; hazard ratio 1.35 (1.21 to 1.50) Intermediate; hazard ratio 1.20 (1.08 to 1.34) Low (reference) Lifestyle No at risk Unfavourable Intermediate Favourable Age (years) Cumulative risk of stroke (%) 0 2 3 5 4 1 50 55 45 45 60 65 70 75 80 80 5087 14 762 30 190 6427 17 920 35 539 7387 20 904 43 264 8327 26 033 60 695 5716 19 635 49 403 989 3843 10 216 Unfavourable; hazard ratio 1.66 (1.45 to 1.89) Intermediate; hazard ratio 1.27 (1.16 to 1.40) Favourable (reference) Fig 2 | Standardised risk of incident stroke according to genetic risk and lifestyle profile. Cox proportional hazards models were adjusted for age and sex, and the genetic risk models included additionally the first 10 principal components of ancestry and genotyping batch to 2.15) for intermediate and unfavourable lifestyle versus favourable lifestyle, respectively. However, among women there was no difference between intermediate and unfavourable lifestyle versus favourable lifestyle: 1.39 (1.21 to 1.61) and 1.36 (1.08 to 1.72), respectively. Genetic risk No at risk Age (years) Cumulative risk of stroke (%) 0 2 3 5 4 1 50 55 60 65 70 75 High; hazard ratio 1.35 (1.21 to 1.50) Intermediate; hazard ratio 1.20 (1.08 to 1.34) Low (reference) 45 80 Discussion Cox proportional hazards models were adjusted for age and sex, and the genetic risk models included additionally the first 10 principal components of ancestry and genotyping batch The reduction of stroke risk by adhering to a healthy lifestyle has been well reported.7  22-25 The Table 2 | Relative and absolute risk of incident stroke according to genetic and lifestyle profiles Genetic risk Lifestyle Favourable Intermediate Unfavourable Low Hazard ratio* (95% CI) 1 (reference) 1.36 (1.14 to 1.63), P=7.3×10−04 1.84 (1.44 to 2.35), P=8.0×10−07 8 year cumulative incidence† (%) (95% CI) 0.54 (0.47 to 0.60) 0.74 (0.63 to 0.85) 0.95 (0.74 to 1.17) Intermediate Hazard ratio* (95% CI) 1.26 (1.09 to 1.46), P=0.002 1.62 (1.37 to 1.92), P=3.2×10−08 1.85 (1.46 to 2.37), P=5.4×10−07 8 year cumulative incidence† (%) (95% CI) 0.67 (0.60 to 0.74) 0.82 (0.71 to 0.93) 0.92 (0.72 to 1.12) High Hazard ratio* (95% CI) 1.44 (1.25 to 1.66), P=7.0×10−07 1.70 (1.44 to 2.01), P=8.1×10−10 2.30 (1.84 to 2.87), P=3.3×10−13 8 year cumulative incidence† (%) (95% CI) 0.78 (0.70 to 0.86) 0.91 (0.78 to 1.04) 1.11 (0.89 to 1.33) *Calculated using Cox proportional hazards models, adjusted for age, sex, first 10 principal components of ancestry, and genotyping batch. †Calculated using the cumulative incidence function as implemented in the “cmprsk” R package. Table 2 | Relative and absolute risk of incident stroke according to genetic and lifestyle profiles Lifestyle the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 5 RESEARCH RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Discussion Downloaded from Table 3 | Multivariable Cox regression analysis of age, sex, and lifestyle factors in relation to risk of stroke, stratified by genetic risk Lifestyle factors Low genetic risk Moderate genetic risk High genetic risk Hazard ratio (95% CI) P value Hazard ratio (95% CI) P value Hazard ratio (95% CI) P value Age, per year 1.08 (1.07 to 1.09) <2×10−16 1.09 (1.08 to 1.10) <2×10−16 1.11 (1.10 to 1.12) <2×10−16 Male sex 1.47 (1.25 to 1.74) 4.6×10−06 1.65 (1.42 to 1.93) 1.2×10−10 1.56 (1.35 to 1.80) 1.4×10−09 Current smoking 2.35 (1.84 to 3.01) 7.3×10−12 2.81 (2.27 to 3.48) <2×10−16 1.87 (1.48 to 2.37) 1.5×10−07 Body mass index ≥30 1.43 (1.20 to 1.71) 8.0×10−05 1.38 (1.17 to 1.63) 1.2×10−04 1.19 (1.02 to 1.40) 0.03 No regular moderate physical activity 1.09 (0.92 to 1.28) 0.32 0.99 (0.85 to 1.63) 0.91 1.09 (0.94 to 1.26) 0.24 Unhealthy diet 1.10 (0.93 to 1.30) 0.27 1.11 (0.77 to 1.05) 0.16 1.21 (1.05 to 1.40) 0.01 | Multivariable Cox regression analysis of age, sex, and lifestyle factors in relation to risk of stroke, stratified by risk lifestyle in detail. Furthermore, to derive a genetic risk score for stroke, we used MEGASTROKE, which is currently the largest genome wide association study of stroke.5 Another distinctive feature of this analysis compared with a previous study is that we also included single nucleotide polymorphisms (SNPs) associated with stroke at a subthreshold level of significance (P<1×10−5).19 This concurs with a previous study examining the predictive utility of genetic risk scores for incident coronary heart disease, which showed that the best performance was achieved by including SNPs that did not necessarily reach the genome wide statistical significance threshold in previous genome wide association studies.32 lifestyle in detail. Comparison with previous studies Therefore, our results may not be generalisable to populations with distinct ancestry. Future studies are needed that test these relations in more diverse populations. Conclusion In the present prospective population based cohort study of 306 473 people we found that genetic and lifestyle factors were independently associated with risk of incident stroke. These findings highlight the potential of lifestyle interventions to reduce risk of stroke across entire populations, even in those at high genetic risk of stroke. Other possibilities for the increased relative risk include potential differences in duration of exposure to unfavourable lifestyle factors. Future studies are needed to evaluate the effect of the duration of exposure to an unfavourable lifestyle profile on the risk of stroke. Comparison with previous studies Across all categories of genetic risk and lifestyle, the risk of incident stroke was higher in men than women. This is an expected finding given the previously consistently shown higher incidence of stroke in men compared with women at the age of most of the UK Biobank participants.26 Our results suggested that the relative risk of incident stroke associated with high genetic risk versus low genetic risk was similar in men and women. A family history and genome wide association studies suggested that genetic susceptibility to stroke is somewhat stronger in women than in men.27 28 The methodological differences of those previous studies and the current study might explain the different conclusions. In the current study, only genetic variants associated with stroke in MEGASTROKE at P<1×10−5 were considered, whereas the other studies evaluated all genome wide variants within the study population or family history, which also includes environmental effects. Our study has several limitations. Firstly, behavioural changes before or after the baseline examinations might have had an effect on the risk estimates. We tried to reduce the effect of behavioural changes that could be related to vascular disease by excluding all those with a history of stroke and by censoring those at the time a myocardial infarction occurred. Secondly, this analysis focused on a narrow range of lifestyle factors, based on the American Heart Association guidelines.10 Expanding the range of lifestyle factors (ie, stress, sleep, alcohol and drug use) and more detailed assessment of diet and physical activity would be of interest for future studies. Thirdly, in the current study we only evaluated stroke of any cause. The effects of lifestyle and genetic variants might differ according to the cause of stroke, although some genetic risk variants and vascular risk factors are shared between two or more causal factors.5 Considerable evidence from previous epidemiological studies also suggests differences in risk factors that are associated with stroke in men compared with women. Women have a higher prevalence of hypertension, whereas men have a higher prevalence of heart disease, diabetes, and unhealthy lifestyle behaviours, including smoking, obesity, and alcohol use.29-31 In the present study we found a higher relative risk of stroke associated with an unfavourable lifestyle in men than women (82% v 36% increased risk, respectively). mber 2018 by guest. Protected by copyright. Finally, in the present study we restricted our analysis to people of European descent. Strengths and limitations of this study The major strengths of the current study include the large sample size of UK Biobank participants, which enabled study of the combination of genetic risk and AUTHOR AFFILIATIONS Discussion Furthermore, to derive a genetic risk score for stroke, we used MEGASTROKE, which is currently the largest genome wide association study of stroke.5 Another distinctive feature of this analysis compared with a previous study is that we also included single nucleotide polymorphisms (SNPs) associated with stroke at a subthreshold level of significance (P<1×10−5).19 This concurs with a previous study examining the predictive utility of genetic risk scores for incident coronary heart disease, which showed that the best performance was achieved by including SNPs that did not necessarily reach the genome wide statistical significance threshold in previous genome wide association studies.32 risk reduction associated with adherence to a healthy lifestyle in the present study was similar across all stratums of genetic risk, which emphasises the benefit for entire populations of adhering to a healthy lifestyle, independent of genetic risk. Among the lifestyle factors, the most statistically significant associations were observed for smoking and body mass index ≥30 kg/m2. on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from N Engl J Med 2016;375:2349-58. doi:10.1056/NEJMoa1605086 9 Sudlow C, Gallacher J, Allen N, et al. UK biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age. PLoS Med 2015;12:e1001779. doi:10.1371/journal.pmed.1001779 9 Sudlow C, Gallacher J, Allen N, et al. UK biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age. PLoS Med 2015;12:e1001779. doi:10.1371/journal.pmed.1001779 10 Benjamin EJ, Blaha MJ, Chiuve SE, et al, American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation 2017;135:e146-603. doi:10.1161/CIR.0000000000000485i 11 Schnier C, Sudlow C. Definitions of acute myocardial infarction (MI) and main MI pathological types for UK Biobank phase 1 outcomes adjudication: UK Biobank; 2017. https://biobank.ctsu.ox.ac.uk/ crystal/docs/alg_outcome_mi.pdf [accessed 1 July 2017].t 11 Schnier C, Sudlow C. Definitions of acute myocardial infarction (MI) and main MI pathological types for UK Biobank phase 1 outcomes adjudication: UK Biobank; 2017. https://biobank.ctsu.ox.ac.uk/ crystal/docs/alg_outcome_mi.pdf [accessed 1 July 2017]. ft k l d 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; HSM has been paid for delivering educational presentations for AstraZeneca; no other 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. 12 Bycroft C, Freeman C, Petkova D, et al. Genome-wide genetic data on ~500 000 UK Biobank participants. BioRxiv 2017 20-07-2017. 13 Rutten-Jacobs LCA, Tozer DJ, Duering M, et al. Genetic Study of White Matter Integrity in UK Biobank (N=8448) and the Overlap With Stroke, Depression, and Dementia. Stroke 2018;49:1340-7. doi:10.1161/STROKEAHA.118.020811 14 McCarthy S, Das S, Kretzschmar W, et al, Haplotype Reference Consortium. A reference panel of 64,976 haplotypes for genotype imputation. Nat Genet 2016;48:1279-83. doi:10.1038/ng.3643 Ethical approval: UK Biobank received ethical approval from the research ethics committee (reference 13/NW/0382). All participants provided informed consent to participate. The present analyses were conducted under UK Biobank application number 19463. 15 O’Connell J, Sharp K, Shrine N, et al. Haplotype estimation for biobank-scale data sets. Nat Genet 2016;48:817-20. doi:10.1038/ng.3583 Data sharing: The genetic and phenotypic UK Biobank data are available on application to the UK Biobank (www.ukbiobank.ac.uk/). on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Healthy diet and lifestyle and risk of stroke in a prospective cohort of women. Neurology 2014;83:1699-704. doi:10.1212/ WNL.0000000000000954 7 Larsson SC, Akesson A, Wolk A. Healthy diet and lifestyle and risk of stroke in a prospective cohort of women. Neurology 2014;83:1699-704. doi:10.1212/ WNL.0000000000000954 Funding: This work was in part supported by a British Heart Foundation programme grant (RG/16/4/32218). LCAR-J was supported by a British Heart Foundation immediate research fellowship (FS/15/61/31626). CLS is chief scientist for UK Biobank. The main sources of funding for CLS’s salary are UK Biobank and the Scottish funding Council. MD received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 666881 (SVDs@target) and from the DFG through the CRC 1123 (B3) and the Munich Cluster for Systems Neurology (EXC 1010 SyNergy). HSM is supported by a National Institute for Health Research (NIHR) senior investigator award, and his work is supported by the Cambridge Universities NIHR Comprehensive Biomedical Research Centre. The funding sources had no role in the design or conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript. The MEGASTROKE project received funding from sources specified at www.megastroke.org/acknowledgments.html. Funding: This work was in part supported by a British Heart Foundation programme grant (RG/16/4/32218). LCAR-J was supported by a British Heart Foundation immediate research fellowship (FS/15/61/31626). CLS is chief scientist for UK Biobank. The main sources of funding for CLS’s salary are UK Biobank and the Scottish funding Council. MD received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 666881 (SVDs@target) and from the DFG through the CRC 1123 (B3) and the Munich Cluster for Systems Neurology (EXC 1010 SyNergy). HSM is supported by a National Institute for Health Research (NIHR) senior investigator award, and his work is supported by the Cambridge Universities NIHR Comprehensive Biomedical Research Centre. The funding sources had no role in the design or conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript. The MEGASTROKE project received funding from sources specified at www.megastroke.org/acknowledgments.html. 8 Khera AV, Emdin CA, Drake I, et al. Genetic Risk, Adherence to a Healthy Lifestyle, and Coronary Disease. N Engl J Med 2016;375:2349-58. doi:10.1056/NEJMoa1605086 8 Khera AV, Emdin CA, Drake I, et al. Genetic Risk, Adherence to a Healthy Lifestyle, and Coronary Disease. on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from 3 Brass LM, Isaacsohn JL, Merikangas KR, Robinette CD. A study of twins and stroke. Stroke 1992;23:221-3. doi:10.1161/01.STR.23.2.221 3 Brass LM, Isaacsohn JL, Merikangas KR, Robinette CD. A study of twins and stroke. Stroke 1992;23:221-3. doi:10.1161/01.STR.23.2.221 4 Flossmann E, Schulz UG, Rothwell PM. Systematic review of methods and results of studies of the genetic epidemiology of ischemic stroke. Stroke 2004;35:212-27. doi:10.1161/01.STR.0000107187.84390.AA 5 Malik R, Chauhan G, Traylor M, et al, AFGen Consortium, Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium, International Genomics of Blood Pressure (iGEN-BP) Consortium, INVENT Consortium, STARNET, BioBank Japan Cooperative Hospital Group, COMPASS Consortium,EPIC-CVD Consortium, EPIC-InterAct Consortium, International Stroke Genetics Consortium (ISGC), METASTROKE Consortium, Neurology Working Group of the CHARGE Consortium, NINDS Stroke Genetics Network (SiGN), UK Young Lacunar DNA Study, MEGASTROKE Consortium, MEGASTROKE Consortium. Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes. Nat Genet 2018;50:524-37. doi:10.1038/ s41588-018-0058-3 g 7Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 8 Edinburgh, Edinburgh, UK 8 9German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Contributors: LCAR-J and MT conceived the study. LCAR-J and MT wrote the first and successive drafts of the manuscript. LCAR-J and MT analysed the data. SCL contributed to study conception and design. All authors revised the manuscript for important intellectual content. LCAR-J and MT had full access to the data and take responsibility for the integrity of the data and the accuracy of the data analysis. LCAR-J is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Contributors: LCAR-J and MT conceived the study. LCAR-J and MT wrote the first and successive drafts of the manuscript. LCAR-J and MT analysed the data. SCL contributed to study conception and design. All authors revised the manuscript for important intellectual content. LCAR-J and MT had full access to the data and take responsibility for the integrity of the data and the accuracy of the data analysis. LCAR-J is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. 6 Colpani V, Baena CP, Jaspers L, et al. Lifestyle factors, cardiovascular disease and all-cause mortality in middle-aged and elderly women: a systematic review and meta-analysis. Eur J Epidemiol 2018;33:831-45. doi:10.1007/s10654-018-0374-z p 7 Larsson SC, Akesson A, Wolk A. on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Summary statistics from the MEGASTROKE meta-analysis of genome wide association studies in stroke and stroke subtypes are available from www.megastroke.org. 16 Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007;81:559-75. doi:10.1086/519795 17 Fine JP, Gray RJ. A Proportional Hazards Model for the Subdistribution of a Competing Risk. J Am Stat Assoc 1999;94:496-509 doi:10.1080/01621459.1999.10474144. 17 Fine JP, Gray RJ. A Proportional Hazards Model for the Subdistribution of a Competing Risk. J Am Stat Assoc 1999;94:496-509 doi:10.1080/01621459.1999.10474144. Transparency: The lead author (LCAR-J) affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; that discrepancies from the study as planned have been explained, and that the paper conforms to transparency policy of the International Committee of Medical Journal Editors uniform requirement for manuscripts submitted to biomedical journals. 18 Fava C, Sjögren M, Olsson S, et al. A genetic risk score for hypertension associates with the risk of ischemic stroke in a Swedish case-control study. Eur J Hum Genet 2015;23:969-74. doi:10.1038/ejhg.2014.212 explained, and that the paper conforms to transparency policy of the International Committee of Medical Journal Editors uniform requirement for manuscripts submitted to biomedical journals. 19 Ibrahim-Verbaas CA, Fornage M, Bis JC, et al. Predicting stroke through genetic risk functions: the CHARGE Risk Score Project. Stroke 2014;45:403-12. doi:10.1161/ STROKEAHA.113.003044f 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 commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/. 20 Tada H, Shiffman D, Smith JG, et al. Twelve-single nucleotide polymorphism genetic risk score identifies individuals at increased risk for future atrial fibrillation and stroke. Stroke 2014;45:2856-62. doi:10.1161/STROKEAHA.114.006072 21 Traylor M, Rutten-Jacobs LC, Thijs V, et al. Genetic Associations With White Matter Hyperintensities Confer Risk of Lacunar Stroke. Stroke 2016;47:1174-9. doi:10.1161/STROKEAHA.115.011625 21 Traylor M, Rutten-Jacobs LC, Thijs V, et al. Genetic Associations With White Matter Hyperintensities Confer Risk of Lacunar Stroke. Stroke 2016;47:1174-9. doi:10.1161/STROKEAHA.115.011625 1 GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;385:117-71. the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 AUTHOR AFFILIATIONS 1German Center for Neurodegenerative diseases (DZNE), Population Health Sciences, Sigmund-Freud-Strasse 27, 53127 Bonn, Germany 1German Center for Neurodegenerative diseases (DZNE), Population Health Sciences, Sigmund-Freud-Strasse 27, 53127 Bonn, Germany doi: 10.1136/bmj.k4168 | BMJ 2018;363:k4168 | the bmj RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from 2Department of Clinical Neurosciences, Stroke Research Group, University of Cambridge, UK 3Unit of Nutritional Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 4Institute for Stroke and Dementia Research (ISD), University Hospital, LMU Munich, Munich, Germany 5Centre for Medical Informatics, Usher Institute of Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, UK 6Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK 7Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 9German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 2Department of Clinical Neurosciences, Stroke Research Group, University of Cambridge, UK 3Unit of Nutritional Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 4Institute for Stroke and Dementia Research (ISD), University Hospital, LMU Munich, Munich, Germany 5Centre for Medical Informatics, Usher Institute of Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, UK 6Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK 7Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 9German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 2 Boehme AK, Esenwa C, Elkind MS. Stroke Risk Factors, Genetics, and Prevention. Circ Res 2017;120:472-95. doi:10.1161/CIRCRESAHA.116.308398 2 Boehme AK, Esenwa C, Elkind MS. Stroke Risk Factors, Genetics, and Prevention. Circ Res 2017;120:472-95. doi:10.1161/CIRCRESAHA.116.308398 1 GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;385:117-71. doi:10.1016/S0140-6736(14)61682-2 No commercial reuse: See rights and reprints http://www.bmj.com/permissions on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from doi:10.1016/S0140-6736(14)61682-2 1 GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;385:117-71. doi:10.1016/S0140-6736(14)61682-2 22 Chiuve SE, Rexrode KM, Spiegelman D, Logroscino G, Manson JE, Rimm EB. Primary prevention of stroke by healthy lifestyle. Circulation 2008;118:947-54. doi:10.1161/CIRCULATIONAHA.108.781062 22 Chiuve SE, Rexrode KM, Spiegelman D, Logroscino G, Manson JE, Rimm EB. Primary prevention of stroke by healthy lifestyle. Circulation 2008;118:947-54. doi:10.1161/CIRCULATIONAHA.108.781062 the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from 23 Larsson SC, Åkesson A, Wolk A. Primary prevention of stroke by a healthy lifestyle in a high-risk group. Neurology 2015;84:2224-8. doi:10.1212/WNL.0000000000001637 24 Zhang Y, Tuomilehto J, Jousilahti P, Wang Y, Antikainen R, Hu G. Lifestyle factors on the risks of ischemic and hemorrhagic stroke. Arch Intern Med 2011;171:1811-8. doi:10.1001/ archinternmed.2011.443 25 Myint PK, Luben RN, Wareham NJ, Bingham SA, Khaw KT. Combined effect of health behaviours and risk of first ever stroke in 20,040 men and women over 11 years’ follow-up in Norfolk cohort of European Prospective Investigation of Cancer (EPIC Norfolk): prospective population study. BMJ 2009;338:b349. doi:10.1136/bmj.b349 26 Seshadri S, Wolf PA. Lifetime risk of stroke and dementia: current concepts, and estimates from the Framingham Study. Lancet Neurol 2007;6:1106-14. doi:10.1016/S1474-4422(07)70291-0 27 Touzé E, Rothwell PM. Sex differences in heritability of ischemic stroke: a systematic review and meta-analysis. Stroke 2008;39:16-23. doi:10.1161/STROKEAHA.107.484618 28 Traylor M, Rutten-Jacobs LC, Holliday EG, et al. Differences in Common Genetic Predisposition to Ischemic Stroke by Age and Sex. Stroke 2015;46:3042-7. doi:10.1161/STROKEAHA.115.009816 29 Andersen KK, Andersen ZJ, Olsen TS. Age- and gender-specific prevalence of cardiovascular risk factors in 40,102 patients with first-ever ischemic stroke: a Nationwide Danish Study. Stroke 2010;41:2768-74. doi:10.1161/STROKEAHA.110.595785f 23 Larsson SC, Åkesson A, Wolk A. Primary prevention of stroke by a healthy lifestyle in a high-risk group. Neurology 2015;84:2224-8. doi:10.1212/WNL.0000000000001637 23 Larsson SC, Åkesson A, Wolk A. Primary prevention of stroke by a healthy lifestyle in a high-risk group. Neurology 2015;84:2224-8. doi:10.1212/WNL.0000000000001637 24 Zhang Y, Tuomilehto J, Jousilahti P, Wang Y, Antikainen R, Hu G. Lifestyle factors on the risks of ischemic and hemorrhagic stroke. Arch Intern Med 2011;171:1811-8. doi:10.1001/ archinternmed.2011.443 30 Reeves MJ, Bushnell CD, Howard G, et al. on 3 December 2018 by guest. Protected b http://www.bmj.com/ BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Sex differences in stroke: epidemiology, clinical presentation, medical care, and outcomes. Lancet Neurol 2008;7:915-26. doi:10.1016/S1474-4422(08)70193-5 25 Myint PK, Luben RN, Wareham NJ, Bingham SA, Khaw KT. Combined effect of health behaviours and risk of first ever stroke in 20,040 men and women over 11 years’ follow-up in Norfolk cohort of European Prospective Investigation of Cancer (EPIC Norfolk): prospective population study. BMJ 2009;338:b349. doi:10.1136/bmj.b349 26 Seshadri S, Wolf PA. Lifetime risk of stroke and dementia: current concepts, and estimates from the Framingham Study. Lancet Neurol 2007;6:1106-14. doi:10.1016/S1474-4422(07)70291-0f 27 Touzé E, Rothwell PM. Sex differences in heritability of ischemic stroke: a systematic review and meta-analysis. Stroke 2008;39:16-23. doi:10.1161/STROKEAHA.107.484618f Supplementary information: additional material Supplementary table: table S3 showing SNPs included in MEGASTROKE risk score Supplementary information: additional material Supplementary table: table S3 showing SNPs included in MEGASTROKE risk score 28 Traylor M, Rutten-Jacobs LC, Holliday EG, et al. Differences in Common Genetic Predisposition to Ischemic Stroke by Age and Sex. Stroke 2015;46:3042-7. doi:10.1161/STROKEAHA.115.009816 No commercial reuse: See rights and reprints http://www.bmj.com/permissions Subscribe: http://www.bmj.com/subscribe
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EDUCAÇÃO EM DIREITOS HUMANOS NO ESTADO DE SÃO PAULO
Revista Espaço do Currículo
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EDUCAÇÃO EM DIREITOS HUMANOS NO ESTADO DE SÃO PAULO EDUCATION IN HUMAN RIGHTS IN THE STATE OF SÃO PAULO EDUCACIÓN EN DERECHOS HUMANOS EN EL ESTADO DE SÃO PAULO Revista Espaço do Currículo ISSN 1983-1579 Doi: 10.15687/rec.v16i1.63713 http://periodicos.ufpb.br/ojs2/index.php Resumo: Este artigo tem o objetivo de contribuir com as permanentes e recentes discussões acerca da educação em direitos humanos e apresenta uma análise do Plano Estadual do Estado de São Paulo de Educação em Direitos Humanos, as questões relacionadas aos Direitos Humanos vêm ganhando destaque em diversos setores da sociedade brasileira, seja de forma declarada, seja na sua negação. Tal debate, portanto, não pode ficar de fora da educação formal, reconhecendo o espaço escolar como um local privilegiado para que se possam realizar momentos de reflexão em torno das concepções e práticas, sem esquecer que a instituição escolar e seu público, estão inseridos em um determinado contexto social, político, econômico e cultural. A política pública desenvolvida para a promoção da educação em direitos humanos para os estudantes de São Paulo. Trata-se de uma pesquisa de natureza qualitativa, alicerçada na Pesquisa bibliográfica. Neste contexto, busca-se através da leitura de documentos sobre direitos humanos, e análise das ações públicas desenvolvidas para implementação da Educação em Direitos Humanos no Currículo Paulista. Priscila Lourenço Soares Santos Licenciada em História Mestranda em Ciências Humanas pela Universidade Santo Amaro e Professora de História na Educação Básica de São Paulo, Brasil. E-mail: priscilalourenco@prof.educacao.sp.gov.br Orcid: https://orcid.org/0000-0001-5346- 9732 Palavras-chave: Políticas Públicas. Educação. Currículo. Diretos Humanos. Palavras-chave: Políticas Públicas. Educação. Currículo. Diretos Humanos. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 1 Recebido em: 15/07/2022 Alterações recebidas em: 22/09/2022 Aceito em: 25/09/2022 Publicação em: 31/01/2023 Como citar este artigo: SANTOS P. L. S. EDUCAÇÃO EM DIREITOS HUMANOS NO ESTADO DE SÃO PAULO. Revista Espaço do Currículo, v. 16, n. 1, p. 1- 13, 2023. ISSN2177-2886. DOI: https://doi.org/10.15687/rec.v16i1.63713. Recebido em: 15/07/2022 Alterações recebidas em: 22/09/2022 Aceito em: 25/09/2022 Publicação em: 31/01/2023 REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 1 SANTOS P. L. S. Abstract: This article aims to contribute to the ongoing and recent discussions about human rights education and presents an analysis of the State Plan of the State of São Paulo for Human Rights Education. Brazilian society, either in a declared way or in its denial. Keywords: Public Policies. Education. Resume. Human Rights. Resumem: Este artículo tiene como objetivo contribuir a las discusiones permanentes y recientes sobre la educación en derechos humanos y presenta un análisis del Plan Estatal del Estado de São Paulo para la Educación en Derechos Humanos, las cuestiones relacionadas con los Derechos Humanos vienen ganando protagonismo en diversos sectores de la sociedad brasileña. , ya sea de forma declarada o en su negación. Tal debate, por lo tanto, no puede quedar fuera de la educación formal, reconociendo el espacio escolar como un lugar privilegiado para momentos de reflexión sobre concepciones y prácticas, sin olvidar que la institución escolar y sus públicos están insertos en un determinado contexto social, político, económico. y contexto cultural. La política pública desarrollada para promover la educación en derechos humanos de los estudiantes de São Paulo. Se trata de una investigación cualitativa, basada en una investigación bibliográfica. En este contexto, se busca a través de la lectura de documentos sobre derechos humanos y análisis de acciones públicas desarrolladas para la implementación de la Educación en Derechos Humanos en el Currículo de São Paulo. Palabras clave: Políticas públicas. Educación. Reanudar. Derechos humanos. EDUCAÇÃO EM DIREITOS HUMANOS NO ESTADO DE SÃO PAULO EDUCATION IN HUMAN RIGHTS IN THE STATE OF SÃO PAULO EDUCACIÓN EN DERECHOS HUMANOS EN EL ESTADO DE SÃO PAULO Such a debate, therefore, cannot be left out of formal education, recognizing the school space as a privileged place so that moments of reflection can be carried out around the concepts and practices, without forgetting that the school institution and its public are inserted in a given social, political, economic and cultural context. The public policy developed to promote human rights education for students in São Paulo. This is a qualitative research, based on bibliographic research. In this context, it is sought through the reading of documents on human rights, and analysis of public actions developed for the implementation of Education in Human Rights in the Paulista Curriculum. Keywords: Public Policies. Education. Resume. Human Rights. ou ela possam vir a sofrer. Os Direitos Humanos nascem justamente das preocupações em relação às formas autoritárias e arbitrárias de tolher a dignidade, a liberdade e as oportunidades de igualdade dos seres humanos em sua convivência social. Lembrando que o fato histórico que desencadeia tal discussão são os Estados nazifascistas, sob o pano de fundo dos acontecimentos da II Guerra Mundial (1939 – 1945), um dos conflitos armados mais devastadores da liberdade e dignidade que o Ocidente vivenciou em sua história, com um saldo de mais de 30 milhões de mortos, considerando estimativas muito otimistas. Cremos, portanto, que os Direitos Humanos não são assuntos marginais e nos negamos a acreditar que a sua defesa e promoção sejam inofensivas e sem resultados práticos. Olhando para o tempo presente, podemos elencar pelo menos duas razões que nos levam a tal consideração. A primeira é a de que, a partir da Declaração de 1948, atualizadas pela Declaração de Viena de 1993, os Direitos Humanos se tornam em um instrumento legal, corroborado internacionalmente, permitindo ações efetivas na sua defesa e promoção, como sanções comerciais e condenações junto à Corte Internacional de Justiça. A segunda razão se deve ao fato de que ao adotar os Direitos Humanos como a referência de ação do Estado, nos ancoramos sob questões que perpassaram por um amplo debate ao nível internacional, ratificados por um documento que conta com mais de 190 países signatários. Ou seja, a construção de seus conceitos e as suas práticas estão assente em discussões que procuram ir além das especificidades culturais de cada sociedade ou nação, porém, afirmando suas autodeterminações e colocando o aspecto da tolerância como um dos principais pilares que os constituem. Nesse bojo, compreendendo as principais questões que perpassaram e perpassam a constituição do conceito de Direitos Humanos, ficará claro que a perspectiva de inclusão dos Direitos Humanos nos currículos escolares não deveres se restringir à criação de mais uma disciplina. Mas sim que eles devem ser um referencial de ação da estrutura escolar, englobando a educação tanto de alunos quanto de professores, funcionários e equipe gestora, ou seja, todos os agentes envolvidos no processo de ensino e aprendizagem. 1. HISTÓRICO Em nossa análise para este artigo, partimos do questionamento da centralidade do Estado de São Paulo no estabelecimento das políticas curriculares para o desenvolvimento da Educação em Direitos Humanos. As questões relacionadas aos Direitos Humanos vêm ganhando destaque em diversos setores da sociedade brasileira, seja de forma declarada, seja na sua negação. Tal debate, portanto, não pode ficar de fora da educação formal, reconhecendo o espaço escolar como um local privilegiado para se poderem realizar momentos de reflexão em torno das concepções e práticas, sem esquecer que a instituição escolar e seu público, estão inseridos em um determinado contexto social, político, econômico e cultural. Quando vamos falar a respeito de valores entre os seres humanos, todo cuidado é pouco para que não os tornemos absolutos. Caso façamos isso, corremos o risco de naturalizar e tornar inerentes características humanas que, na conformação das estruturas das sociedades, são frutos de constructos sociais e dependem de complexas relações intersubjetivas e coletivas, aspectos que variam no tempo e no espaço. Nesse sentido, compreendemos que os Direitos Humanos englobam os valores humanos, pois defendem a garantia da dignidade humana contra qualquer forma de opressão, para que o sujeito portador de direitos possa usufruir de sua liberdade em igualdade de condições e oportunidades na sua atuação em sociedade, sempre na busca de uma convivência pacífica e harmônica. É somente na garantia desses direitos que podemos exigir do cidadão e da cidadã uma atuação consciente e responsável, possibilitando um sujeito que saiba defender os seus direitos, pois reconhece que é somente a partir de sua garantia que se efetivará as suas próprias condições de liberdade. Tais preceitos dos Direitos Humanos estão embasados em dois documentos: a Declaração Universal dos Direitos Humanos, aprovada pela ONU em 1948, e a Declaração da Conferência Mundial de Direitos Humanos, aprovada em 1993, em Viena. Documentos dos quais o Brasil é um dos signatários. Assim, mais do que uma carta de intenções, as declarações são instrumentos jurídicos que visam garantir a defesa e a promoção dos direitos do cidadão ou da cidadã, contra as violações e arbitrariedades que ele REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 2 2. DIREITOS HUMANOS EM SÃO PAULO Nesta fase do estudo, foi realizada uma primeira busca de informações sobre estado de São Paulo, concernentes em relação aos Direitos Humanos oficialmente. Para desenvolver a análise, além de contar a documentação oficial e leitura de bibliografia. Assim também irei apresentar ações desenvolvidas pelo estado de São Paulo sobre a temática, para estabelecer uma linha de trabalho com a Educação em Direitos Humanos. Importante ressaltar que a questão das discussões sobre Direitos Humanos ainda está muito no início mesmo com todas as legislações vigentes no Brasil, sendo necessário este trabalho de construção de uma política pública como diz: Daí a importância educativa, preventiva, inibidora e transformadora das políticas públicas de direitos humanos, as quais se encontram em estado de germinação, desenvolvimento, implementação e diagnóstico por parte do Estado brasileiro, em diversos âmbitos de atuação, através dos governos federal, estaduais e municipais. (BITTAR, 2018, P. 16) No âmbito da Secretaria da Justiça e da Defesa da Cidadania foi instituído o Observatório de Direitos Humanos, para monitorar as violações, denunciadas pela sociedade civil, na abrangência estadual. Ou seja, criou-se um instrumento de controle da sociedade civil sobre o Estado, para ele poder possa garantir cada vez mais a defesa dos direitos de cidadania. Com relação à Secretaria de Estado de Educação se mostra preocupado com as ações a serem desenvolvidas, buscando um plano de ação no sistema de ensino que reforce o caráter transversal e interdisciplinar, em que os Direitos Humanos sejam um eixo estruturador, tendo como objetivo a construção de valores democráticos voltados para a cultura dos Direitos Humanos. Segundo Candau a educação escolar não pode ser pensando e construída com uma perspectiva meramente instrumental. A educação escolar não pode ser reduzida a um produto que se negocia na lógica do mercado; nem ter como referência quase que exclusivamente a aquisição de determinados “conteúdos”, por mais socialmente reconhecidos que sejam. Deve ter como horizonte a construção de uma cidadania participativa, a formação de sujeitos de direito, o desenvolvimento da vocação humana de todas as pessoas nela implicadas. (CANDAU, 2012, p. 721) Assim da mesma forma devemos buscar a compreensão sobre educação em Direitos Humanos nas escolas em seus currículos e nas políticas públicas que estão sendo construídas para o desenvolvimento dela. ou ela possam vir a sofrer. Para desenvolver a análise mbém irei apresentar ações des ma linha de trabalho com a Educa s sobre Direitos Humanos aind , sendo necessário este trabalho preventiva, inibidora e trans umanos, as quais se encontram implementação e diagnóstico mbitos de atuação, através dos go 2018, P. 16) SANTOS P. L. S. direitos humanos, a educação por meio dos direitos humanos e a educação para os direitos humanos”. (ROSENDO, 2018, p. 470). Assim é importante este olhar para educação formal e a não formal dos estudantes, pois estão em processo de formação com cidadão. Para esta pesquisa realizei uma análise dos documentos presentes no site da Secretaria de Educação do Estado de São Paulo, e selecionei livros que trabalham com a temática Educação em Direitos Humanos com base a formação formal dos estudantes da educação básica. ou ela possam vir a sofrer. Contudo, vale lembrar que as questões colocadas sob a rubrica de Educação em Direitos Humanos não se restringem apenas à educação formal, inclui também a educação não formal, a educação dos agentes de justiça, segurança e a educação e mídia, conforme colocado no Plano Nacional de Educação em Direitos Humanos, de 2003. A proposição da Educação de Direitos Humanos, assim, tem uma abrangência que ultrapassa questões curriculares das escolas e se compromete com todos os níveis da vida social, sendo, então, uma preocupação de todos os cidadãos e cidadãs interessados numa convivência justa e igualitária, quer seja na sua defesa na sua promoção. Segundo Haddad a DUDH estipula que a educação deve ter como objetivo ampliar a personalidade humana e fortalecer os direitos de todos. Em outras palavras, não basta que todos tenham acesso à educação. Também é necessário que essa educação promova a individualidade de cada pessoa, sua personalidade e fortaleça seus direitos declarados pela DUDH, assim ele afirma: A educação é um elemento fundamental para a realização dessa vocação humana não apenas a educação escolar, mas a educação no seu sentido amplo, a educação pensa da num sistema geral, que implica na educação escolar, mas que não se basta nela, porque o processo educativo começa como nascimento extermina apenas no momento da morte do ser humano. (HADDAD, 2004, p.1) Mais uma vez destacamos que não é apenas a partir de documentos e decretos que se transformam pensamentos e atitudes, no entanto, eles são instrumentos mais incisivos de cobrança perante os poderes constituídos, na medida em que a sociedade civil participa de forma ativa em suas elaborações, ao menos é o que se espera de seu envolvimento nesses processos. É de suma importância, assim, que a sociedade civil se organize e cobre do Estado que suas ações estejam baseadas em princípios que respeitem a igualdade de oportunidades e o respeito à diferença, que constitui essa nossa sociedade tão plural e diversificada. Segundo Rosendo “A educação e formação em direitos humanos incluem: a educação sobre os REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 3 S. manos e a educação para os dire har para educação formal e a dadão. Para esta pesquisa real ação do Estado de São Paulo, e Humanos com base a forma usca de informações sobre estad nte. 2. DIREITOS HUMANOS EM SÃO PAULO O que propomos aqui são análises dos documentos nacionais e demais literatura sobre a EDH, não podendo ser considerado como um manual, mas sim como ponto de partida para que profissionais da educação envolvidos no projeto de Escola de Tempo Integral entendam o significado desta ação e cumpram com êxito o seu papel de ministrar oficinas eletivas sobre esta temática para a construção de um conhecimento para a rede pública de ensino paulista. Assim, trata-se de uma tarefa e missão pretensiosas e temos a plena consciência de que a sociedade brasileira exige muito mais do que apenas uma séria discussão em torno dos tantos problemas enfrentados em nosso dia a dia. Contudo, sabemos também que não conseguiremos efetivar tais medidas e políticas públicas se a sociedade não se envolver e abraçar os esforços que encaminhamos para essas questões, não apenas no auxílio para implementar políticas públicas que sejam eficazes, mas, sobretudo, que a sociedade civil possa ser os nossos olhos e ouvidos, cobrando e controlando quaisquer desvios que observarem na administração de recursos e ações. Esse também é um papel que o cidadão brasileiro deve começar a exercer em sua cidadania, o de fiscalizar as ações desenvolvidas pelo Estado, no intuito de que ele seja efetivamente direcionado para o cumprimento de seu papel, o de garantir uma qualidade de vida e dignidade à sua população. No entendimento de Silva (2016), a educação é dedicada a isso. A emancipação fundamenta-se no processo de conscientização e responsabilização das pessoas diante de condições de opressão, domínio, exclusão social e política, com o objetivo de que as pessoas demonstrem sua raiva e intolerância diante do desrespeito ao próximo e da valorização da vida em sua plenitude. Na mesma medida, a educação paulista vem contribuindo para que se aprofundem na educação básica, junto à sociedade civil, os debates concernentes às questões relacionadas aos Direitos Humanos que vêm ganhando destaque em diversos setores da sociedade brasileira, seja de forma declarada, seja na sua negação. Tal debate, portanto, não pode ficar de fora da educação formal, reconhecendo o espaço escolar como um local privilegiado para se poderem realizar momentos de reflexão em torno das concepções e práticas, sem esquecer que a instituição escolar e seu público, estão inseridos em um determinado contexto social, político, econômico e cultural. 2. DIREITOS HUMANOS EM SÃO PAULO Esta análise busca sintetizar todas as ideias aqui colocadas, procurando refletir e dialogar com os documentos paulistas e ações desenvolvidas pela secretaria de educação, o Currículo Paulista e seus materiais de apoio, podemos expandi-las para todas Ciências Humanas Aplicadas para os professores e estudantes, assim uma cultura que efetive uma sociedade democrática, em que o respeito e a tolerância sejam os princípios que norteiam a convivência social estão presentes no material do Currículo em Ação. Certamente de que isso se dá das mais diversificadas maneiras, e não se efetivará apenas discutindo tais temas em sala de aula, mas sim procurando incutir formas práticas, em que os valores dos Direitos Humanos se conectem de forma intrínseca ao cotidiano vivido dos profissionais das escolas estaduais de São Paulo. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 4 O tema Educação em Direitos Humanos (EDH) se configura como um tema transversal, mas consideramos importante que todo o currículo esteja integrado a essa proposta. E que toda a escola e professores possam contribuir para a construção de uma educação que perceba os indivíduos como sujeitos de direitos, colocando a dignidade da pessoa humana no centro de referência de todas as ações desencadeadas por estes atores. Há uma clara necessidade do Brasil em avançar na EDH. Dentre os agentes que podem empenhar para este propósito está a educação básica. Dessa forma, as unidades de ensino poderão contribuir para valorização da diversidade, para o respeito ao outro, para conscientização e ampliação da cidadania. Há uma compreensão de que a prática da EDH ultrapassa até mesmo os muros da escola. Sabemos dos desafios a serem enfrentados, mas sabemos da importância da incorporação da EDH nas políticas educacionais e principalmente no cotidiano das instituições escolares. Umas das prerrogativas para o desenvolvimento da EDH na educação básica será relacionamento da escola com a comunidade local, reconhecendo suas fragilidades, debatendo e buscando soluções para os problemas comuns que afetam, não somente os estudantes, mas suas famílias e o contexto no qual está inserido. Entendemos que os processos educacionais voltados para os direitos humanos deverão contribuir para a desnaturalização em relação a situações que desumanizam os sujeitos, que retiram a cidadania, que violam os direitos fundamentais do cidadão. 2. DIREITOS HUMANOS EM SÃO PAULO O atual Currículo Paulista do Estado de São Paulo está em consonância com a perspectiva da EDH, sendo que desde o Ensino Fundamental dos Anos Finais até a última série do Ensino Médio, em todas as disciplinas, os professores estão trabalhando os seus aspectos institucionais, históricos, práticos e aspectos teóricos e conceituais do tema. Em relação ao Ensino Médio, destacamos a área de Ciências Humanas, que possui maior ênfase em relação à apresentação de temas importantes para a EDH. Nunca podemos esquecer que currículo, escolhas de culturalmente limitadas pelo tempo escolar e a capacidade do estudante estão presentes nesta instituição. O primeiro problema que deve ser enfrentado pelos agentes que lidam com essa tarefa é baseado na esfera política e social, onde deve-se reconhecer que todos são levados em conta por sua personalidade, pois nem todos os estudantes leem tudo ou o mesmo conteúdo. Portanto, é preciso pensar nos objetivos a serem alcançados, ou seja, o que, como, como e por que ensinar, quais valores devem ser transmitidos, quais métodos devem ser utilizados na seleção. Nestas questões, estarão os fundamentos da especificação curricular. Ao analisar o Currículo de Ciências Humanas (Filosofia Geografia, História e Sociologia), encontramos os temas como: democracia e justiça social, o estado, ética, racismo, meio ambiente, construção da identidade, gênero, migração e Diversidade: O estrangeiro do ponto de vista sociológico, o significado de ser cidadão ontem e hoje, os direitos civis, políticos, sociais e humanos, as diferentes formas de violência, a desigualdade social e a própria desumanização do outro. Encontramos pontualmente, no Currículo de História, ao longo do Ensino Fundamental dos Anos Finais e Ensino Médio, os estudos sobre o Iluminismo, Revolução Francesa, Golpe e Ditadura Militar, remetem a discussões sobre Direitos Humanos e sua violação. Desse modo, são de conhecimento dos educandos da rede de ensino do Estado de São Paulo, conteúdos curriculares referentes ao tema Direitos Humanos presentes para ser desenvolvidos com os estudantes durante a educação formal. Muito importante a presenças dos conteúdos para os estudantes, entretanto, faz-se importante reconhecer que os princípios e conceitos defendidos nos temas trabalhados na EDH dizem respeito não somente aos educandos e professores das disciplinas, mas de toda a comunidade escolar, sobretudo no que tange aos dispositivos legais que estão estabelecidos pela Legislação vigente e devem ser de conhecimento de todos os agentes envolvidos no processo educacional: professores, funcionários, corpo gestor. 2. DIREITOS HUMANOS EM SÃO PAULO Quando analisamos conseguimos observar que houve um grande avanço no Brasil nos últimos anos sobre esta temática, principalmente a partir da elaboração e publicação do Plano Nacional de Educação em Direitos Humanos (PNEDH/2006), que define princípios norteadores para a educação em Direitos Humanos, inclusive para a educação básica, objeto em questão. Neste caso, chamamos a atenção para trechos do documento que afirma que “a prática escolar deve ser orientada para a educação em direitos humanos, assegurando o seu caráter transversal e a relação dialógica entre os diversos atores sociais.” REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 5 SANTOS P. L. S. Entendemos dessa forma, que a Educação em Direitos Humanos deve permear os currículos da Educação Básica, não sendo necessário o seu atendimento em formato disciplinar. Deve estar contida nos objetivos declarados junto ao projeto político pedagógico, de modo a favorecer práticas educacionais democráticas no cotidiano das unidades escolares. O Plano Nacional de Educação em Direitos Humanos (PNEDH) que preconiza que: [...] a educação em direitos humanos deve abarcar questões concernentes aos campos da educação formal, à escola, aos procedimentos pedagógicos, às agendas e instrumentos que possibilitem uma ação pedagógica conscientizadora e libertadora, voltada para o respeito e valorização da diversidade, aos conceitos de sustentabilidade e de formação da cidadania ativa (BRASIL, 2009, p. 23). [...] a educação em direitos humanos deve abarcar questões concernentes aos campos da educação formal, à escola, aos procedimentos pedagógicos, às agendas e instrumentos que possibilitem uma ação pedagógica conscientizadora e libertadora, voltada para o respeito e valorização da diversidade, aos conceitos de sustentabilidade e de formação da cidadania ativa (BRASIL, 2009, p. 23). Compreendemos assim, como versa o PNEDH/2006, que fazem parte da perspectiva da Educação em Direitos Humanos, a inclusão no currículo de temáticas como Gênero, Identidade, Raça e Etnia, Sexualidade, Diversidade, entre outros, bem como as inúmeras formas de discriminação e violações de direitos na sociedade. Outro documento importante e que merece destaque são as Diretrizes Nacionais para a Educação Básica: diversidade e inclusão, quando em seu capítulo 9, trata da Educação em Direitos, enfatizando a importância da educação para mudança e transformação social pautada nos princípios de da dignidade humana, igualdade de direitos, reconhecimentos e valorização das diversidades, laicidade do Estado, democracia na educação, transversalidade, vivência, globalidade e sustentabilidade socioambiental. 3. CURRÍCULO ESTADUAL E DIREITOS HUMANOS No estado de São Paulo nos anos de 2017 e 2018, o Conselho Estadual de Defesa dos Direitos da Pessoa Humana do Estado de São Paulo (CONDEPE) em parceria com órgãos públicos, universidades públicas e movimentos sociais empreenderam esforços para a construção de um Plano de Educação em Direitos Humanos. A iniciativa tinha como propósito central a elaboração de metas e objetivos para o desenvolvimento de políticas públicas de educação em Direitos Humanos. Encontramos em nossa verificação que as DEEDH-SP são condizentes com o Currículo Paulista, para as etapas do Ensino Fundamental Anos Finais e do Ensino Médio. O documento define um rol de competências, habilidades e objetos de conhecimento que aborda temáticas diretamente relacionadas aos Direitos Humanos, reiterando os pressupostos da Base Nacional Comum Curricular (BNCC) e da legislação nacional referente à Educação em Direitos Humanos (Decreto nº 7.037/2009, Parecer CNE/CP nº 8/2012 e Resolução CNE/CP nº 1/2012), dos quais destacamos duas Competências Gerais: 7. Argumentar com base em fatos, dados e informações confiáveis, para formular, negociar e defender ideias, pontos de vista e decisões comuns que respeitem e promovam os direitos humanos, a consciência socioambiental e o consumo responsável em âmbito local, regional e global, com posicionamento ético em relação ao cuidado de si mesmo, dos outros e do planeta. 9. Exercitar a empatia, o diálogo, a resolução de conflitos e a cooperação, fazendo-se respeitar e promovendo o respeito ao outro e aos direitos humanos, com acolhimento e valorização da diversidade de indivíduos e de grupos sociais, seus saberes, identidades, culturas e potencialidades, sem preconceitos de qualquer natureza. (SÃO PAULO, 2019, p.30.) 9. Exercitar a empatia, o diálogo, a resolução de conflitos e a cooperação, fazendo-se respeitar e promovendo o respeito ao outro e aos direitos humanos, com acolhimento e valorização da diversidade de indivíduos e de grupos sociais, seus saberes, identidades, culturas e potencialidades, sem preconceitos de qualquer natureza. (SÃO PAULO, 2019, p.30.) Educar os estudantes do século XXI, formar cidadãos globais que enfrentem o planeta em que vivem, buscando um desenvolvimento humano completo são desafios que exigem ações de todos os atores envolvidos no processo de educação. Há algum tempo são utilizados testes que buscam medir a inteligência dos alunos testando suas habilidades cognitivas. presentes em nossa sociedade, além do fomento e promoção da cultura de paz. Em relação à formação dos professores da rede, a Secretaria de Estado da Educação, encontramos em pesquisa que a formação e realizada através da Escola de Formação e Aperfeiçoamento de Professores (EFAPE), com validação dos técnicos de Ciências Humanas da Coordenadoria de Gestão da Educação Básica (CGEB), promove desde 2013, o curso Introdução aos Direitos Humanos e Estatuto da Criança e Adolescente (ECA) para Educadores na modalidade EAD. Segunda Silva formação dos professores sobre Direitos Humanos, deve refletir sua realidade para sala de aula: Frente a isso, o desafio dos educadores é descobrir, entre os muitos métodos utilizados atualmente, qual deles é o que melhor se adapta à realidade social dos alunos e quais os estimularão de forma efetiva. (SILVA, 2021, p.17) Segundo o regulamento do curso promovido pela Secretaria de Educação ele teve duração de 60 horas, sendo dividido em 10 módulos com duração de 6 horas cada. O público-alvo são professores, professores mediadores escolares (PMEC), diretores, vice-diretores, professores coordenadores (PC) e supervisores, conselheiros tutelares dos municípios do Estado de São Paulo, profissionais que atuam no âmbito da SEDUC e demais servidor de apoio escolar. 2. DIREITOS HUMANOS EM SÃO PAULO Estes devem estar aptos quanto ao conhecimento do sistema de garantia de direitos, da rede de proteção às crianças e aos adolescentes e da perspectiva da convivência humana democrática, evidenciando no contexto escolar formas e estratégias (práticas) que visam o combate às desigualdades REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 6 questão de Direitos Humanos. Encontramos entre as competências específicas os aprendizados essenciais que devem ser assegurados aos estudantes por um componente específico, ou por um conjunto de componentes curriculares que integram determinada área. Uma competência implica na mobilização de conceitos, procedimentos, habilidades, práticas cognitivas e socioemocionais, atitudes e valores para a resolução de demandas da vida cotidiana. Na competência específica número 4 da área de Linguagens para os Anos Finais do Ensino Fundamental, por exemplo, é salientado: 4. Utilizar diferentes linguagens para defender pontos de vista que respeitem o outro e promovam os direitos humanos, a consciência socioambiental e o consumo responsável em âmbito local, regional e global, atuando criticamente frente a questões do mundo contemporâneo. (SÃO PAULO, 2019, p.96) [..] Essa competência trata da necessidade da promoção dos Direitos Humanos e possui relação com a competência específica número 6 de Língua Portuguesa, que trata da importância da análise crítica de conteúdos que violem os Direitos Humanos: 6. Analisar informações, argumentos e opiniões manifestados em interações sociais e nos meios de comunicação, posicionando-se ética e criticamente em relação a conteúdos discriminatórios que ferem direitos humanos e ambientais. (SÃO PAULO, 2019, p.30.) (SÃO PAULO, 2019, p.109) Por meio de exemplos como estes, é possível verificar que a educação em Direitos Humanos é uma temática transversal que é abordada por diferentes áreas do Currículo Paulista dos Anos Finais do Ensino Fundamental. Isso se reflete tanto nas competências maiores de diferentes áreas, quanto nas habilidades de componentes curriculares específicos. Em relação ao Ensino Médio, indo ao encontro do que está previsto na Resolução nº 03 de 21 de novembro de 2018, que atualizou as Diretrizes Curriculares Nacionais para o Ensino Médio, dentre seus princípios específicos, o Currículo pressupõe que sejam assegurados: IV - respeito aos direitos humanos como direito universal; V - compreensão da diversidade e realidade dos sujeitos, das formas de produção e de trabalho e das culturas. (SÃO PAULO, 2020, p.32) O que pode ser visto em uma série de habilidades de diferentes componentes e em competências específicas da área de Ciências Humanas e Sociais Aplicadas e da área de Linguagens e suas Tecnologias. Como exemplo, duas competências específicas da área de Linguagens para o Ensino Médio que abordam a temática dos Direitos Humanos: 2. 3. CURRÍCULO ESTADUAL E DIREITOS HUMANOS Assim o currículo das escolas pode ser o campo um diálogo pedagógico entre o que se aprende e o que se ensina, sempre considerando a convivência e a formação de pessoas muito diferentes. (ARROYO, 2011). Desta forma as reflexões sobre Direitos Humanos presente neste documento formal têm grande importância para formação dos estudantes. Ao analisar o Currículo Paulista para as etapas dos Anos Finais do Ensino Fundamental e Ensino Médio, que está pautado em competências visa observar se ele apresenta esta visão e reflexão para a REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 7 SANTOS P. L. S. questão de Direitos Humanos. questão de Direitos Humanos. 90) Tal população é composta de uma diversidade étnica e cultural que inclui comunidades indígenas, comunidades quilombolas, autodeclarados negros e pardos, populações prisionais, adolescentes em cumprimento de medidas socioeducativas, comunidades do campo (acampados, assentados e comunidades tradicionais), estudantes migrantes internacionais, estudantes itinerantes etc., é preciso criar possibilidades para que esses educandos desenvolvam suas habilidades. Dessa forma, os conteúdos curriculares devem ser adaptados, considerando as especificidades de cada estudante, respeitando, assim, as potencialidades e dificuldades individuais – ritmos diferentes de aprendizagem – por meio de estratégias diversificadas de ensino. (SÃO PAULO, 2020, p. 90) O segundo objetivo desta Diretriz Estaduais de Educação em Direitos Humanos do Estado de São Paulo, evidencia que, para promoção de uma “educação com qualidade social, acesso e permanência” é importante “desenvolver o respeito a diversas nacionalidades e suas culturas no currículo escolar”. Assim sendo, é importante pontuar que uma das bases do Currículo Paulista é o princípio da equidade, que visa assegurar a inclusão de todos os estudantes nas escolas e a garantia do direito a uma educação pública e de qualidade, como prevê a Lei de Diretrizes e Bases da Educação. Já na apresentação do Currículo Paulista da etapa do Ensino Fundamental é enfatizado que para garantia da equidade é necessário “respeitar a diversidade cultural, a socioeconômica, a étnico-racial, a de gênero e os socioculturais presentes no território estadual”. (SÃO PAULO, 2019, p.27) Diferentes componentes curriculares possuem competências e habilidades que abordam temáticas a este respeito e buscam inserir no repertório manifestações culturais de nacionalidades diferentes. Destacamos aqui a oitava competência específica de História, que vai ao encontro desta perspectiva: 8. Compreender a história e a cultura africana, afro-brasileira, imigrante e indígena, bem como suas contribuições para o desenvolvimento social, cultural, econômico, científico, tecnológico e político e tratar com equidade as diferentes culturas. (SÃO PAULO, 2019, p. 461) Neste sentido, citamos também uma competência específica e uma habilidade de Ciências Humanas e Sociais Aplicadas do Ensino Médio, que dentro da categoria Território e Fronteira vai trabalhar a compreensão das relações de poder que envolvem os processos migratórios e suas implicações para os direitos humanos: Competência Específica: 2. Analisar a formação de territórios e fronteiras em diferentes tempos e espaços, mediante a compreensão das relações de poder que determinam as territorialidades e o papel geopolítico dos Estados-nações. questão de Direitos Humanos. Compreender os processos identitários, conflitos e relações de poder que permeiam as práticas sociais de linguagem, respeitando as diversidades e a pluralidade de ideias e posições, e atuar socialmente com base em princípios e valores assentados na democracia, na igualdade e nos Direitos Humanos, exercitando o autoconhecimento, a empatia, o diálogo, a resolução de conflitos e a cooperação, e combatendo preconceitos de qualquer natureza. (SÃO PAULO, 2020, p. 84) 3. Utilizar diferentes linguagens (artísticas, corporais e verbais) para exercer, com autonomia e colaboração, protagonismo e autoria na vida pessoal e coletiva, de forma crítica, criativa, ética e solidária, defendendo pontos de vista que respeitem o outro e promovam os Direitos Humanos, a consciência socioambiental e o consumo responsável, em âmbito local, regional e global. (SÃO PAULO, 2020, p. 89) No que diz respeito às Diretrizes Estaduais de Educação em Direitos Humanos do Estado de São Paulo, tem como eixo orientador Educação Básica, coloca em seu primeiro objetivo a necessidade de acompanhamento dos educandos com dificuldades de aprendizagem, adolescentes em cumprimento de medida socioeducativa e adolescentes ou crianças em medida protetiva. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 8 Neste sentido, é importante situar que o Currículo Paulista da etapa do Ensino Médio prevê que, para sua implementação nas redes de ensino do Estado de São Paulo deve-se pressupor um compromisso com a garantia da Educação Básica para “segmentos da população paulista que, devido às distintas características socioculturais e/ou às diversas situações de vulnerabilidades, requerem atendimento educacional inclusivo para efetivar seu direito à educação”. (SÃO PAULO, 2020, p. 90) Ainda especifica as características de grupos que compõem esta população, colocando sobre a necessidade da realização de adaptações curriculares considerando cada caso individualmente: Tal população é composta de uma diversidade étnica e cultural que inclui comunidades indígenas, comunidades quilombolas, autodeclarados negros e pardos, populações prisionais, adolescentes em cumprimento de medidas socioeducativas, comunidades do campo (acampados, assentados e comunidades tradicionais), estudantes migrantes internacionais, estudantes itinerantes etc., é preciso criar possibilidades para que esses educandos desenvolvam suas habilidades. Dessa forma, os conteúdos curriculares devem ser adaptados, considerando as especificidades de cada estudante, respeitando, assim, as potencialidades e dificuldades individuais – ritmos diferentes de aprendizagem – por meio de estratégias diversificadas de ensino. (SÃO PAULO, 2020, p. para os direitos humanos. Os regimes políticos e a “produção” dá moral. Geografia: As correntes migratórias, a produção e circulação de mercadorias e suas marcas na paisagem; conflitos socioespaciais e organização territorial. História: Processos migratórios, suas motivações e desdobramentos (questões étnicas, xenofobia e conflitos territoriais). Geografia: As correntes migratórias, a produção e circulação de mercadorias e suas marcas na paisagem; conflitos socioespaciais e organização territorial. História: Processos migratórios, suas motivações e desdobramentos (questões étnicas, xenofobia e conflitos territoriais). Sociologia: Processos de gentrificação em territorialidades urbanas: xenofobia, migrações, conflitos socioespaciais e territoriais. (SÃO PAULO, 2020, p. 182) Sociologia: Processos de gentrificação em territorialidades urbanas: xenofobia, migrações, conflitos socioespaciais e territoriais. (SÃO PAULO, 2020, p. 182) O Currículo esclarece que a promoção da equidade pressupõe o uso de respostas adequadas “ao público atendido nas modalidades da Educação Especial, Educação de Jovens e Adultos, Educação do Campo, Educação Escolar Indígena e Educação Escolar Quilombola, segundo as necessidades locais”. (SÃO PAULO, 2020, p. 27) Encontramos na análise dos materiais produzidos pela Secretaria Educação para as escolas, explícita sobre a repressão e a resistência à ditadura civil - militar, devem ter sua distribuição assegurada aos estudantes. Neste sentido, destacamos que uma série de habilidades do Currículo Paulista trabalham habilidades e objetos de conhecimentos relacionados à ditadura civil - militar. Os materiais, Currículo em Ação cadernos de atividades voltados para estudantes que são distribuídos para toda a rede pública de ensino e que são produzidos pela própria Secretaria da Educação, trabalham diretamente todas as habilidades do Currículo Paulista. No caso dos Anos Finais do Ensino Fundamental, o contexto de repressão da ditadura civil – militar é trabalhado diretamente no caderno de História do 9º ano no 3º e 4º bimestres, cujas habilidades implicam em: (EF09HI19) Identificar e compreender o processo que resultou na ditadura civil- militar no Brasil e discutir a emergência de questões relacionadas à memória e à justiça sobre os casos de violação dos direitos humanos. (EF09HI19) Identificar e compreender o processo que resultou na ditadura civil- militar no Brasil e discutir a emergência de questões relacionadas à memória e à justiça sobre os casos de violação dos direitos humanos. (EF09HI20) Discutir os processos de resistência e as propostas de reorganização da sociedade brasileira durante a ditadura civil-militar. (EF09HI20) Discutir os processos de resistência e as propostas de reorganização da sociedade brasileira durante a ditadura civil-militar. questão de Direitos Humanos. Habilidade: (EM13CHS201) Analisar e caracterizar as dinâmicas das populações, das mercadorias e do capital nos diversos continentes, com destaque para a mobilidade e a fixação de pessoas, grupos humanos e povos, em função de eventos naturais, políticos, econômicos, sociais, religiosos e culturais, de modo a compreender e posicionar-se criticamente em relação a esses processos e às possíveis relações entre eles. Objetos de Conhecimento: Filosofia: A reflexão ética: as exigências morais do homem moderno. As exigências morais da contemporaneidade e as implicações REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 9 SANTOS P. L. S. para os direitos humanos. Os regimes políticos e a “produção” dá moral. para os direitos humanos. Os regimes políticos e a “produção” dá moral. Objetos de conhecimento: Os anos 1960: revolução cultural; A ditadura civil- militar e os processos de resistência; as questões indígena e negra e a ditadura. (SÃO PAULO, 2019, p. 483) No Currículo do Ensino Médio, a temática também é abordada pela área de Ciências Humanas e Sociais Aplicadas, e dentre as competências específicas e habilidades que colaboram com seu desenvolvimento destacamos a questão do combate às formas de injustiça por meio do respeito aos Direitos Humanos: Competência Específica: 5. Identificar e combater as diversas formas de injustiça, preconceito e violência, adotando princípios éticos, democráticos, inclusivos e solidários, e respeitando os Direitos Humanos. Habilidade: (EM13CHS503) Identificar diversas formas de violência (física, simbólica, psicológica etc.), suas principais vítimas, suas causas sociais, psicológicas e afetivas, seus significados e usos políticos, sociais e culturais, discutindo e avaliando mecanismos para combatê-las, com base em argumentos éticos. Objetos de Conhecimento: Objetos de Conhecimento: Filosofia: Comportamentos opressores e modos de violência: pressupostos e implicações da opressão, da violência e indiferença em relação aos fenômenos sociais. O totalitarismo e o terrorismo como ameaça a democracia e aos Direitos Humanos. Geografia: Representação cartográfica da violência. O discurso da violência nas campanhas políticas, propagandas ideológicas, redes sociais e no uso político de REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 10 História: O uso institucional (político, social e cultural) da violência: regimes ditatoriais e totalitários, golpes de Estado e terrorismo, Apartheid na África do Sul e segregação étnico-racial no mundo. Sociologia: Formas e dimensões da violência: física, psicológica e simbólica. (SÃO PAULO, 2020, p. 483) As temáticas que devem ser abordadas nos currículos e materiais didáticos de modo a assegurar uma abordagem transversal na abordagem dos Direitos Humanos. Como já esclarecido, a Educação em Direitos Humanos é uma temática transversal que é abordada por diferentes áreas do Currículo Paulista, no entanto, a diretriz cita uma série de Leis Federais, que também teriam de ser tratadas pelo Currículo e pelos materiais. Assim sendo, é importante situar que o que fora colocado no objetivo em questão está em consonância com o que aparece na própria Base Nacional Comum Curricular (BNCC), que orientou o Currículo Paulista. A BNCC prevê que os seguintes temas transversais estejam presentes nos currículos do Ensino Fundamental e do Ensino Médio, sendo eles contemplados nos objetos de aprendizagem na área de Ciências Humanas e Sociais Aplicadas, Ciências da Natureza e suas Tecnologias, e na área de Linguagens e suas Tecnologias. Encontramos em nossa analise curricular das eletivas sugeridas pela Secretaria de Educação do Estado de São Paulo no cardápio de sugestões aos professores da rede, uma eletiva como o nome de direitos e Direitos e cidadania foi criada pelo Instituto Auschwitz a partir do projeto Cidadania e democracia desde a escola no âmbito do programa Inova, para trabalhar com estudantes do 6º ano do Ensino Fundamental até 3º ano do Ensino Médio nas escolas estaduais. Objetos de Conhecimento: Está eletiva se apresenta com objetivo segundo o material trabalhar com as atitudes e os comportamentos habituais dos estudantes e desenvolve conteúdo para a compreensão dos valores e procedimentos que sustentam a confiança cívica entre cidadãos numa democracia, tais como a capacidade de se colocar no lugar do outro, a necessidade de aprender a dialogar no espaço público e a importância de construir argumentos fundamentados para a defesa de suas posições com autonomia dos estudantes que realizam estas aulas. Em sua justificativa para seu desenvolvimento pedagógico está baseada na presente Base Nacional Comum Curricular, através das metodologias aplicadas em seu conteúdo com as dinâmicas, análise de notícias e vídeos, rodas de conversa e pesquisa, envolvendo temas contemporâneos e pertinentes à realidade dos estudantes. Com o material produzido pelo Instituto Auschwitz os professores da rede têm acesso à proposta pedagógica com um material disponibilizado no site da instituição para realização das atividades semestralmente, finalizando com uma atividade de encerramento chamada pela Secretaria de Educação como culminância. As sugestões envolvem projetos artísticos (6º e 7º), de comunicação social (8º e 9º) e de ações cidadãs (Ensino Médio) presentes nos materiais elaborados. Devemos sempre refletir sobre “a educação é uma prática social e política cujas ferramentas são elementos simbólicos, produzidos e manuseados pela subjetividade e mediados pela cultura.” (SEVERINO, 2002, p. 72), contarmos avanços, dificuldades e desafios sobre a Educação em Direitos Humanos para a educação básica para a formação inicial dos estudantes de São Paulo. CONSIDERAÇÕES FINAIS Em relação ao Currículo dos Anos Finais do Ensino Fundamental e do Ensino Médio da educação básica, essas diante do exposto, é possível concluir que as diretrizes relacionadas ao Currículo estão alinhadas ao que é nele proposto em relação à Educação para os Direitos Humanos. Os seus objetivos estão em consonância com os pressupostos do Currículo e com os seus Materiais produzidos para a implementação do Currículo do Estado de São Paulo. Por fim, acreditamos que seja importante debater e refletir a questão da Educação em Direitos REV. 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Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em: https://periodicos.ufpb.br/index.php/rec/article/view/ufpb.1983-1579.2018v3n11.40385. Acesso em: 14 set. 2022. SEVERINO, Antonio Joaquim. Educação, sujeito e história. 1. ed. São Paulo: Olho D’Água. 2002. SILVA, Phamella Rodrigues da. Direitos humanos e educação com ênfase na formação de professores. 2021. 25 f. Trabalho de Conclusão de Curso (Graduação em Pedagogia) – Universidade Federal de Uberlândia, Uberlândia, 2021. competências socioemocionais e ética ambiental. Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em: https://periodicos.ufpb.br/index.php/rec/article/view/ufpb.1983-1579.2018v3n11.40385. Acesso em: 14 set. 2022. SEVERINO A t i J i Ed ã j it hi tó i d Sã P l Olh D’Á competências socioemocionais e ética ambiental. Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em: https://periodicos.ufpb.br/index.php/rec/article/view/ufpb.1983-1579.2018v3n11.40385. Acesso em: 14 set. 2022. competências socioemocionais e ética ambiental. Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em: https://periodicos.ufpb.br/index.php/rec/article/view/ufpb.1983-1579.2018v3n11.40385. Acesso em: 14 set. 2022. SEVERINO, Antonio Joaquim. Educação, sujeito e história. 1. ed. São Paulo: Olho D’Água. 2002. EVERINO, Antonio Joaquim. Educação, sujeito e história. 1. ed. São Paulo: Olho D’Água. 2002 SILVA, Phamella Rodrigues da. Direitos humanos e educação com ênfase na formação de professores. 2021. 25 f. Trabalho de Conclusão de Curso (Graduação em Pedagogia) – Universidade Federal de Uberlândia, Uberlândia, 2021. SILVA, Phamella Rodrigues da. Direitos humanos e educação com ênfase na formação de professores. 2021. 25 f. Trabalho de Conclusão de Curso (Graduação em Pedagogia) – Universidade Federal de Uberlândia, Uberlândia, 2021. Esta obra está licenciada com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional. Esta obra está licenciada com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 13
https://openalex.org/W1964282709
https://zenodo.org/records/1273479/files/article.pdf
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A low-cost test approach for embedded RF passive circuits
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https://openalex.org/W2551832670
https://boris.unibe.ch/99946/1/art%253A10.1007%252FJHEP11%25282016%2529112.pdf
English
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Search for the Standard Model Higgs boson produced by vector-boson fusion and decaying to bottom quarks in s = 8 $$ \sqrt{s}=8 $$ TeV pp collisions with the ATLAS detector
˜The œJournal of high energy physics/˜The œjournal of high energy physics
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Published for SISSA by Springer Received: June 8, 2016 Revised: September 28, 2016 Accepted: November 10, 2016 Published: November 21, 2016 Received: June 8, 2016 Revised: September 28, 2016 Accepted: November 10, 2016 Published: November 21, 2016 Received: June 8, 2016 Revised: September 28, 2016 Accepted: November 10, 2016 Published: November 21, 2016 Search for the Standard Model Higgs boson produced by vector-boson fusion and decaying to bottom quarks in √s = 8 TeV pp collisions with the ATLAS detector JHEP11(2016)112 The ATLAS collaboration E-mail: atlas.publications@cern.ch E-mail: atlas.publications@cern.ch Abstract: A search with the ATLAS detector is presented for the Standard Model Higgs boson produced by vector-boson fusion and decaying to a pair of bottom quarks, using 20.2 fb−1 of LHC proton-proton collision data at √s = 8 TeV. The signal is searched for as a resonance in the invariant mass distribution of a pair of jets containing b-hadrons in vector-boson-fusion candidate events. The yield is measured to be −0.8 ± 2.3 times the Standard Model cross-section for a Higgs boson mass of 125 GeV. The upper limit on the cross-section times the branching ratio is found to be 4.4 times the Standard Model cross- section at the 95% confidence level, consistent with the expected limit value of 5.4 (5.7) in the background-only (Standard Model production) hypothesis. Keywords: Hadron-Hadron scattering (experiments), Higgs physics, proton-proton scat- tering ArXiv ePrint: 1606.02181 doi:10.1007/JHEP11(2016)112 doi:10.1007/JHEP11(2016)112 Contents 1 Introduction 1 2 The ATLAS detector 3 3 Data and simulation samples 3 4 Object reconstruction 4 5 Event pre-selection 5 6 Multivariate analysis 5 7 Invariant mass spectrum of the two b-jets 6 8 Sources of systematic uncertainty 9 8.1 Experimental uncertainties 10 8.2 Modelling uncertainties on the mbb shape of the non-resonant background 10 8.3 Theoretical uncertainties 10 9 Statistical procedure and results 11 10 Cut-based analysis 13 11 Summary 15 The ATLAS collaboration 20 Contents 1 Introduction 1 2 The ATLAS detector 3 3 Data and simulation samples 3 4 Object reconstruction 4 5 Event pre-selection 5 6 Multivariate analysis 5 7 Invariant mass spectrum of the two b-jets 6 8 Sources of systematic uncertainty 9 8.1 Experimental uncertainties 10 8.2 Modelling uncertainties on the mbb shape of the non-resonant background 10 8.3 Theoretical uncertainties 10 9 Statistical procedure and results 11 10 Cut-based analysis 13 11 Summary 15 The ATLAS collaboration 20 Contents 1 Introduction 1 2 The ATLAS detector 3 3 Data and simulation samples 3 4 Object reconstruction 4 5 Event pre-selection 5 6 Multivariate analysis 5 7 Invariant mass spectrum of the two b-jets 6 8 Sources of systematic uncertainty 9 8.1 Experimental uncertainties 10 8.2 Modelling uncertainties on the mbb shape of the non-resonant background 10 8.3 Theoretical uncertainties 10 9 Statistical procedure and results 11 10 Cut-based analysis 13 11 Summary 15 The ATLAS collaboration 20 1 3 3 4 5 5 6 9 10 10 10 11 13 JHEP11(2016)112 The ATLAS collaboration 1 Introduction Since the ATLAS and CMS collaborations reported the observation [1, 2] of a new par- ticle with a mass of about 125 GeV and with properties consistent with those expected for the Higgs boson in the Standard Model (SM) [3–5], more precise measurements have strengthened the hypothesis that the new particle is indeed the Higgs boson [6–9]. These measurements were performed primarily in the bosonic decay modes of the new particle: H →γγ, ZZ, W +W −. It is essential to study whether it also directly decays into fermions as predicted by the SM. Recently CMS and ATLAS reported evidence for the H →τ +τ − decay mode at a significance level of 3.4 and 4.5 standard deviations, respectively [10–12], and the combination of these results qualifies as an observation [13]. However, the H →b¯b decay mode has not yet been observed [14–19], and the only direct evidence of its existence so far has been obtained by the CDF and D0 collaborations [14] at the Tevatron collider. – 1 – q q ¯b W/Z q H b W/Z q Figure 1. An example Feynman diagram illustrating vector-boson-fusion production of the Higgs boson and its decay to a b¯b pair. JHEP11(2016)112 Figure 1. An example Feynman diagram illustrating vector-boson-fusion production of the Higgs boson and its decay to a b¯b pair. The production processes of Higgs bosons at the LHC include gluon fusion (gg →H, denoted ggF), vector-boson fusion (qq →qqH, denoted VBF), Higgs-strahlung (q¯q′ → WH, ZH, denoted WH/ZH or jointly V H), and production in association with a top- quark pair (gg →t¯tH, denoted t¯tH). While an inclusive observation of the SM Higgs boson decaying to a b¯b pair is difficult in hadron collisions because of the overwhelming background from multijet production, the V H, VBF, and t¯tH processes offer viable options for the observation of the b¯b decay channel. As reported in refs. [15–19], the leptonic decays of vector bosons, the kinematic properties of the production process, and the identification of top quarks are used to reduce the background for V H, VBF, and t¯tH, respectively. This article presents a search for VBF production of the SM Higgs boson in the b¯b decay mode (VBF signal or VBF Higgs hereafter) using data recorded with the ATLAS detector in proton-proton collisions at a centre-of-mass energy √s = 8 TeV. 1ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upwards. Cylindrical coordinates (r, φ) are used in the transverse plane, φ being the azimuthal angle around the z-axis. The pseudorapidity is defined in terms of the polar angle θ as η = −ln tan(θ/2). Angular distance is measured in units of ∆R ≡ p (∆η)2 + (∆φ)2. 2 The ATLAS detector The ATLAS experiment uses a multi-purpose particle detector [20] with a forward- backward symmetric cylindrical geometry and a near 4π coverage in solid angle.1 It consists of an inner tracking detector (ID) surrounded by a thin superconducting solenoid providing a 2 T magnetic field, electromagnetic and hadronic calorimeters, and a muon spectrome- ter (MS). The ID consists of silicon pixel and microstrip tracking detectors covering the pseudorapidity range |η| < 2.5, and a transition radiation detector in the region |η| < 2.0. Lead/liquid-argon (LAr) sampling calorimeters in the region |η| < 3.2 provide electro- magnetic energy measurements with high granularity. A hadron (steel/scintillator-tile) calorimeter covers the range |η| < 1.7. The end-cap and forward regions are instrumented with LAr calorimeters for both the electromagnetic and hadronic energy measurements up to |η| = 4.9. The MS surrounds the calorimeters and is based on three large air-core toroid superconducting magnets with eight coils each. It includes a system of tracking chambers covering |η| < 2.7 and fast detectors for triggering in the range |η| < 2.4. The ATLAS trigger system [21] consists of three levels: the first (L1) is a hardware-based system, and the second and third levels are software-based systems which are collectively referred to as the high-level trigger (HLT). JHEP11(2016)112 1 Introduction The signal is searched for as a resonance in the invariant mass distribution (mbb) of a pair of jets containing b-hadrons (b-jets) in vector-boson-fusion candidates. Events are selected by requiring four energetic jets generated from the qqH →qqb¯b process as illustrated in figure 1: two light- quark jets (VBF jets) at a small angle with respect to the beam line and two b-jets from the Higgs boson decay in more central regions. Higgs bosons are colour singlets with no colour line to the bottom quarks; thus little QCD radiation and hadronic activity is expected between the two VBF jets, creating a rapidity gap between them. This feature is used to distinguish signal events from multijet events, which form the dominant background with a non-resonant contribution to the mbb distribution. Another relevant background source arises from the decay of a Z boson to b¯b in association with two jets (Z →b¯b or Z hereafter). This results in a resonant contribution to the mbb distribution. To improve the sensitivity, a multivariate analysis (MVA) is used to exploit the topol- ogy of the VBF Higgs final state. An alternative analysis is performed using kinematic cuts and the mbb distribution. The selected sample contains a minor contribution from Higgs boson events produced via the ggF process in association with two jets. These events ex- hibit an mbb distribution similar to that of VBF Higgs events, and are treated as signal in this analysis. The possible contribution of V H production to the signal was also studied but found to be negligible compared to VBF and ggF Higgs production for this analysis. – 2 – 3 Data and simulation samples The data used in this analysis were collected by the ATLAS experiment at a centre-of- mass energy of 8 TeV during 2012, and correspond to an integrated luminosity of 20.2 fb−1 recorded in stable beam conditions and with all relevant sub-detectors providing high- quality data. Events are primarily selected by a trigger requiring four jets with transverse momentum pT > 15 GeV at L1 and pT > 35 GeV in the HLT, two of which must be identified as b-jets by a dedicated HLT b-tagging algorithm (HLT b-jets). This trigger was available during the entire 2012 data-taking period. Two triggers designed to enhance the acceptance for VBF H →b¯b events (VBF Higgs triggers) were added during the 2012 data-taking period. They require either three L1 jets with pT > 15 GeV where one jet is in the forward region (|η| > 3.2), or two L1 jets in the forward region with pT > 15 GeV. These criteria are completed by the requirement of at least one HLT b-jet with pT > 35 GeV. The VBF Higgs triggers were used for a data sample corresponding to an integrated luminosity of 4.4 fb−1, resulting in an approximately 25% increase of the signal acceptance. VBF and ggF Higgs boson signal events and Z boson background events are modelled by Monte Carlo (MC) simulations. The signal samples with a Higgs boson mass of 125 GeV are generated by Powheg [22–24], which calculates the VBF and ggF Higgs production processes up to next-to-leading order (NLO) in αS. Samples of Z boson + jets events – 3 – are generated using MadGraph5 [25], where the associated jets are produced via strong or electroweak (EW) processes including VBF, and the matrix elements are calculated for up to and including three partons at leading order. For all simulated samples, the NLO CT10 parton distribution functions (PDF) [26] are used. The parton shower and the hadronisation are modelled by Pythia8 [27], with the AU2 set of tuned parameters [28, 29] for the underlying event. The VBF Higgs predictions are normalised to a cross-section calculation that in- cludes full NLO QCD and EW corrections and approximate next-to-next-to-leading-order (NNLO) QCD corrections [30]. The NLO EW corrections also affect the pT shape of the Higgs boson [31]. The pT shape is reweighted, based on the shape difference between Hawk calculations without and with NLO EW corrections included [32, 33]. 3 Data and simulation samples JHEP11(2016)112 The overall normalisation of the ggF process is taken from a calculation at NNLO in QCD that includes soft-gluon resummation up to next-to-next-to-leading logarithmic terms (NNLL) [30]. Corrections to the shape of the generated pT distribution of Higgs bosons are applied to match the distribution from the NNLO calculation with the NNLL corrections provided by the Hres program [34, 35]. In this calculation, the effects of finite masses of the top and bottom quarks are included and dynamic renormalisation and factorisation scales are used. A reweighting is derived such that the inclusive Higgs pT spectrum matches the Hres prediction, and the Higgs pT spectrum of events with at least two jets matches the the Minlo hjj [36] prediction, the most recent calculation in this phase space. The ATLAS simulation [37] of the detector is used for all MC events based on the Geant4 program [38] except for the response of the calorimeters, for which a parameterised simulation [39] is used. All simulated events are generated with a range of minimum-bias interactions overlaid on the hard-scattering interaction to account for multiple pp inter- actions that occur in the same or neighbouring bunch crossings (pile-up). The simulated events are processed with the same reconstruction algorithms as the data. Corrections are applied to the simulated samples to account for differences between data and simulation in the trigger and reconstruction efficiencies and in pile-up contributions. 5 Event pre-selection Events with exactly four jets, each with pT > 50 GeV and |η| < 4.5, are retained. The four jets are ordered in η such that η1 < η2 < η3 < η4. The jets associated with η1 and η4 are labelled as VBF jets (or J1 and J2). The other two jets associated with η2 and η3 (Higgs jets or b1 and b2) are required to be within the tracker acceptance (|η| < 2.5), and to be identified as b-jets. The two Higgs jets must be matched to the HLT b-jets for events satisfying the primary trigger; for events satisfying the VBF Higgs triggers, one of the two Higgs jets is required to be matched to an HLT b-jet. The 50 GeV cut on jet pT shapes the mbb distribution for non-resonant backgrounds, creating a peak near 130 GeV, which makes the extraction of a signal difficult. This shaping is removed by requiring the pT of the b¯b system to exceed 100 GeV. Table 1 summarises the acceptances of these pre-selection criteria, for the VBF and ggF Higgs MC events [30, 47] and the Z MC events. For the pre-selected events, corrections are applied to improve the b-jet energy mea- surements. If muons with pT > 4 GeV and |η| < 2.5 are found within a b-jet, the four- momentum of the muon closest to the jet axis is added to that of the jet (after correcting for the expected energy deposited by the muon in the calorimeter material). Such muons are reconstructed by combining measurements from the ID and MS systems, and are re- quired to satisfy tight muon identification quality criteria [48]. In addition, a pT-dependent correction of up to 5% is applied to account for biases in the response due to resolution effects. This correction is determined from simulated WH/ZH events following ref. [15]. 4 Object reconstruction Charged-particle tracks are reconstructed with a pT threshold of 400 MeV. Event vertices are formed from these tracks and are required to have at least three tracks. The primary vertex is chosen as the vertex with the largest Σ p2 T of the associated tracks. Jets are reconstructed from topological clusters of energy deposits, after noise sup- pression, in the calorimeters [40] using the anti-kt algorithm [41] with a radius parameter R = 0.4. Jet energies are corrected for the contribution of pile-up interactions using a jet-area-based technique [42], and calibrated using pT- and η-dependent correction factors determined from MC simulations and in-situ data measurements of Z+jet, γ+jet and mul- tijet events [43, 44]. To suppress jets from pile-up interactions, which are mainly at low pT, a jet vertex tagger [45], based on tracking and vertexing information, is applied to jets with pT < 50 GeV and |η| < 2.4. – 4 – Process Cross-section × BR [pb] Acceptance VBF H →b¯b 0.9 6.9 ×10−3 ggF H →b¯b 11.1 4.2 ×10−4 Z →b¯b + 1, 2, or 3 partons 5.9 ×102 3.1 ×10−4 Table 1. Cross-sections times branching ratios (BRs) used for the VBF and ggF H →b¯b and Z →b¯b MC generation, and acceptances of the pre-selection criteria for simulated samples. Table 1. Cross-sections times branching ratios (BRs) used for the VBF and ggF H →b¯b and Z →b¯b MC generation, and acceptances of the pre-selection criteria for simulated samples. Table 1. Cross-sections times branching ratios (BRs) used for the VBF and ggF H →b¯b and Z →b¯b MC generation, and acceptances of the pre-selection criteria for simulated samples. The b-jets are identified (b-tagged) by exploiting the relatively long lifetime and large mass of b-hadrons. The b-tagging methods are based on the presence of tracks with a large impact parameter with respect to the primary vertex, and secondary decay vertices. This information is combined into a single neural-network discriminant [46]. This analysis uses a b-tagging criterion that, in simulated t¯t events, provides an average efficiency of 70% for b-jets and a c-jet (light-jet) mis-tag rate less than 20% (1%). JHEP11(2016)112 6 Multivariate analysis A Boosted Decision Tree [49, 50] (BDT) method, as implemented in the Toolkit for Multi- variate Data Analysis package [51], is used to exploit the characteristics of VBF production. The BDT is trained to discriminate between VBF Higgs signal events and non-resonant – 5 – background events modelled using the data in the sideband regions of the mbb distribution (70 < mbb < 90 GeV and 150 < mbb < 190 GeV). The input variables of the BDT are chosen to exploit the difference in topologies between signal events and background events while keeping them as uncorrelated as possible with mbb, to ensure that the sideband regions provide a good description of the non-resonant background in the signal region. In order of decreasing discrimination power, which is determined by removing variables one by one from the analysis, the variables are: the jet widths of VBF jets having |η| < 2.1 (the jet width is defined as the pT-weighted angular distance of the jet constituents from the jet axis, and is set to zero if |η| > 2.1), which differs on average for quark and gluon jets; the scalar sum of the pT of additional jets with pT > 20 GeV in the region |η| < 2.5, Σpjets T ; the invariant mass of the two VBF jets, mJJ; the η separation between the two VBF jets, ∆ηJJ; the maximum |η| of the two VBF jets, max(|ηJ1|, |ηJ2|); the separation between the |η| average of the VBF jets and that of the Higgs jets, (|ηJ1| + |ηJ2|)/2 −(|ηb1| + |ηb2|)/2; and the cosine of the polar angle of the cross product of the VBF jets momenta, cos θ, which is sensitive to the production mechanism. JHEP11(2016)112 Figures 2 and 3 show the distributions of the BDT input variables in the data and the simulated samples for the VBF H →b¯b, ggF H →b¯b, and Z →b¯b events that satisfy the pre-selection criteria. The BDT responses to the pre-selected data and simulated events are compared in figure 4. As expected, the BDT response to the VBF Higgs signal sample is significantly different from its response to the data, which are primarily multijet events, and also from its response to the Z and ggF Higgs samples. 7 Invariant mass spectrum of the two b-jets The signal is estimated using a fit to the mbb distribution in the range 70 < mbb < 300 GeV. The contributions to the distribution include H →b¯b events, from either VBF or ggF production; Z →b¯b events produced in association with jets; and non-resonant processes such as multijet, t¯t, single top, and W+jets production. In order to better exploit the MVA discrimination power, the fit is performed simultaneously in four categories based on the BDT output. The boundaries of the four categories, shown in table 2, were optimised by minimising the relative statistical uncertainties, pNsig + Nbg/Nsig, where Nsig and Nbg are the expected numbers of signal and background events, respectively. Table 2 shows, for each category, the total number of events observed in the data and the number of Higgs events expected from the VBF and ggF production processes, along with the number of Z events expected in the entire mass range. The categories in table 2 are listed in order of increasing sensitivity. The shapes of the mbb distributions for Higgs and Z boson events are taken from simu- lation. Their shapes in the four categories are found to be comparable; therefore the inclu- sive shapes are used. The mbb shapes for VBF and ggF Higgs boson events are similar, as expected. In order to minimise the effects of the limited MC sample size, the resulting mbb histograms for Higgs and Z events are smoothed using the 353QH algorithm [52]. The mbb distributions used in the fit are shown in figure 5. The Higgs yield is left free to vary. The Z yield is constrained to the SM prediction within its theoretical uncertainty (see section 8.3). 7 Invariant mass spectrum of the two b-jets – 6 – w = Calorimeter width J1, J2 0 0.05 0.1 0.15 0.2 0.25 0.3 (1/N) dN/dw 0.1 0.2 0.3 0.4 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (a) [GeV] jets T p Σ w = 0 20 40 60 80 100 120 140 (1/N) dN/dw -2 10 -1 10 1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (b) [GeV] JJ w = m 0 500 1000 1500 2000 2500 3000 3500 (1/N) dN/dw 0.05 0.1 0.15 0.2 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (c) Figure 2. Distributions of the BDT input variables from the data (points) and the simulated samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms) and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the jet widths for the VBF jets having |η| < 2.1 (the jet width is set at zero if |η| > 2.1); (b) the scalar sum of the pT of additional jets with pT > 20 GeV in the region |η| < 2.5, Σpjets T (the peak at zero represents events without additional jets); and (c) the invariant mass of the two VBF jets, mJJ. w = Calorimeter width J1, J2 0 0.05 0.1 0.15 0.2 0.25 0.3 (1/N) dN/dw 0.1 0.2 0.3 0.4 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H JHEP11(2016)112 (a) a) [GeV] JJ w = m 0 500 1000 1500 2000 2500 3000 3500 (1/N) dN/dw 0.05 0.1 0.15 0.2 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (c) [GeV] jets T p Σ w = 0 20 40 60 80 100 120 140 (1/N) dN/dw -2 10 -1 10 1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (b) (c) (b) gure 2. 7 Invariant mass spectrum of the two b-jets Distributions of the BDT input variables from the data (points) and the simulated mples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms) Figure 2. Distributions of the BDT input variables from the data (points) and the simulated samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms) and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the jet widths for the VBF jets having |η| < 2.1 (the jet width is set at zero if |η| > 2.1); (b) the scalar sum of the pT of additional jets with pT > 20 GeV in the region |η| < 2.5, Σpjets T (the peak at zero represents events without additional jets); and (c) the invariant mass of the two VBF jets, mJJ. Process Pre-selection Category I Category II Category III Category IV (−0.08 to 0.01) (0.01 to 0.06) (0.06 to 0.09) (> 0.09) VBF H →b¯b 130 39 33 23 19 ggF H →b¯b 94 31 8.5 3.8 1.6 Z →b¯b 3700 1100 350 97 49 Data 554302 176073 46912 15015 6493 T bl 2 E t d b f t f VBF d F H b¯b d Z b¯b d th Process Pre-selection Category I Category II Category III Category IV (−0.08 to 0.01) (0.01 to 0.06) (0.06 to 0.09) (> 0.09) VBF H →b¯b 130 39 33 23 19 ggF H →b¯b 94 31 8.5 3.8 1.6 Z →b¯b 3700 1100 350 97 49 Data 554302 176073 46912 15015 6493 Table 2. Expected numbers of events for VBF and ggF H →b¯b and Z →b¯b processes, and the observed numbers of events in data with 70 < mbb < 300 GeV, after the pre-selection criteria are applied, in the four categories of the BDT response. The categories are listed in order of increasing sensitivity. The values in the parentheses represent the boundaries of each BDT category. Table 2. Expected numbers of events for VBF and ggF H →b¯b and Z →b¯b processes, and the observed numbers of events in data with 70 < mbb < 300 GeV, after the pre-selection criteria are applied, in the four categories of the BDT response. The categories are listed in order of increasing sensitivity. The values in the parentheses represent the boundaries of each BDT category. 7 Invariant mass spectrum of the two b-jets JJ η ∆ w = 0 1 2 3 4 5 6 7 8 9 (1/N) dN/dw 0.05 0.1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (a) |) J2 η |,| J1 η w = max(| 0 1 2 3 4 5 6 (1/N) dN/dw 0.05 0.1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (b) (1/N) dN/dw JHEP11(2016)112 (a) (b) θ w = cos -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 (1/N) dN/dw 0 0.05 0.1 0.15 0.2 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (d) * J η w = -2 -1 0 1 2 3 4 (1/N) dN/dw 0.05 0.1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (c) (d) (c) Figure 3. Distributions of the BDT input variables from the data (points) and the simulated samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms) and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the η separation between the two VBF jets, ∆ηJJ; (b) the maximum |η| of the two VBF jets, max(|ηJ1|, |ηJ2|); (c) the separation between the |η| average of the VBF jets and that of the Higgs jets, η∗ J = (|ηJ1| + |ηJ2|)/2 −(|ηb1| + |ηb2|)/2; and (d) the cosine of the polar angle of the cross product of the VBF jets momenta, cos θ. A data-driven method is used to model the mbb distribution of the non-resonant back- ground. Data in the sidebands of the mbb distribution are fit simultaneously to a function which is then interpolated to the signal region. The analytic forms considered are Bernstein polynomials [53], combinations of exponential functions, and combinations of Bernstein polynomials and exponential functions with various numbers of coefficients, and functions with a χ2 probability greater than 0.05, that do not introduce a bias, are selected. For each form, the minimum number of coefficients is determined by performing an F-test, and the corresponding function is chosen as a candidate function. 7 Invariant mass spectrum of the two b-jets – 7 – JJ η ∆ w = 0 1 2 3 4 5 6 7 8 9 (1/N) dN/dw 0.05 0.1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (a) |) J2 η |,| J1 η w = max(| 0 1 2 3 4 5 6 (1/N) dN/dw 0.05 0.1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (b) * J η w = -2 -1 0 1 2 3 4 (1/N) dN/dw 0.05 0.1 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (c) θ w = cos -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 (1/N) dN/dw 0 0.05 0.1 0.15 0.2 ATLAS -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H (d) Figure 3. Distributions of the BDT input variables from the data (points) and the simulated samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms) and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the η separation between the two VBF jets, ∆ηJJ; (b) the maximum |η| of the two VBF jets, max(|ηJ1|, |ηJ2|); (c) the separation between the |η| average of the VBF jets and that of the Higgs jets, η∗ J = (|ηJ1| + |ηJ2|)/2 −(|ηb1| + |ηb2|)/2; and (d) the cosine of the polar angle of the cross product of the VBF jets momenta, cos θ. 7 Invariant mass spectrum of the two b-jets The fitted signal strength is measured for each candidate function using toy samples. The function giving the smallest bias is used as the nominal distribution. The function giving the second smallest bias is – 8 – w = BDT response -0.3 -0.2 -0.1 0 0.1 (1/N) dN/dw 0.05 0.1 0.15 -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H ATLAS Figure 4. Distributions of the BDT response to the data (points) and to the simulated samples for VBF H →b¯b events (shaded histogram), ggF H →b¯b events (open dashed histogram) and Z →b¯b events (open solid histogram). The pre-selection criteria are applied to these samples. w = BDT response -0.3 -0.2 -0.1 0 0.1 (1/N) dN/dw 0.05 0.1 0.15 -1 = 8 TeV, 20.2 fb s data b b → Z b b → ggF H b b → VBF H ATLAS JHEP11(2016)112 Figure 4. Distributions of the BDT response to the data (points) and to the simulated samples for VBF H →b¯b events (shaded histogram), ggF H →b¯b events (open dashed histogram) and Z →b¯b events (open solid histogram). The pre-selection criteria are applied to these samples. [GeV] bb m 40 60 80 100 120 140 160 180 Entries / 5 GeV 2 4 6 8 10 ATLAS Simulation Category IV b b → Z b b → ggF H b b → VBF H Figure 5. Simulated invariant mass distributions of two b-jets from decays of Higgs bosons, summed for VBF (shaded histogram) and ggF (open dashed histogram) production, as well as from decays of Z bosons (open solid histogram), normalised to the expected contributions in category IV, which gives the highest sensitivity. [GeV] bb m 40 60 80 100 120 140 160 180 Entries / 5 GeV 2 4 6 8 10 ATLAS Simulation Category IV b b → Z b b → ggF H b b → VBF H Figure 5. Simulated invariant mass distributions of two b-jets from decays of Higgs bosons, summed for VBF (shaded histogram) and ggF (open dashed histogram) production, as well as from decays of Z bosons (open solid histogram), normalised to the expected contributions in category IV, which gives the highest sensitivity. taken as an alternative distribution, and is used to estimate the systematic uncertainty due to the choice of analytic function. 7 Invariant mass spectrum of the two b-jets The shapes of the mbb distributions are observed to be different in the four categories. Bernstein polynomials of different degrees, fourth-order in category I and third-order in the higher-sensitivity categories, are found to best describe the mbb shape of the non-resonant background. The nominal and alternative functions are summarised in table 3. 8 Sources of systematic uncertainty This section discusses sources of systematic uncertainty: experimental uncertainties, un- certainties on the modelling of the non-resonant background, and theoretical uncertainties – 9 – category I category II category III category IV Nominal 4th Pol. 3rd Pol. 3rd Pol. 3rd Pol. Alternative 2nd Pol. × exponential 3 exponentials 2 exponentials exponential Table 3. Nominal and alternative functions describing the non-resonant background in the four BDT categories. The fourth-, third-, and second-order Bernstein polynomials are referred to as 4th Pol., 3rd Pol., and 2nd Pol. on the Higgs and Z processes. The uncertainties can affect the normalisation and the kinematic distributions individually or both together. JHEP11(2016)112 8.2 Modelling uncertainties on the mbb shape of the non-resonant backgroun The uncertainties on the shape of the mbb distribution for the non-resonant background is the largest source of systematic uncertainty, contributing about 80% to the total uncer- tainty on the Higgs yield. The dominant contributions to this source come from the limited number of events in the mbb sidebands of the data used for the fit to the nominal function, and from the choice of the function. For the latter, an alternative function is chosen for each BDT region, as described in section 7 and listed in table 3. Pseudo-data are generated using the nominal functions and are fit simultaneously in the four BDT categories with nominal and alternative functions. The bin-by-bin differences in the background yield pre- dicted by the two alternative descriptions are used to estimate, by means of an eigenvector decomposition, the corresponding systematic uncertainties. 8.1 Experimental uncertainties The dominant experimental uncertainties on the Higgs signal yield arise from the statistical uncertainty due to the finite size of the MC samples, the jet energy scale uncertainty, and the b-jet triggering and tagging, contributing 15%, 10–20%, and 10% respectively, to the total uncertainty on the Higgs yield. Limited MC sizes affect the normalisation via the acceptance of the signal events and the shape of the signal mbb distribution. Several sources contribute to the uncertainty on the jet energy scale [44]. They include the in situ jet calibration, pile-up-dependent corrections and the flavour composition of jets in different event classes. The shape of the mbb distribution for the Higgs signal and the Z background is affected by the jet energy scale uncertainty. Moreover, the change in the jet energy modifies the value of the BDT output and can cause migration of events between BDT categories. The b-jet trigger and tagging efficiencies are another source of systematic uncertainty, contributing 10% to the total uncertainty. They are calibrated using multijet events containing a muon and t¯t events, respectively [54]. The uncertainty on the jet energy resolution contributes about 4%. The uncertainty on the integrated luminosity, 1.9% [55], is included, but is negligible compared to the other uncertainties mentioned above. 8.3 Theoretical uncertainties The uncertainties on the MC modelling of the Higgs signal events contribute about 10% to the total uncertainty on the Higgs yield. The sources for these uncertainties are higher order – 10 – QCD corrections, the modelling of the underlying event and the parton shower, the PDFs, and the H →b¯b branching ratio. An uncertainty on higher order QCD corrections for the cross-sections and acceptances is estimated by varying the factorisation and renormalisation scales, µF and µR, independently by a factor of two around the nominal values [31] with the constraint 0.5 ≤µF/µR ≤2. Higher order corrections to the pT spectrum of the Higgs boson (described in section 3) are an additional source of the modelling uncertainties. This uncertainty is estimated by comparing the results between LO and NLO calculations for VBF production and by varying the factorisation and renormalisation scales for ggF production. Uncertainties related to the simulation of the underlying event and the parton shower are estimated by comparing distributions obtained using Powheg+Pythia8 and Powheg+Herwig [56]. The uncertainties on the acceptance due to uncertainties in the PDFs are estimated by studying the change in the acceptance when different PDF sets such as MSTW2008NLO [57] and NNPDF2.3 [58] are used or the CT10 PDF set parameters are varied within their uncertainties. The largest variation in acceptance is taken as a systematic uncertainty. The uncertainty on the H →b¯b branching ratio, 3.2% [47], is also accounted for. JHEP11(2016)112 The uncertainty on higher order QCD corrections to the Z →b¯b yield is estimated by varying the factorisation and renormalisation scales around the nominal value in the manner described above. It is found to be about 40-50%, depending on the BDT category, out of which about 25% is correlated. These correlated and uncorrelated uncertainties are used to constrain the Z yield in the fit. This process results in about 20-25% to the total uncertainty on the Higgs yield. 9 Statistical procedure and results A statistical fitting procedure based on the RooStats framework [59, 60] is used to estimate the Higgs signal strength, µ, from the data, where µ is the ratio of the measured signal yield to the SM prediction. A binned likelihood function is constructed as the product of Poisson-probability terms of the bins in the mbb distributions, and of the four different BDT categories. The impact of systematic uncertainties on the signal and background expectations, presented in section 8, is described by a vector of nuisance parameters (NPs), ⃗θ. The expected numbers of signal and background events in each bin and category are func- tions of ⃗θ. For each NP with an a priori constraint, the prior is taken into account as a Gaussian constraint in the likelihood. The NPs associated with uncertainties in the shape and normalisation of the non-resonant background events, which do not have priors, are determined from the data. The test statistic qµ is constructed according to the profile-likelihood ratio: qµ = 2 ln(L(µ, ⃗θµ)/L(ˆµ,⃗ˆθ)), (9.1) (9.1) where ˆµ and ⃗ˆθ are the parameters that maximise the likelihood, and ⃗θµ are the nuisance parameter values that maximise the likelihood for a given µ. This test statistic is used where ˆµ and ⃗ˆθ are the parameters that maximise the likelihood, and ⃗θµ are the nuisance parameter values that maximise the likelihood for a given µ. This test statistic is used – 11 – Source of uncertainty Uncertainty on µ MVA Cut-based Experimental uncertainties Detector-related +0.2/−0.3 +1.6/−1.2 MC statistics ±0.4 ±0.1 Theoretical uncertainties MC signal modelling ±0.1 ±1.3 Z yield +0.6/−0.5 ±1.4 Non-resonant background modelling Choice of function ±1.0 ±1.0 Sideband statistics ±1.7 ±3.7 Statistical uncertainties ±1.3 Total ±2.3 +4.6/−4.4 Source of uncertainty Uncertainty on µ MVA Cut-based Experimental uncertainties Detector-related +0.2/−0.3 +1.6/−1.2 MC statistics ±0.4 ±0.1 Theoretical uncertainties MC signal modelling ±0.1 ±1.3 Z yield +0.6/−0.5 ±1.4 Non-resonant background modelling Choice of function ±1.0 ±1.0 Sideband statistics ±1.7 ±3.7 Statistical uncertainties ±1.3 Total ±2.3 +4.6/−4.4 Table 4. Summary of uncertainties on the Higgs signal strength for the MVA analysis, and for the cut-based analysis. They are estimated at the central values of the signal strength, µ = −0.8 and −5.2 for the MVA and cut-based analyses, respectively. The two systematic uncertainties accounting for non-resonant background modelling are strongly correlated. Their combined value for the MVA analysis is 1.8. JHEP11(2016)112 Table 4. 9 Statistical procedure and results Summary of uncertainties on the Higgs signal strength for the MVA analysis, and for the cut-based analysis. They are estimated at the central values of the signal strength, µ = −0.8 and −5.2 for the MVA and cut-based analyses, respectively. The two systematic uncertainties accounting for non-resonant background modelling are strongly correlated. Their combined value for the MVA analysis is 1.8. both to measure the compatibility of the background-only model with the data, and to determine exclusion intervals using the CLS method [61, 62]. both to measure the compatibility of the background-only model with the data, and to determine exclusion intervals using the CLS method [61, 62]. The robustness of the fit is validated by generating pseudo-data and estimating the number of signal events for various values of µ. The results of the fit in the four categories are shown in figure 6. The Z yield is constrained to the SM prediction within its theoretical uncertainty, using four independent constraints in the four BDT regions (uncorrelated terms) and a common constraint (correlated term) as described in section 8.3. The ratios of Z yields to the SM predictions (µZ) are found to be compatible in all of the four BDT regions. Combined over the four categories, the fit further constrains µZ to 0.7 ± 0.2. The combined Higgs signal strength is −0.8±2.3, where the uncertainty includes both the statistical (±1.3) and systematic (+1.8/−1.9) components. The breakdown of the systematic uncertainty on the estimated signal strength is given in table 4. The correlation coefficient between the combined µ and the combined µZ is found to be 0.22. In the absence of a signal, the limit on the Higgs signal strength at 95% confidence level (CL) is expected to be 5.4. When Standard Model production is assumed, the expected limit is found to be 5.7. The observed limit is 4.4. The compatibility between the measured Z yield and its SM prediction is alternatively tested by removing its a priori constraint from the fit. In this case a value of µZ = 0.3±0.3 is extracted from the fit, to be compared to the theory prediction of 1.0±0.4. The absence of the Z constraint modifies the combined Higgs signal strength slightly, to −0.5 ± 2.3. 9 Statistical procedure and results – 12 – Entries / 4 GeV 1000 2000 3000 4000 5000 6000 7000 8000 data Z component Higgs component non-resonant component (bkg) ATLAS Category I -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -100 0 100 200 (a) Entries / 4 GeV 200 400 600 800 1000 1200 1400 1600 1800 2000 data Z component Higgs component non-resonant component (bkg) ATLAS Category II -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -50 0 50 100 (b) Entries / 4 GeV 100 200 300 400 500 600 700 data Z component Higgs component non-resonant component (bkg) ATLAS Category III -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -50 0 50 100 (c) Entries / 4 GeV 50 100 150 200 250 300 data Z component Higgs component non-resonant component (bkg) ATLAS Category IV -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -40 -20 0 20 40 (d) Figure 6. Results of the profile-likelihood fit to the mbb distributions in the four BDT categories. The points represent the data, and the histograms represent the non-resonant background, Z, and Higgs contributions. In the lower panels, the data after subtraction of the non-resonant back- ground (points) are compared with the fit to the Z (open histogram) and Higgs (shaded histogram) contributions. 9 Statistical procedure and results Entries / 4 GeV 1000 2000 3000 4000 5000 6000 7000 8000 data Z component Higgs component non-resonant component (bkg) ATLAS Category I -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -100 0 100 200 (a) Entries / 4 GeV 200 400 600 800 1000 1200 1400 1600 1800 2000 data Z component Higgs component non-resonant component (bkg) ATLAS Category II -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -50 0 50 100 (b) Entries / 4 GeV JHEP11(2016)112 (a) (b) Entries / 4 GeV 100 200 300 400 500 600 700 data Z component Higgs component non-resonant component (bkg) ATLAS Category III -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -50 0 50 100 (c) Entries / 4 GeV 50 100 150 200 250 300 data Z component Higgs component non-resonant component (bkg) ATLAS Category IV -1 = 8 TeV, 20.2 fb s [GeV] bb m 100 150 200 250 Data - Bkg -40 -20 0 20 40 (d) (d) (c) Figure 6. Results of the profile-likelihood fit to the mbb distributions in the four BDT categories. The points represent the data, and the histograms represent the non-resonant background, Z, and Higgs contributions. In the lower panels, the data after subtraction of the non-resonant back- ground (points) are compared with the fit to the Z (open histogram) and Higgs (shaded histogram) contributions. 10 Cut-based analysis An alternative analysis is performed based on kinematic cuts. While the MVA performs a simultaneous fit to the mbb distributions of the four samples categorised by the BDT response, the cut-based analysis performs a fit to one mbb distribution of the entire sample in the mass range between 70 GeV and 300 GeV. Events are required to satisfy kinematic criteria featuring the VBF Higgs final state. Events must not have any additional jet with pT > 25 GeV and |η| < 2.4, and must satisfy |∆ηJJ| > 3.0 and mJJ > 650 GeV. Figure 7 shows the mbb distribution of 32906 events in the data that satisfy the selection criteria. The number of signal events in the data is expected to be 68.8, with about 15% coming from ggF production. This can be compared to 158.9 events in the MVA, as obtained by summing the corresponding numbers in table 2 over the four categories, where about 28% comes from ggF production. – 13 – Entries / 4 GeV 500 1000 1500 2000 2500 3000 data Z component Higgs component non-resonant component (bkg) ATLAS -1 = 8 TeV, 20.2 fb s Cut-based [GeV] bb m 100 150 200 250 300 Data - Bkg 100 − 50 − 0 50 100 Figure 7. Distribution of mbb for events selected in the cut-based analysis. The points represent the data, and the histograms represent the non-resonant background, Z, and Higgs contributions. In the lower panel, the data after subtraction of the non-resonant background (points) are compared with the fit to the Z (open histogram) and Higgs (shaded histogram) contributions. The Higgs yield extracted from the fit is consistent with zero. Entries / 4 GeV 500 1000 1500 2000 2500 3000 data Z component Higgs component non-resonant component (bkg) ATLAS -1 = 8 TeV, 20.2 fb s Cut-based [GeV] bb m 100 150 200 250 300 Data - Bkg 100 − 50 − 0 50 100 JHEP11(2016)112 Figure 7. Distribution of mbb for events selected in the cut-based analysis. The points represent the data, and the histograms represent the non-resonant background, Z, and Higgs contributions. In the lower panel, the data after subtraction of the non-resonant background (points) are compared with the fit to the Z (open histogram) and Higgs (shaded histogram) contributions. The Higgs yield extracted from the fit is consistent with zero. The cut-based analysis uses an unbinned maximum likelihood fit. 11 Summary A search for the Standard Model Higgs boson produced by vector-boson fusion and de- caying into a pair of bottom quarks is presented. The dataset analysed corresponds to an integrated luminosity of 20.2 fb−1 from pp collisions at √s = 8 TeV, recorded by the ATLAS experiment during Run 1 of the LHC. Events are selected using the distinct fi- nal state of the VBF H →b¯b signal, which is the presence of four energetic jets: two b-jets from the Higgs boson decay in the central region of the detector and two jets in the forward/backward region. To improve the sensitivity, a multivariate analysis is used, exploiting the topology of the VBF Higgs final state and the properties of jets. The signal yield is estimated by performing a fit to the invariant mass distribution of the two b-jets in the range 70 < mbb < 300 GeV and assuming a Higgs boson mass of 125 GeV. The ratio of the Higgs signal yield to the SM prediction is measured to be µ = −0.8 ± 1.3(stat.)+1.8 −1.9(syst.) = −0.8 ± 2.3. The upper limit on µ is observed to be µ = 4.4 at the 95% CL, which should be compared to the expected limits of 5.4 in the background-only hypothesis and 5.7 if Standard Model production is assumed. An alterna- tive analysis is performed using kinematic selection criteria and provides consistent results: µ = −5.2+4.6 −4.4 and a 95% CL upper limit of 5.4. JHEP11(2016)112 10 Cut-based analysis The resonance shapes of the mbb distributions for the Higgs and Z events are determined by a fit to a Bukin function [63] using MC events. The analytic functions describing the non-resonant background are studied by using events that satisfy the pre-selection criteria described in section 5. A fourth-order polynomial is chosen as the nominal function and a fifth-order polynomial is chosen as the alternative function. The Higgs yield is left free to vary, but the Z yield is fixed to its SM prediction. The robustness of the fit is validated by generating pseudo-data and constructing pulls of the estimated number of Higgs events for various values of µ. The fit results are presented in figure 7. The Higgs signal strength is measured to be µ = −5.2 ± 3.7(stat.)+2.7 −2.5(syst.), where the statistical uncertainty includes the statistical uncertainty on the non-resonant background modelling (see table 4). The sources of systematic uncertainty are the same as those for the MVA analysis as described in section 8 and are summarised in table 4. The uncertainties on µ are estimated as the changes in µ when the sources are varied within their uncertainties. Higher-order corrections to the Z samples and to the signal samples, the choice of function describing the non-resonant background, and the jet energy scale are the dominant sources of systematic uncertainty, each contributing about 40–50% to the total systematic uncertainty on the Higgs signal strength. The magnitudes of experimental and theoretical uncertianties are scaled with the central value of µ, as illustrated in table 4 except for the case of the MC statistical uncertainty. This is due to the fact that the MVA divides the MC samples into four categories, and uses the signal mbb distribution directly in the fit as a template while the cut-based analysis uses an interpolated function. The upper limit on the strength is found to be 5.4 at the 95% CL, which can be compared to the expected limit values of 8.5 in the background-only hypothesis and 9.5 if Standard Model production is assumed. These results are consistent with those of the MVA. As expected, the cut-based analysis is less sensitive than the MVA. – 14 – Acknowledgments We thank CERN for the very successful operation of the LHC, as well as the support staff from our institutions without whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Aus- tralia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Fed- eration; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, FP7, Horizon 2020 and Marie Sk lodowska-Curie Actions, European Union; Investissements d’Avenir Labex and Idex, ANR, R´egion Auvergne and Fondation Partager le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co- financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; – 15 – Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leve hulme Trust, United Kingdom. Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Lever- hulme Trust, United Kingdom. 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Brandt8, G. Brandt56, O. Brandt60a, U. Bratzler157, B. Brau88, J.E. Brau117, R. Brenner165, S. Bressler172, T.M. Bristow48, D. Britton55, D. Britzger44, F.M. Brochu30, R. Brenner165, S. Bressler172, T.M. Bristow48, D. Britton55, D. Britzger44, F.M. Brochu30, I. Brock23, R. Brock92, G. Brooijmans37, T. Brooks79, W.K. Brooks34b, J. Brosamer16, JHEP11(2016)112 E. Brost117, J.H Broughton19, P.A. Bruckman de Renstrom41, D. Bruncko145b, R. Brune A. Bruni22a, G. Bruni22a, BH Brunt30, M. Bruschi22a, N. Bruscino23, P. Bryant33, L. Bryngemark83, T. Buanes15, Q. Buat143, P. Buchholz142, A.G. Buckley55, I.A. Buda F. Buehrer50, M.K. Bugge120, O. Bulekov99, D. Bullock8, H. Burckhart32, S. Burdin76, C.D. Burgard50, B. Burghgrave109, K. Burka41, S. Burke132, I. Burmeister45, E. Busato36, D. B¨uscher50, V. B¨uscher85, P. Bussey55, J.M. The ATLAS collaboration Butler24, C.M. Buttar55, J.M. Butterworth80, P. Butti108, W. Buttinger27, A. Buzatu55, A.R. Buzykaev110,c, S. Cabrera Urb´an167, D. Caforio129, V.M. Cairo39a,39b, O. Cakir4a, N. Calace51, P. Calafiura16, A. Calandri87, G. Calderini82, P. Calfayan101, L.P. Caloba26a, D. Calvet36, S. Calvet36, T.P. Calvet87, R. Camacho Toro33, S. Camarda32, P. Camarri134a,134b, D. Cameron120, R. Caminal Armadans166, C. Camincher57, S. Campana32, M. Campanelli80, A. Camplani93 A. Campoverde149, V. Canale105a,105b, A. Canepa160a, M. Cano Bret35e, J. Cantero115, R. Cantrill127a, T. Cao42, M.D.M. Capeans Garrido32, I. Caprini28b, M. Caprini28b, M. Capua39a,39b, R. Caputo85, R.M. Carbone37, R. Cardarelli134a, F. Cardillo50, I. Car M. Capua39a,39b, R. Caputo85, R.M. Carbone37, R. Cardarelli134a, F. Cardillo50, I. Carli130, M. Capua39a,39b, R. Caputo85, R.M. Carbone37, R. Cardarelli134a, F. Cardillo50, T. Carli32, G. Carlino105a, L. Carminati93a,93b, S. Caron107, E. Carquin34b, . Carli32, G. Carlino105a, L. Carminati93a,93b, S. G.D. Carrillo-Montoya32, J.R. Carter30, J. Carvalho127a,127c, D. Casadei19, M.P. Casado13,h M. Casolino13, D.W. Casper163, E. Castaneda-Miranda146a, R. Castelijn108, A. Castelli108, V. Castillo Gimenez167, N.F. Castro127a,i, A. Catinaccio32, J.R. Catmore120, A. Cattai32, J. Caudron85, V. Cavaliere166, E. Cavallaro13, D. Cavalli93a, M. Cavalli-Sforza13, V. Cavasinni125a,125b, F. Ceradini135a,135b, L. Cerda Alberich167, B.C. Cerio47, A.S. Cerqueira26 A. Cerri150, L. Cerrito78, F. Cerutti16, M. Cerv32, A. Cervelli18, S.A. Cetin20d, A. Chafaq136a, D. Chakraborty109, S.K. Chan59, Y.L. Chan62a, P. Chang166, J.D. Chapman30, D.G. Charlton19 A. Chatterjee51, C.C. Chau159, C.A. Chavez Barajas150, S. Che112, S. Cheatham74, A. Chatterjee , C.C. Chau , C.A. Chavez Barajas , S. Che , S. Cheatham , A. Chegwidden92, S. Chekanov6, S.V. Chekulaev160a, G.A. Chelkov67,j, M.A. Chelstowska91, C. Chen66, H. Chen27, K. Chen149, S. Chen35c, S. Chen156, X. Chen35f, Y. Chen69, H.C. Cheng91, H.J Cheng35a, Y. Cheng33, A. Cheplakov67, E. Cheremushkina131, R. Cherkaoui El Moursli136e, V. Chernyatin27,∗, E. Cheu7, L. Chevalier137, V. Chiarella49, G. Chiarelli125a,125b, G. Chiodini75a, A.S. Chisholm19, A. Chitan28b, M.V. Chizhov67, K. Choi63, A.R. Chomont36, S. Chouridou9, B.K.B. Chow101, V. Christodoulou80, D. Chromek-Burckhart32, J. Chudoba128, A.J. Chuinard89, J.J. Chwastowski41, L. Chytka116, G. Ciapetti133a,133b, A.K. Ciftci4a, D. Cinca55, V. Cindro77, I.A. Cioara23, A. Ciocio16, F. Cirotto105a,105b, Z.H. Citron172, M. Citterio93a, M. Ciubancan28b, A. Clark51, B.L. Clark59, M.R. Clark37, P.J. Clark48, R.N. Clarke16, C. Clement147a,147b, Y. Coadou87, M. Cobal164a,164c, A. Coccaro51, J. Cochran66, L. Coffey25, L. Colasurdo107, B. Cole37, A.P. Colijn108, J. Collot57, T. Colombo32, G. Compostella102, P. Conde Mui˜no127a,127b, E. Coniavitis50, S.H. Connell146b, I.A. Connelly79, V. Consorti50, S. Constantinescu28b, G. Conti32, F. Conventi105a,k, M. Cooke16, B.D. Cooper80, A.M. Cooper-Sarkar121, K.J.R. Cormier159, T. Cornelissen175, M. Corradi133a,133b, F. Corriveau89,l, A. The ATLAS collaboration Dann86, M. Danninger168, M. Dano Hoffmann137, V. Dao50, G. Darbo52a, M. Davies154, P. Davison80, E. Dawe90, I. Dawson140, R.K. Daya-Ishmukhametova88, K. De8, , , , , y , , R. de Asmundis105a, A. De Benedetti114, S. De Castro22a,22b, S. De Cecco82, N. De Groot107, JHEP11(2016)112 P. de Jong108, H. De la Torre84, F. De Lorenzi66, A. De Maria56, D. De Pedis133a, A. De Salvo133a, U. De Sanctis150, A. De Santo150, J.B. De Vivie De Regie118, W.J. Dearnaley74, R. Debbe27, C. Debenedetti138, D.V. Dedovich67, N. Dehghanian3, I. Deigaard108, M. Del Gaudio39a,39b, J. Del Peso84, T. Del Prete125a,125b, D. Delgove118, F. Deliot137, C.M. Delitzsch51, M. Deliyergiyev77, A. Dell’Acqua32, L. Dell’Asta24, M. Dell’Orso125a,125b, M. Della Pietra105a,k, D. della Volpe51, M. Delmastro5, P.A. Delsart57, C. Deluca108, D.A. DeMarco159, S. Demers176, M. Demichev67, A. Demilly82, S.P. Denisov131, D. Denysiuk137, D. Derendarz41, J.E. Derkaoui136 F. Derue82, P. Dervan76, K. Desch23, C. Deterre44, K. Dette45, P.O. Deviveiros32, A. Dewhurst13 S. Dhaliwal25, A. Di Ciaccio134a,134b, L. Di Ciaccio5, W.K. Di Clemente123, C. Di Donato133a,13 A. Di Girolamo32, B. Di Girolamo32, B. Di Micco135a,135b, R. Di Nardo32, A. Di Simone50, R. Di Sipio159, D. Di Valentino31, C. Diaconu87, M. Diamond159, F.A. Dias48, M.A. Diaz34a, E.B. Diehl91, J. Dietrich17, S. Diglio87, A. Dimitrievska14, J. Dingfelder23, P. Dita28b, S. Dita28b, F. Dittus32, F. Djama87, T. Djobava53b, J.I. Djuvsland60a, M.A.B. do Vale26c, D. Dobos32, M. Dobre28b, C. Doglioni83, T. Dohmae156, J. Dolejsi130, Z. Dolezal130, B.A. Dolgoshein99,∗, M. Dobre28b, C. Doglioni83, T. Dohmae156, J. Dolejsi130, Z. Dolezal130, B.A. Dolgoshein99,∗, M. Donadelli26d, S. Donati125a,125b, P. Dondero122a,122b, J. Donini36, J. Dopke132, A. Doria105 M.T. Dova73, A.T. Doyle55, E. Drechsler56, M. Dris10, Y. Du35d, J. Duarte-Campderros154, E. Duchovni172, G. Duckeck101, O.A. Ducu96,m, D. Duda108, A. Dudarev32, E.M. Duffield16, L. Duflot118, L. Duguid79, M. D¨uhrssen32, M. Dumancic172, M. Dunford60a, H. Duran Yildiz4a, M. D¨uren54, A. Durglishvili53b, D. Duschinger46, B. Dutta44, M. Dyndal44, C. Eckardt44, K.M. Ecker102, R.C. Edgar91, N.C. Edwards48, T. Eifert32, G. Eigen15, K. Einsweiler16, T. Ekelof165, M. El Kacimi136c, V. Ellajosyula87, M. Ellert165, S. Elles5, F. Ellinghaus175, ot169, N. Ellis32, J. Elmsheuser27, M. Elsing32, D. Emeliyanov132, Y. Enari156, A.A. Elliot169, N. Ellis32, J. Elmsheuser27, M. Elsing32, D. Emeliyanov132, Y. Enari156, O.C. Endner85, M. Endo119, J.S. Ennis170, J. Erdmann45, A. Ereditato18, G. Ernis175, J. Ernst2 dner85, M. Endo119, J.S. Ennis170, J. Erdmann45, A. Ereditato18, G. Ernis175, J. Ernst2, M. Ernst27, S. Errede166, E. Ertel85, M. Escalier118, H. Esch45, C. Escobar126, B. Esposito49, A.I. Etienvre137, E. The ATLAS collaboration Corso-Radu163, A. Chegwidden92, S. Chekanov6, S.V. Chekulaev160a, G.A. Chelkov67,j, M.A. Chelstowska91, C. Chen66, H. Chen27, K. Chen149, S. Chen35c, S. Chen156, X. Chen35f, Y. Chen69, H.C. Cheng9 H.J Cheng35a, Y. Cheng33, A. Cheplakov67, E. Cheremushkina131, R. Cherkaoui El Moursli136e, V. Chernyatin27,∗, E. Cheu7, L. Chevalier137, V. Chiarella49, G. Chiarelli125a,125b, G. Chiodini75a, A.S. Chisholm19, A. Chitan28b, M.V. Chizhov67, K. Choi63, A.R. Chomont36, S. Chouridou9, A.S. Chisholm , A. Chitan , M.V. Chizhov , K. Choi , A.R. Chomont , S. Chouridou , B.K.B. Chow101, V. Christodoulou80, D. Chromek-Burckhart32, J. Chudoba128, A.J. Chuinard89, J.J. Chwastowski41, L. Chytka116, G. Ciapetti133a,133b, A.K. Ciftci4a, D. Cinca55, V. Cindro77, I.A. Cioara23, A. Ciocio16, F. Cirotto105a,105b, Z.H. Citron172, M. Citterio93a, M. Ciubancan28b, A. Clark51, B.L. Clark59, M.R. Clark37, P.J. Clark48, R.N. Clarke16, C. Clement147a,147b, Y. Coadou87, M. Cobal164a,164c, A. Coccaro51, J. Cochran66, L. Coffey25, L. Colasurdo107, B. Cole37, A.P. Colijn108, J. Collot57, T. Colombo32, G. Compostella102, P. Conde Mui˜no127a,127b, E. Coniavitis50, S.H. Connell146b, I.A. Connelly79, V. Consorti50, S. Constantinescu28b, G. Conti32, F. Conventi105a,k, M. Cooke16, B.D. Cooper80, A.M. Cooper-Sarkar121, K.J.R. Cormier159, T. Cornelissen175, M. Corradi133a,133b, F. Corriveau89,l, A. Corso-Radu163, B.K.B. Chow101, V. Christodoulou80, D. Chromek-Burckhart32, J. Chudoba128, A.J. Chuinard89, y I.A. Cioara23, A. Ciocio16, F. Cirotto105a,105b, Z.H. Citron172, M. Citterio93a, M. Ciubancan28b, A. Clark51, B.L. Clark59, M.R. Clark37, P.J. Clark48, R.N. Clarke16, C. Clement147a,147b, , , , , , , Y. Coadou87, M. Cobal164a,164c, A. Coccaro51, J. Cochran66, L. Coffey25, L. Colasurdo107, B. Cole37, A.P. Colijn108, J. Collot57, T. Colombo32, G. Compostella102, P. Conde Mui˜no127a,12 B. Cole , A.P. Colijn , J. Collot , T. Colombo , G. Compostella , P. Conde Muino E. Coniavitis50, S.H. Connell146b, I.A. Connelly79, V. Consorti50, S. Constantinescu28b, G. Conti32, F. Conventi105a,k, M. Cooke16, B.D. Cooper80, A.M. Cooper-Sarkar121, K.J.R. Cormier159, T. Cornelissen175, M. Corradi133a,133b, F. Corriveau89,l, A. Corso-Radu16 – 21 – A. Cortes-Gonzalez13, G. Cortiana102, G. Costa93a, M.J. Costa167, D. Costanzo140, G. Cottin30, , , , , , G. Cowan79, B.E. Cox86, K. Cranmer111, S.J. Crawley55, G. Cree31, S. Cr´ep´e-Renaudin57, , , , y , , p , F. Crescioli82, W.A. Cribbs147a,147b, M. Crispin Ortuzar121, M. Cristinziani23, V. Croft107, G. Crosetti39a,39b, T. Cuhadar Donszelmann140, J. Cummings176, M. Curatolo49, J. C´uth85, C. Cuthbert151, H. Czirr142, P. Czodrowski3, G. D’amen22a,22b, S. D’Auria55, M. D’Onofrio76, M.J. Da Cunha Sargedas De Sousa127a,127b, C. Da Via86, W. Dabrowski40a, T. Dado145a, T. Dai91, O. Dale15, F. Dallaire96, C. Dallapiccola88, M. Dam38, J.R. Dandoy33, N.P. Dang50, A.C. Daniells19, N.S. The ATLAS collaboration Etzion154, H. Evans63, A. Ezhilov124, F. Fabbri22a,22b, L. Fabbri22a,22b, G. Facini33, R.M. Fakhrutdinov131, S. Falciano133a, R.J. Falla80, J. Faltova130, Y. Fang35a, cini33, R.M. Fakhrutdinov131, S. Falciano133a, R.J. Falla80, J. Faltova130, Y. Fang35a, nti93a,93b, A. Farbin8, A. Farilla135a, C. Farina126, T. Farooque13, S. Farrell16, M. Fanti93a,93b, A. Farbin8, A. Farilla135a, C. Farina126, T. Farooque13, S. Farrell16, S.M. Farrington170, P. Farthouat32, F. Fassi136e, P. Fassnacht32, D. Fassouliotis9, M. Faucci Giannelli79, A. Favareto52a,52b, W.J. Fawcett121, L. Fayard118, O.L. Fedin124,n, M. Faucci Giannelli79, A. Favareto52a,52b, W.J. Fawcett121, L. Fayard118, O.L. Fedin124,n, L. Feremenga8, P. Fernandez Martinez167, S. Fernandez Perez13, J. Ferrando55, A. Ferrari165, L. Feremenga8, P. Fernandez Martinez167, S. Fernandez Perez13, J. Ferrando55, A. Ferrari165, P. Ferrari108, R. Ferrari122a, D.E. Ferreira de Lima60b, A. Ferrer167, D. Ferrere51, C. Ferretti A. Ferretto Parodi52a,52b, F. Fiedler85, A. Filipˇciˇc77, M. Filipuzzi44, F. Filthaut107, M. Fincke-Keeler169, K.D. Finelli151, M.C.N. Fiolhais127a,127c, L. Fiorini167, A. Firan4 A. Fischer2, C. Fischer13, J. Fischer175, W.C. Fisher92, N. Flaschel44, I. Fleck142, P. Fleischmann91, G.T. Fletcher140, R.R.M. Fletcher123, T. Flick175, A. Floderus83, L.R. Flores Castillo62a, M.J. Flowerdew102, G.T. Forcolin86, A. Formica137, A. Forti86, – 22 – A.G. Foster19, D. Fournier118, H. Fox74, S. Fracchia13, P. Francavilla82, M. Franchini22a,22b, D. Francis32, L. Franconi120, M. Franklin59, M. Frate163, M. Fraternali122a,122b, D. Freeborn8 S.M. Fressard-Batraneanu32, F. Friedrich46, D. Froidevaux32, J.A. Frost121, C. Fukunaga157, E. Fullana Torregrosa85, T. Fusayasu103, J. Fuster167, C. Gabaldon57, O. Gabizon175, Gabrielli22a,22b, A. Gabrielli16, G.P. Gach40a, S. Gadatsch32, S. Gadomski51, A. Gabrielli22a,22b, A. Gabrielli16, G.P. Gach40a, S. Gadatsch32, S. Gadomski51, G. Gagliardi52a,52b, L.G. Gagnon96, P. Gagnon63, C. Galea107, B. Galhardo127a,127c, Gagliardi52a,52b, L.G. Gagnon96, P. Gagnon63, C. Galea107, B. Galhardo127a,127c, E.J. Gallas121, B.J. Gallop132, P. Gallus129, G. Galster38, K.K. Gan112, J. Gao35b,87, Y. Gao J. Gallas121, B.J. Gallop132, P. Gallus129, G. Galster38, K.K. Gan112, J. Gao35b,87, Y. Gao48, Y.S. Gao144,f, F.M. Garay Walls48, C. Garc´ıa167, J.E. Garc´ıa Navarro167, M. Garcia-Sciveres16, Y.S. Gao144,f, F.M. Garay Walls48, C. Garc´ıa167, J.E. Garc´ıa Navarro167, M. Garcia-Sciveres16, A. Gaudiello52a,52b, G. Gaudio122a, B. Gaur142, L. Gauthier96, I.L. Gavrilenko97, C. Gay168, A. Gaudiello52a,52b, G. Gaudio122a, B. Gaur142, L. Gauthier96, I.L. Gavrilenko97, C. Gay168, Gaycken23, E.N. Gazis10, Z. Gecse168, C.N.P. Gee132, Ch. Geich-Gimbel23, M. Geisen85, G. Gaycken23, E.N. Gazis10, Z. Gecse168, C.N.P. Gee132, Ch. Geich-Gimbel23, M. Geisen85, JHEP11(2016)112 M.P. Geisler60a, C. Gemme52a, M.H. Genest57, C. Geng35b,o, S. Gentile133a,133b, S. George79, M.P. Geisler60a, C. Gemme52a, M.H. Genest57, C. Geng35b,o, S. Gentile133a,133b, S. George79, D. Gerbaudo13, A. Gershon154, S. Ghasemi142, H. Ghazlane136b, M. Ghneimat23, B. The ATLAS collaboration Giacobbe22 S. Giagu133a,133b, P. Giannetti125a,125b, B. Gibbard27, S.M. Gibson79, M. Gignac168, M. Gilchriese16, T.P.S. Gillam30, D. Gillberg31, G. Gilles175, D.M. Gingrich3,d, N. Giokaris9, M.P. Giordani164a,164c, F.M. Giorgi22a, F.M. Giorgi17, P.F. Giraud137, P. Giromini59, D. Giugni93a, F. Giuli121, C. Giuliani102, M. Giulini60b, B.K. Gjelsten120, S. Gkaitatzis155, Gkialas155, E.L. Gkougkousis118, L.K. Gladilin100, C. Glasman84, J. Glatzer32, I. Gkialas155, E.L. Gkougkousis118, L.K. Gladilin100, C. Glasman84, J. Glatzer32, P.C.F. Glaysher48, A. Glazov44, M. Goblirsch-Kolb102, J. Godlewski41, S. Goldfarb91, C.F. Glaysher48, A. Glazov44, M. Goblirsch-Kolb102, J. Godlewski41, S. Goldfarb91, T. Golling51, D. Golubkov131, A. Gomes127a,127b,127d, R. Gon¸calo127a, J. Goncalves Pinto Firmino Da Costa137, L. Gonella19, A. Gongadze67, S. Gonz´alez de la Hoz167 G. Gonzalez Parra13, S. Gonzalez-Sevilla51, L. Goossens32, P.A. Gorbounov98, H.A. Gordon2 I. Gorelov106, B. Gorini32, E. Gorini75a,75b, A. Goriˇsek77, E. Gornicki41, A.T. Goshaw47, C. G¨ossling45, M.I. Gostkin67, C.R. Goudet118, D. Goujdami136c, A.G. Goussiou139, N. Govender146b,p, E. Gozani153, L. Graber56, I. Grabowska-Bold40a, P.O.J. Gradin57, N. Govender146b,p, E. Gozani153, L. Graber56, I. Grabowska-Bold40a, P.O.J. Gradin57, P.M. Gravila28e, H.M. Gray32, E. Graziani135a, Z.D. Greenwood81,q, C. Grefe23, K. Gregers P.M. Gravila28e, H.M. Gray32, E. Graziani135a, Z.D. Greenwood81,q, C. Grefe23, K. Gregersen8 I.M. Gregor44, P. Grenier144, K. Grevtsov5, J. Griffiths8, A.A. Grillo138, K. Grimm74, S. Grinstein13,r, Ph. Gris36, J.-F. Grivaz118, S. Groh85, J.P. Grohs46, E. Gross172, J. Grosse-Knetter56, G.C. Grossi81, Z.J. Grout150, L. Guan91, W. Guan173, J. Guenther129, J. Grosse-Knetter56, G.C. Grossi81, Z.J. Grout150, L. Guan91, W. Guan173, J. Guenther129, F. Guescini51, D. Guest163, O. Gueta154, E. Guido52a,52b, T. Guillemin5, S. Guindon2, U. Gul55, C Gumpert32 J Guo35e Y Guo35b,o S Gupta121 G Gustavino133a,133b P Gutierrez114 F. Guescini51, D. Guest163, O. Gueta154, E. Guido52a,52b, T. Guillemin5, S. Guindon2, U. Gul55, C. Gumpert32, J. Guo35e, Y. Guo35b,o, S. Gupta121, G. Gustavino133a,133b, P. Gutierrez114, N.G. Gutierrez Ortiz80, C. Gutschow46, C. Guyot137, C. Gwenlan121, C.B. Gwilliam76, Gutierrez Ortiz80, C. Gutschow46, C. Guyot137, C. Gwenlan121, C.B. Gwilliam76, A. Haas111, C. Haber16, H.K. Hadavand8, N. Haddad136e, A. Hadef87, P. Haefner23, geb¨ock23, Z. Hajduk41, H. Hakobyan177,∗, M. Haleem44, J. Haley115, G. Halladjian92, S. Hageb¨ock23, Z. Hajduk41, H. Hakobyan177,∗, M. Haleem44, J. Haley115, G. Halladjian92, G.D. Hallewell87, K. Hamacher175, P. Hamal116, K. Hamano169, A. Hamilton146a, G.N. Ha D. Hallewell87, K. Hamacher175, P. Hamal116, K. Hamano169, A. Hamilton146a, G.N. Hamity140, P.G. Hamnett44, L. Han35b, K. Hanagaki68,s, K. Hanawa156, M. Hance138, B. Haney123, P.G. Hamnett44, L. Han35b, K. Hanagaki68,s, K. Hanawa156, M. Hance138, B. Haney123, P. Hanke60a, R. Hanna137, J.B. Hansen38, J.D. Hansen38, M.C. Hansen23, P.H. The ATLAS collaboration Hansen38, P. Hanke60a, R. Hanna137, J.B. Hansen38, J.D. Hansen38, M.C. Hansen23, P.H. Hansen38, K. Hara161, A.S. Hard173, T. Harenberg175, F. Hariri118, S. Harkusha94, R.D. Harrington48, P.F. Harrison170, F. Hartjes108, N.M. Hartmann101, M. Hasegawa69, Y. Hasegawa141, A. Hasib1 P.F. Harrison170, F. Hartjes108, N.M. Hartmann101, M. Hasegawa69, Y. Hasegawa141, A. Hasib114, R.J. Hawkings32, D. Hayden92, C.P. Hays121, J.M. Hays78, H.S. Hayward76, S.J. Haywood132, R.J. Hawkings32, D. Hayden92, C.P. Hays121, J.M. Hays78, H.S. Hayward76, S.J. Haywood132, S.J. Head19, T. Heck85, V. Hedberg83, L. Heelan8, S. Heim123, T. Heim16, B. Heinemann16, J.J. Heinrich101, L. Heinrich111, C. Heinz54, J. Hejbal128, L. Helary24, S. Hellman147a,147b, C. Helsens32, J. Henderson121, R.C.W. Henderson74, Y. Heng173, S. Henkelmann168, A.M. Henriques Correia32, S. Henrot-Versille118, G.H. Herbert17, Y. Hern´andez Jim´enez167, G. Herten50, R. Hertenberger101, L. Hervas32, G.G. Hesketh80, N.P. Hessey108, J.W. Hetherl Herten50, R. Hertenberger101, L. Hervas32, G.G. Hesketh80, N.P. Hessey108, J.W. Hetherly42, R. Hickling78, E. Hig´on-Rodriguez167, E. Hill169, J.C. Hill30, K.H. Hiller44, S.J. Hillier19, – 23 – I. Hinchliffe16, E. Hines123, R.R. Hinman16, M. Hirose158, D. Hirschbuehl175, J. Hobbs149, N. Hod160a, M.C. Hodgkinson140, P. Hodgson140, A. Hoecker32, M.R. Hoeferkamp106, F. Hoenig101, D. Hohn23, T.R. Holmes16, M. Homann45, T.M. Hong126, B.H. Hooberman166, W.H. Hopkins117, Y. Horii104, A.J. Horton143, J-Y. Hostachy57, S. Hou152, A. Hoummada136 J. Howarth44, M. Hrabovsky116, I. Hristova17, J. Hrivnac118, T. Hryn’ova5, A. Hrynevich95, C. Hsu146c, P.J. Hsu152,t, S.-C. Hsu139, D. Hu37, Q. Hu35b, Y. Huang44, Z. Hubacek129, F. Hubaut87, F. Huegging23, T.B. Huffman121, E.W. Hughes37, G. Hughes74, M. Huhtin T.A. H¨ulsing85, P. Huo149, N. Huseynov67,b, J. Huston92, J. Huth59, G. Iacobucci O. Igonkina108,u, T. Iizawa171, Y. Ikegami68, M. Ikeno68, Y. Ilchenko11,v, D. Iliadis155, N. Ilic14 T. Ince102, G. Introzzi122a,122b, P. Ioannou9,∗, M. Iodice135a, K. Iordanidou37, V. Ippolito59 JHEP11(2016)112 M. Ishino70, M. Ishitsuka158, R. Ishmukhametov112, C. Issever121, S. Istin20a, F. Ito161, J.M. Iturbe Ponce86, R. Iuppa134a,134b, W. Iwanski41, H. Iwasaki68, J.M. Izen43, V. Izzo105a, S. Jabbar3, B. Jackson123, M. Jackson76, P. Jackson1, V. Jain2, K.B. Jakobi85, K. Jakobs50, S. Jakobsen32, T. Jakoubek128, D.O. Jamin115, D.K. Jana81, E. Jansen80, R. Jansky64, Janssen23, M. Janus56, G. Jarlskog83, N. Javadov67,b, T. Jav˚urek50, F. Jeanneau137, J. Janssen23, M. Janus56, G. Jarlskog83, N. Javadov67,b, T. Jav˚urek50, F. Jeanneau137, anty16, J. Jejelava53a,w, G.-Y. Jeng151, D. Jennens90, P. Jenni50,x, J. Jentzsch45, C. Jeske170, L. Jeanty16, J. Jejelava53a,w, G.-Y. Jeng151, D. Jennens90, P. Jenni50,x, J. Jentzsch45, C. J´ez´equel5, H. Ji173, J. Jia149, H. Jiang66, Y. Jiang35b, S. Jiggins80, J. Jimenez Pena167, S. J´ez´equel5, H. Ji173, J. The ATLAS collaboration Jia149, H. Jiang66, Y. Jiang35b, S. Jiggins80, J. Jimenez Pena167, Jin35a, A. Jinaru28b, O. Jinnouchi158, P. Johansson140, K.A. Johns7, W.J. Johnson139, S. Jin35a, A. Jinaru28b, O. Jinnouchi158, P. Johansson140, K.A. Johns7, W.J. Johnson139, Jon-And147a,147b, G. Jones170, R.W.L. Jones74, S. Jones7, T.J. Jones76, J. Jongmanns60a, K. Jon-And147a,147b, G. Jones170, R.W.L. Jones74, S. Jones7, T.J. Jones76, J. Jongmann P.M. Jorge127a,127b, J. Jovicevic160a, X. Ju173, A. Juste Rozas13,r, M.K. K¨ohler172, M. Jorge127a,127b, J. Jovicevic160a, X. Ju173, A. Juste Rozas13,r, M.K. K¨ohler172, aczmarska41, M. Kado118, H. Kagan112, M. Kagan144, S.J. Kahn87, E. Kajomovitz47, A. Kaczmarska41, M. Kado118, H. Kagan112, M. Kagan144, S.J. Kahn87, E. Kajomovitz47, C.W. Kalderon121, A. Kaluza85, S. Kama42, A. Kamenshchikov131, N. Kanaya156, S. Kanet C.W. Kalderon121, A. Kaluza85, S. Kama42, A. Kamenshchikov131, N. Kanaya156, S. Kaneti30, L. Kanjir77, V.A. Kantserov99, J. Kanzaki68, B. Kaplan111, L.S. Kaplan173, A. Kapliy33, 77, V.A. Kantserov99, J. Kanzaki68, B. Kaplan111, L.S. Kaplan173, A. Kapliy33, D. Kar146c, K. Karakostas10, A. Karamaoun3, N. Karastathis10, M.J. Kareem56, E. Karentzos1 D. Kar146c, K. Karakostas10, A. Karamaoun3, N. Karastathis10, M.J. Kareem56, E. Karentzos10 A.N. Karyukhin131, K. Kasahara161, L. Kashif173, R.D. Kass112, A. Kastanas15, Y. Kataoka156, A.N. Karyukhin131, K. Kasahara161, L. Kashif173, R.D. Kass112, A. Kastanas15, Y. Kataoka156, C. Kato156, A. Katre51, J. Katzy44, K. Kawagoe72, T. Kawamoto156, G. Kawamura56, S. Kazama156, V.F. Kazanin110,c, R. Keeler169, R. Kehoe42, J.S. Keller44, J.J. Kempster S. Kazama156, V.F. Kazanin110,c, R. Keeler169, R. Kehoe42, J.S. Keller44, J.J. Kempster79, K Kentaro104, H. Keoshkerian159, O. Kepka128, B.P. Kerˇsevan77, S. Kersten175, R.A. Keye F. Khalil-zada12, A. Khanov115, A.G. Kharlamov110,c, T.J. Khoo51, V. Khovanskiy98, mov67, J. Khubua53b,y, S. Kido69, H.Y. Kim8, S.H. Kim161, Y.K. Kim33, N. Kimura155, E. Khramov67, J. Khubua53b,y, S. Kido69, H.Y. Kim8, S.H. Kim161, Y.K. Kim33, N. Kimur O.M. Kind17, B.T. King76, M. King167, S.B. King168, J. Kirk132, A.E. Kiryunin102, T. Kishimoto69, D. Kisielewska40a, F. Kiss50, K. Kiuchi161, O. Kivernyk137, E. Kladiva145b, M.H. Klein37, M. Klein76, U. Klein76, K. Kleinknecht85, P. Klimek147a,147b, A. Klimentov27, R. Klingenberg45, J.A. Klinger140, T. Klioutchnikova32, E.-E. Kluge60a, P. Kluit108, S. Kluth102 J. Knapik41, E. Kneringer64, E.B.F.G. Knoops87, A. Knue55, A. Kobayashi156, D. Kobayashi158 T. Kobayashi156, M. Kobel46, M. Kocian144, P. Kodys130, T. Koffas31, E. Koffeman108, T. Koi144, H. Kolanoski17, M. Kolb60b, I. Koletsou5, A.A. Komar97,∗, Y. Komori156, T. Kondo68, N. Kondrashova44, K. K¨oneke50, A.C. K¨onig107, T. Kono68,z, R. Konoplich111,aa, N. Konstantinidis80, R. Kopeliansky63, S. Koperny40a, L. K¨opke85, A.K. Kopp50, K. Korcyl41, K. Kordas155, A. Korn80, A.A. Korol110,c, I. Korolkov13, E.V. Korolkova140, O. The ATLAS collaboration Kortner102, N. Konstantinidis80, R. Kopeliansky63, S. Koperny40a, L. Kopke85, A.K. Kopp50, K. Korcyl41, K. Kordas155, A. Korn80, A.A. Korol110,c, I. Korolkov13, E.V. Korolkova140, O. Kortner102, S. Kortner102, T. Kosek130, V.V. Kostyukhin23, A. Kotwal47, A. Kourkoumeli-Charalampidi155, C. Kourkoumelis9, V. Kouskoura27, A.B. Kowalewska41, R. Kowalewski169, T.Z. Kowalski40a, C. Kozakai156, W. Kozanecki137, A.S. Kozhin131, V.A. Kramarenko100, G. Kramberger77, D. Krasnopevtsev99, M.W. Krasny82, A. Krasznahorkay32, J.K. Kraus23, A. Kravchenko27, M. Kretz60c, J. Kretzschmar76, K. Kreutzfeldt54, P. Krieger159, K. Krizka33, K. Kroeninger45, H. Kroha102, J. Kroll123, J. Kroseberg23, J. Krstic14, U. Kruchonak67, H. Kr¨uger23, K. Kordas155, A. Korn80, A.A. Korol110,c, I. Korolkov13, E.V. Korolkova140, O. Kortner102, – 24 – N. Krumnack66, A. Kruse173, M.C. Kruse47, M. Kruskal24, T. Kubota90, H. Kucuk80, S. Kuday4 , , , , , , y J.T. Kuechler175, S. Kuehn50, A. Kugel60c, F. Kuger174, A. Kuhl138, T. Kuhl44, V. Kukhtin67, , , g , g , , , R. Kukla137, Y. Kulchitsky94, S. Kuleshov34b, M. Kuna133a,133b, T. Kunigo70, A. Kupco128, H. Kurashige69, Y.A. Kurochkin94, V. Kus128, E.S. Kuwertz169, M. Kuze158, J. Kvita116, T. Kwan169, D. Kyriazopoulos140, A. La Rosa102, J.L. La Rosa Navarro26d, L. La Rotonda39 C. Lacasta167, F. Lacava133a,133b, J. Lacey31, H. Lacker17, D. Lacour82, V.R. La E. Ladygin67, R. Lafaye5, B. Laforge82, T. Lagouri176, S. Lai56, S. Lammers63, W. Lampl7, E. Lan¸con137, U. Landgraf50, M.P.J. Landon78, V.S. Lang60a, J.C. Lange13, A.J. L F. Lasagni Manghi22a,22b, M. Lassnig32, P. Laurelli49, W. Lavrijsen16, A.T. Law138, P. Laycock76, g g , g , , j , , y T. Lazovich59, M. Lazzaroni93a,93b, B. Le90, O. Le Dortz82, E. Le Guirriec87, E.P. Le Quilleuc13 JHEP11(2016)112 M. LeBlanc169, T. LeCompte6, F. Ledroit-Guillon57, C.A. Lee27, S.C. Lee152, L. Lee1, G. Lefebvre82, M. Lefebvre169, F. Legger101, C. Leggett16, A. Lehan76, G. Lehmann Miotto32, X. Lei7, W.A. Leight31, A. Leisos155,ab, A.G. Leister176, M.A.L. Leite26d, R. Leitner130, D. Lellouch172, B. Lemmer56, K.J.C. Leney80, T. Lenz23, B. Lenzi32, R. Leone7, S. Leone125a,125 C. Leonidopoulos48, S. Leontsinis10, G. Lerner150, C. Leroy96, A.A.J. Lesage137, C.G. Lester30, M. Levchenko124, J. Levˆeque5, D. Levin91, L.J. Levinson172, M. Levy19, D. Lewis78, A.M. Leyko2 M. Leyton43, B. Li35b,o, H. Li149, H.L. Li33, L. Li47, L. Li35e, Q. Li35a, S. Li47, X. Li86, Y. Li142 Z. Liang35a, B. Liberti134a, A. Liblong159, P. Lichard32, K. Lie166, J. Liebal23, W. Liebig15, A. Limosani151, S.C. Lin152,ac, T.H. Lin85, B.E. Lindquist149, A.E. Lionti51, E. Lipeles123, A. Lipniacka15, M. Lisovyi60b, T.M. Liss166, A. Lister168, A.M. Litke138, B. Liu152,ad, D. Liu152 H. The ATLAS collaboration Liu91, H. Liu27, J. Liu87, J.B. Liu35b, K. Liu87, L. Liu166, M. Liu47, M. Liu35b, Y.L. Liu35b, Y. Liu35b, M. Livan122a,122b, A. Lleres57, J. Llorente Merino35a, S.L. Lloyd78, F. Lo Sterzo152, E. Lobodzinska44, P. Loch7, W.S. Lockman138, F.K. Loebinger86, A.E. Loevschall-Jensen38, K.M. Loew25, A. Loginov176, T. Lohse17, K. Lohwasser44, M. Lokajicek128, B.A. Long24, B. Lopez Paredes140, I. Lopez Paz13, A. Lopez Solis82, J. Lorenz101, N. Lorenzo Martinez63, M. Losada21, P.J. L¨osel101, X. Lou35a, A. Lounis118, J. Love6, P.A. Love74, H. Lu62a, N. Lu91, H.J. Lubatti139, C. Luci133a,133b, A. Lucotte57, C. Luedtke50, F. Luehring63, W. Lukas64, L. Luminari133a, O. Lundberg147a,147b, B. Lund-Jensen148, P.M. Luzi82, D. Lynn27, R. Lysa E. Lytken83, V. Lyubushkin67, H. Ma27, L.L. Ma35d, Y. Ma35d, G. Maccarrone49, A. Macchiolo102, C.M. Macdonald140, B. Maˇcek77, J. Machado Miguens123,127b, D. R. Madar36, H.J. Maddocks165, W.F. Mader46, A. Madsen44, J. Maeda69, S. Maeland15, Madar36, H.J. Maddocks165, W.F. Mader46, A. Madsen44, J. Maeda69, S. Maeland15, Maeno27, A. Maevskiy100, E. Magradze56, J. Mahlstedt108, C. Maiani118, C. Maidantchik26a, T. Maeno27, A. Maevskiy100, E. Magradze56, J. Mahlstedt108, C. Maiani118, C. Maidantchik A. Maier102, T. Maier101, A. Maio127a,127b,127d, S. Majewski117, Y. Makida68, N. Makovec118, A.A. Maier102, T. Maier101, A. Maio127a,127b,127d, S. Majewski117, Y. Makida68, N. Makovec B. Malaescu82, Pa. Malecki41, V.P. Maleev124, F. Malek57, U. Mallik65, D. Malon6, C. Malone144, I. Mandi´c77, J. Maneira127a,127b, L. Manhaes de Andrade Filho26b, J. Manjarres Ramos160b, S. Manzoni93a,93b, L. Mapelli32, G. Marceca29, L. March51, G. Marchiori82, M. Marcis S. Marti-Garcia167, B. Martin92, T.A. Martin170, V.J. Martin48, B. Martin dit Lat M. Martinez13,r, S. Martin-Haugh132, V.S. Martoiu28b, A.C. Martyniuk80, M. Marx139, – 25 – J.A. Mcfayden80, G. Mchedlidze56, S.J. McMahon132, R.A. McPherson169,l, M. Medinnis44, y , , , , S. Meehan139, S. Mehlhase101, A. Mehta76, K. Meier60a, C. Meineck101, B. Meirose43, , , , , , , D. Melini167, B.R. Mellado Garcia146c, M. Melo145a, F. Meloni18, A. Mengarelli22a,22b, S. Menke102, E. Meoni162, S. Mergelmeyer17, P. Mermod51, L. Merola105a,105b, C. Meroni93a F.S. Merritt33, A. Messina133a,133b, J. Metcalfe6, A.S. Mete163, C. Meyer85, C. Meyer123, J-P. Meyer137, J. Meyer108, H. Meyer Zu Theenhausen60a, F. Miano150, R.P. Middleton132, S. Miglioranzi52a,52b, L. Mijovi´c23, G. Mikenberg172, M. Mikestikova128, M. Mikuˇz77, M. Milesi9 A. Milic64, D.W. Miller33, C. Mills48, A. Milov172, D.A. Milstead147a,147b, A.A. Minaenko13 , , , , , g , L.M. Mir13, K.P. Mistry123, T. Mitani171, J. Mitrevski101, V.A. Mitsou167, A. Miucci51, P.S. Miyagawa140, J.U. Mj¨ornmark83, T. Moa147a,147b, K. Mochizuki96, S. Mohapatra37, JHEP11(2016)112 S. Molander147a,147b, R. The ATLAS collaboration Moles-Valls23, R. Monden70, M.C. Mondragon92, K. M¨onig44, J. Monk38, E. Monnier87, A. Montalbano149, J. Montejo Berlingen32, F. Monticelli73, S. Monzani93a,93b, R.W. Moore3, N. Morange118, D. Moreno21, M. Moreno Ll´acer56, P. Morettini52a, D. Mori143 T. Mori156, M. Morii59, M. Morinaga156, V. Morisbak120, S. Moritz85, A.K. Morley151, Mornacchi32, J.D. Morris78, S.S. Mortensen38, L. Morvaj149, M. Mosidze53b, J. Moss144, G. Mornacchi32, J.D. Morris78, S.S. Mortensen38, L. Morvaj149, M. Mosidze53b, J. Moss144, K. Motohashi158, R. Mount144, E. Mountricha27, S.V. Mouraviev97,∗, E.J.W. Moyse88, Motohashi158, R. Mount144, E. Mountricha27, S.V. Mouraviev97,∗, E.J.W. Moyse88, S. Muanza87, R.D. Mudd19, F. Mueller102, J. Mueller126, R.S.P. Mueller101, T. Mueller30, Muanza87, R.D. Mudd19, F. Mueller102, J. Mueller126, R.S.P. Mueller101, T. Mueller30, D. Muenstermann74, P. Mullen55, G.A. Mullier18, F.J. Munoz Sanchez86, J.A. Murillo Quija stermann74, P. Mullen55, G.A. Mullier18, F.J. Munoz Sanchez86, J.A. Murillo Quijada19, W.J. Murray170,132, H. Musheghyan56, M. Muˇskinja77, A.G. Myagkov131,ae, M. Myska129, rray170,132, H. Musheghyan56, M. Muˇskinja77, A.G. Myagkov131,ae, M. Myska129, B.P. Nachman144, O. Nackenhorst51, K. Nagai121, R. Nagai68,z, K. Nagano68, Y. Nagasaka6 B.P. Nachman144, O. Nackenhorst51, K. Nagai121, R. Nagai68,z, K. Nagano68, Y. Nagasaka61, K. Nagata161, M. Nagel50, E. Nagy87, A.M. Nairz32, Y. Nakahama32, K. Nakamura68, T. Nakamura156, I. Nakano113, H. Namasivayam43, R.F. Naranjo Garcia44, R. Narayan11, T. Nakamura156, I. Nakano113, H. Namasivayam43, R.F. Naranjo Garcia44, R. Narayan11, D.I. Narrias Villar60a, I. Naryshkin124, T. Naumann44, G. Navarro21, R. Nayyar7, H.A. Neal91, P.Yu. Nechaeva97, T.J. Neep86, P.D. Nef144, A. Negri122a,122b, M. Negrini22a, S. Nektarijevic107, P.Yu. Nechaeva97, T.J. Neep86, P.D. Nef144, A. Negri122a,122b, M. Negrini22a, S. Nektarijevic107, M.S. Neubauer166, M. Neumann175, R.M. Neves111, P. Nevski27, P.R. Newman19, D.H. Nguyen6 T. Nguyen Manh96, R.B. Nickerson121, R. Nicolaidou137, J. Nielsen138, A. Nikiforov17, V. Nikolaenko131,ae, I. Nikolic-Audit82, K. Nikolopoulos19, J.K. Nilsen120, P. Nilsson27, Y. Ninomiya156, A. Nisati133a, R. Nisius102, T. Nobe156, L. Nodulman6, M. Nomachi119 miya156, A. Nisati133a, R. Nisius102, T. Nobe156, L. Nodulman6, M. Nomachi119, I. Nomidis31, T. Nooney78, S. Norberg114, M. Nordberg32, N. Norjoharuddeen121, is31, T. Nooney78, S. Norberg114, M. Nordberg32, N. Norjoharuddeen121, orodova46, S. Nowak102, M. Nozaki68, L. Nozka116, K. Ntekas10, E. Nurse80, F. Nuti90, O. Novgorodova46, S. Nowak102, M. Nozaki68, L. Nozka116, K. Ntekas10, E. Nurse80 O’grady7, D.C. O’Neil143, A.A. O’Rourke44, V. O’Shea55, F.G. Oakham31,d, H. Oberlack102, F. O’grady7, D.C. O’Neil143, A.A. O’Rourke44, V. O’Shea55, F.G. Oakham31,d, H. Oberlack1 T. Obermann23, J. Ocariz82, A. Ochi69, I. Ochoa37, J.P. Ochoa-Ricoux34a, S. Oda72, S. Odak Obermann23, J. Ocariz82, A. Ochi69, I. Ochoa37, J.P. Ochoa-Ricoux34a, S. Oda72, S. Odaka68, H. Ogren63, A. The ATLAS collaboration Oh86, S.H. Oh47, C.C. Ohm16, H. Ohman165, H. Oide32, H. Okawa161, Ogren63, A. Oh86, S.H. Oh47, C.C. Ohm16, H. Ohman165, H. Oide32, H. Okawa161, Okumura33, T. Okuyama68, A. Olariu28b, L.F. Oleiro Seabra127a, S.A. Olivares Pino48, Y. Okumura33, T. Okuyama68, A. Olariu28b, L.F. Oleiro Seabra127a, S.A. Olivares Pino48 D. Oliveira Damazio27, A. Olszewski41, J. Olszowska41, A. Onofre127a,127e, K. Onogi104, D. Oliveira Damazio27, A. Olszewski41, J. Olszowska41, A. Onofre127a,127e, K. Onogi104, U.E. Onyisi11,v, M.J. Oreglia33, Y. Oren154, D. Orestano135a,135b, N. Orlando62b, R.S. Orr159, B. Osculati52a,52b, R. Ospanov86, G. Otero y Garzon29, H. Otono72, M. Ouchrif136d, Osculati52a,52b, R. Ospanov86, G. Otero y Garzon29, H. Otono72, M. Ouchrif136d, F. Ould-Saada120, A. Ouraou137, K.P. Oussoren108, Q. Ouyang35a, M. Owen55, R.E. Owen F. Ould-Saada120, A. Ouraou137, K.P. Oussoren108, Q. Ouyang35a, M. Owen55, R.E. Ow M. Pag´aˇcov´a50, S. Pagan Griso16, F. Paige27, P. Pais88, K. Pajchel120, G. Palacino160b, M. Pag´aˇcov´a50, S. Pagan Griso16, F. Paige27, P. Pais88, K. Pajchel120, G. Palacino160b g , g , g , , j , S. Palestini32, M. Palka40b, D. Pallin36, A. Palma127a,127b, E.St. Panagiotopoulou10, S. Palestini32, M. Palka40b, D. Pallin36, A. Palma127a,127b, E.St. Panagiotopoulou10, , A. Palma127a,127b, E.St. Panagiotopoulou10, g E. Pandini82, J.G. Panduro Vazquez79, P. Pani147a,147b, S. Panitkin27, D. Pantea28b, C.E. Pandini82, J.G. Panduro Vazquez79, P. Pani147a,147b, S. Panitkin27, D. Pantea28b Paolozzi51, Th.D. Papadopoulou10, K. Papageorgiou155, A. Paramonov6, L. Paolozzi51, Th.D. Papadopoulou10, K. Papageorgiou155, A. Paramonov6, Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a,52b, D. Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a,52b, A. Parsons37, U. Parzefall50, V.R. Pascuzzi159, E. Pasqualucci133a, S. Passaggio52a, D. Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a,52b J.A. Parsons37, U. Parzefall50, V.R. Pascuzzi159, E. Pasqualucci133a, S. Passaggio52a, J.A. Parsons37, U. Parzefall50, V.R. Pascuzzi159, E. Pasqualucci133a, S. Passaggio52a, Fr. Pastore79, G. P´asztor31,ag, S. Pataraia175, J.R. Pater86, T. Pauly32, J. Pearce169, – 26 – B. Pearson114, L.E. Pedersen38, M. Pedersen120, S. Pedraza Lopez167, R. Pedro127a,127b, , , , p , , S V Peleganchuk110,c D Pelikan165 O Penc128 C Peng35a H Peng35b J Penwell63 S.V. Peleganchuk110,c, D. Pelikan165, O. Penc128, C. Peng35a, H. Peng35b, J. Penwell63, g g g B.S. Peralva26b, M.M. Perego137, D.V. Perepelitsa27, E. Perez Codina160a, L. Perini93a,93b, H. Pernegger32, S. Perrella105a,105b, R. Peschke44, V.D. Peshekhonov67, K. Peters44, R.F.Y. Peters86, B.A. Petersen32, T.C. Petersen38, E. Petit57, A. Petridis1, C. Petridou155, P. Petroff118, E. Petrolo133a, M. Petrov121, F. Petrucci135a,135b, N.E. The ATLAS collaboration Pettersson88, A. Peyau R. Pezoa34b, P.W. Phillips132, G. Piacquadio144, E. Pianori170, A. Picazio88, E. Piccaro78, M. Piccinini22a,22b, M.A. Pickering121, R. Piegaia29, J.E. Pilcher33, A.D. Pilk M. Pitt172, L. Plazak145a, M.-A. Pleier27, V. Pleskot85, E. Plotnikova67, P. Plucinski92, D. Pluth M. Pitt172, L. Plazak145a, M.-A. Pleier27, V. Plesk M. Pitt 7 , L. Plazak 5 , M.-A. Pleier 7, V. Pleskot85, E. Plotnikova67, P. Plucinski9 , D. Pluth R. Poettgen147a,147b, L. Poggioli118, D. Pohl23, G. Polesello122a, A. Poley44, A. Policicchio39a,39b JHEP11(2016)112 R. Polifka159, A. Polini22a, C.S. Pollard55, V. Polychronakos27, K. Pomm`es32, L. Pontecorvo133a, B.G. Pope92, G.A. Popeneciu28c, D.S. Popovic14, A. Poppleton32, S. Pospisil129, K. Potamianos I.N. Potrap67, C.J. Potter30, C.T. Potter117, G. Poulard32, J. Poveda32, V. Pozdnyakov67, M.E. Pozo Astigarraga32, P. Pralavorio87, A. Pranko16, S. Prell66, D. Price86, L.E. Price6, M. Primavera75a, S. Prince89, M. Proissl48, K. Prokofiev62c, F. Prokoshin34b, S. Protopopescu27 J. Proudfoot6, M. Przybycien40a, D. Puddu135a,135b, D. Puldon149, M. Purohit27,ai, P. Puzo118, J. Qian91, G. Qin55, Y. Qin86, A. Quadt56, W.B. Quayle164a,164b, M. Queitsch-Maitland86, D. Quilty55, S. Raddum120, V. Radeka27, V. Radescu60b, S.K. Radhakrishnan149, P. Radloff117, P. Rados90, F. Ragusa93a,93b, G. Rahal178, J.A. Raine86, S. Rajagopalan27, M. Rammensee32 dos90, F. Ragusa93a,93b, G. Rahal178, J.A. Raine86, S. Rajagopalan27, M. Rammensee32, C. Rangel-Smith165, M.G. Ratti93a,93b, F. Rauscher101, S. Rave85, T. Ravenscroft55, ngel-Smith165, M.G. Ratti93a,93b, F. Rauscher101, S. Rave85, T. Ravenscroft55, I. Ravinovich172, M. Raymond32, A.L. Read120, N.P. Readioff76, M. Reale75a,75b, D.M. Rebuzzi122a,122b, A. Redelbach174, G. Redlinger27, R. Reece138, K. Reeves43, L. Rehnisch17, D.M. Rebuzzi122a,122b, A. Redelbach174, G. Redlinger27, R. Reece138, K. Reeves43, L. Rehn J. Reichert123, H. Reisin29, C. Rembser32, H. Ren35a, M. Rescigno133a, S. Resconi93a, O.L. Rezanova110,c, P. Reznicek130, R. Rezvani96, R. Richter102, S. Richter80, E. Richter-Was40b, O.L. Rezanova110,c, P. Reznicek130, R. Rezvani96, R. Richter102, S. Richter80, E. Richter-Was40b O.L. Rezanova110,c, P. Reznicek130, R. Rezvani96, R. Richter102, S. Richter80, E. Richter O. Ricken23, M. Ridel82, P. Rieck17, C.J. Riegel175, J. Rieger56, O. Rifki114, M. Rijssenb A. Rimoldi122a,122b, M. Rimoldi18, L. Rinaldi22a, B. Risti´c51, E. Ritsch32, I. Riu13, A. Rimoldi122a,122b, M. Rimoldi18, L. Rinaldi22a, B. Risti´c51, E. Ritsch32, I. Riu13, F. Rizatdinova115, E. Rizvi78, C. Rizzi13, S.H. Robertson89,l, A. Robichaud-Veronneau89 Robinson30, J.E.M. Robinson44, A. Robson55, C. Roda125a,125b, Y. Rodina87, D. Robinson30, J.E.M. Robinson44, A. Robson55, C. Roda125a,125b, Y. Rodina87, driguez Perez13, D. Rodriguez Rodriguez167, S. Roe32, C.S. Rogan59, O. Røhne120, A. Rodriguez Perez13, D. Rodriguez Rodriguez167, S. Roe32, C.S. Rogan59, O. Røhne120, A. Romaniouk99, M. Romano22a,22b, S.M. Romano Saez36, E. The ATLAS collaboration Simard5, Lj. Simic14, jacki14, J. Silva127a,127d, S.B. Silverstein147a, V. Simak129, O. Simard5, Lj. Simic14, S. Simion118, E. Simioni85, B. Simmons80, D. Simon36, M. Simon85, P. Sinervo159, N.B. Sinev117, M. Sioli22a,22b, G. Siragusa174, S.Yu. Sivoklokov100, J. Sj¨olin147a,147b, T.B. Sjursen15, , , , , , , M. Sioli22a,22b, G. Siragusa174, S.Yu. Sivoklokov100, J. Sj¨olin147a,147b, T.B. Sjursen15, Skinner74, H.P. Skottowe59, P. Skubic114, M. Slater19, T. Slavicek129, M. Slawinska108, M.B. Skinner74, H.P. Skottowe59, P. Skubic114, M. Slater19, T. Slavicek129, M. Slawins 162 130 172 5 15 145 M.B. Skinner , H.P. Skottowe , P. Skubic , M. Slater , T. Slavicek , M. Slawinska , K. Sliwa162, R. Slovak130, V. Smakhtin172, B.H. Smart5, L. Smestad15, J. Smiesko145a, K. Sliwa162, R. Slovak130, V. Smakhtin172, B.H. Smart5, L. Smestad15, J. Smiesko145a, R.W. Smith37, M. Smizanska74, K. Smolek129, A.A. Snesarev97, S. Snyder27, R. Sobie169,l, R.W. Smith37, M. Smizanska74, K. Smolek129, A.A. Snesarev97, S. Snyder27, R. Sobie169,l, y F. Socher46, A. Soffer154, D.A. Soh152, G. Sokhrannyi77, C.A. Solans Sanchez32, M. Solar129 E.Yu. Soldatov99, U. Soldevila167, A.A. Solodkov131, A. Soloshenko67, O.V. Solovyanov131, E.Yu. Soldatov99, U. Soldevila167, A.A. Solodkov131, A. Soloshenko67, O.V. Solovyanov131, 124 50 162 35b 16 129 12 D. South44, B.C. Sowden79, S. Spagnolo75a,75b, M. Spalla125a,125b, M. Spangenberg170, an`o79, D. Sperlich17, F. Spettel102, R. Spighi22a, G. Spigo32, L.A. Spiller90, M. Spousta130, F. Span`o79, D. Sperlich17, F. Spettel102, R. Spighi22a, G. Spigo32, L.A. Spiller90, M. Sp R.D. St. Denis55,∗, A. Stabile93a, R. Stamen60a, S. Stamm17, E. Stanecka41, R.W. Stanek6, St. Denis55,∗, A. Stabile93a, R. Stamen60a, S. Stamm17, E. Stanecka41, R.W. Stanek6, C. Stanescu135a, M. Stanescu-Bellu44, M.M. Stanitzki44, S. Stapnes120, E.A. Starchenko131, Stanescu135a, M. Stanescu-Bellu44, M.M. Stanitzki44, S. Stapnes120, E.A. Starchenko131, H. Stark33, J. Stark57, P. Staroba128, P. Starovoitov60a, S. St¨arz32, R. Staszewski41, G.H. Stark33, J. Stark57, P. Staroba128, P. Starovoitov60a, S. St¨arz32, R. Staszewski41, Steinberg27, B. Stelzer143, H.J. Stelzer32, O. Stelzer-Chilton160a, H. Stenzel54, G.A. Stewart55, P. Steinberg27, B. Stelzer143, H.J. Stelzer32, O. Stelzer-Chilton160a, H. Stenzel54, G.A. Stewa J.A. Stillings23, M.C. Stockton89, M. Stoebe89, G. Stoicea28b, P. Stolte56, S. Stonjek102, A. Stillings23, M.C. Stockton89, M. Stoebe89, G. Stoicea28b, P. Stolte56, S. Stonjek102, A.R. Stradling8, A. Straessner46, M.E. Stramaglia18, J. Strandberg148, S. Strandberg147a, R. Stradling8, A. Straessner46, M.E. Stramaglia18, J. Strandberg148, S. Strandberg147a,147b, A. Strandlie120, M. Strauss114, P. Strizenec145b, R. Str¨ohmer174, D.M. Strom117, A. Strandlie120, M. Strauss114, P. Strizenec145b, R. Str¨ohmer174, D.M. Strom117, y g g y R. Subramaniam81, S. Suchek60a, Y. Sugaya119, M. Suk129, V.V. Sulin97, S. Sultansoy4c, R. The ATLAS collaboration Schaarschmidt172, P. Schacht102, B.M. Schachtner101, D. Schaefer32, R. Schaefer44, , , , , , J. Schaarschmidt172, P. Schacht102, B.M. Schachtner101, D. Schaefer32, R. Schaefer44, J. Schaeffer85, S. Schaepe23, S. Schaetzel60b, U. Sch¨afer85, A.C. Schaffer118, D. Schaile101, D. Schamberger149, V. Scharf60a, V.A. Schegelsky124, D. Scheirich130, M. Schernau163, R.D. Schamberger149, V. Scharf60a, V.A. Schegelsky124, D. Scheirich130, M. Schernau163, Schiavi52a,52b, S. Schier138, C. Schillo50, M. Schioppa39a,39b, S. Schlenker32, C. Schiavi52a,52b, S. Schier138, C. Schillo50, M. Schioppa39a,39b, S. Schlenker32, R. Schmidt-Sommerfeld102, K. Schmieden32, C. Schmitt85, S. Schmitt44, S. Schmitz85, K.R. Schmidt-Sommerfeld102, K. Schmieden32, C. Schmitt85, S. Schmitt44, S. Schmitz85, B. Schneider160a, U. Schnoor50, L. Schoeffel137, A. Schoening60b, B.D. Schoenrock92, E. Scho Schneider160a, U. Schnoor50, L. Schoeffel137, A. Schoening60b, B.D. Schoenrock92, E. Schopf23, M. Schott85, J. Schovancova8, S. Schramm51, M. Schreyer174, N. Schuh85, M.J. Schultens23, M. Schott85, J. Schovancova8, S. Schramm51, M. Schreyer174, N. Schuh85, M.J. Schultens23, A. Schwartzman144, T.A. Schwarz91, Ph. Schwegler102, H. Schweiger86, Ph. Schwemling137, A. Schwartzman144, T.A. Schwarz91, Ph. Schwegler102, H. Schweiger86, Ph. Schwemling137, R. Schwienhorst92, J. Schwindling137, T. Schwindt23, G. Sciolla25, F. Scuri125a,125b, F. Scu JHEP11(2016)112 Searcy91, P. Seema23, S.C. Seidel106, A. Seiden138, F. Seifert129, J.M. Seixas26a, J. Searcy91, P. Seema23, S.C. Seidel106, A. Seiden138, F. Seifert129, J.M. Seixas26a, khniaidze105a, K. Sekhon91, S.J. Sekula42, D.M. Seliverstov124,∗, N. Semprini-Cesari22a,22b, G. Sekhniaidze105a, K. Sekhon91, S.J. Sekula42, D.M. Seliverstov124,∗, N. Semprini-Cesari22a G. Sekhniaidze , K. Sekhon , S.J. Sekula , D.M. Seliverstov , N. Semprini Cesari C. Serfon120, L. Serin118, L. Serkin164a,164b, M. Sessa135a,135b, R. Seuster169, H. Severini114, C. Serfon120, L. Serin118, L. Serkin164a,164b, M. Sessa135a,135b, R. Seuster169, H. Severini114, Sfiligoj77, F. Sforza32, A. Sfyrla51, E. Shabalina56, N.W. Shaikh147a,147b, L.Y. Shan35a, T. Sfiligoj77, F. Sforza32, A. Sfyrla51, E. Shabalina56, N.W. Shaikh147a,147b, L.Y. Shan35a R. Shang166, J.T. Shank24, M. Shapiro16, P.B. Shatalov98, K. Shaw164a,164b, S.M. Shaw86 Shang166, J.T. Shank24, M. Shapiro16, P.B. Shatalov98, K. Shaw164a,164b, S.M. Shaw86, A. Shcherbakova147a,147b, C.Y. Shehu150, P. Sherwood80, L. Shi152,aj, S. Shimizu69, Shcherbakova147a,147b, C.Y. Shehu150, P. Sherwood80, L. Shi152,aj, S. Shimizu69, Shimmin163, M. Shimojima103, M. Shiyakova67,ak, A. Shmeleva97, D. Shoaleh Saadi96, C.O. Shimmin163, M. Shimojima103, M. Shiyakova67,ak, A. Shmeleva97, D. Shoaleh Saadi M.J. Shochet33, S. Shojaii93a,93b, S. Shrestha112, E. Shulga99, M.A. Shupe7, P. Sicho128, J. Shochet33, S. Shojaii93a,93b, S. Shrestha112, E. Shulga99, M.A. Shupe7, P. Sicho128, M. Sickles166, P.E. Sidebo148, O. Sidiropoulou174, D. Sidorov115, A. Sidoti22a,22b, F. Siegert46, A.M. Sickles166, P.E. Sidebo148, O. Sidiropoulou174, D. Sidorov115, A. Sidoti22a,22b, F. Siege Dj. Sijacki14, J. Silva127a,127d, S.B. Silverstein147a, V. Simak129, O. The ATLAS collaboration Romero Adam167, N. Rompotis139, A. Romaniouk99, M. Romano22a,22b, S.M. Romano Saez36, E. Romero Adam167, N. Rompotis139, M. Ronzani50, L. Roos82, E. Ros167, S. Rosati133a, K. Rosbach50, P. Rose138, O. Rosen Ronzani50, L. Roos82, E. Ros167, S. Rosati133a, K. Rosbach50, P. Rose138, O. Rosenthal142, N.-A. Rosien56, V. Rossetti147a,147b, E. Rossi105a,105b, L.P. Rossi52a, J.H.N. Rosten30, -A. Rosien56, V. Rossetti147a,147b, E. Rossi105a,105b, L.P. Rossi52a, J.H.N. Rosten30, R. Rosten139, M. Rotaru28b, I. Roth172, J. Rothberg139, D. Rousseau118, C.R. Royon137, Rosten139, M. Rotaru28b, I. Roth172, J. Rothberg139, D. Rousseau118, C.R. Royon137, Rozanov87, Y. Rozen153, X. Ruan146c, F. Rubbo144, M.S. Rudolph159, F. R¨uhr50, A. Rozanov87, Y. Rozen153, X. Ruan146c, F. Rubbo144, M.S. Rudolph159, F. R¨uhr50, A. Ruiz-Martinez31, Z. Rurikova50, N.A. Rusakovich67, A. Ruschke101, H.L. Russell139, Ruiz-Martinez31, Z. Rurikova50, N.A. Rusakovich67, A. Ruschke101, H.L. Russell139, P. Rutherfoord7, N. Ruthmann32, Y.F. Ryabov124, M. Rybar166, G. Rybkin118, S. Ryu6, J.P. Rutherfoord7, N. Ruthmann32, Y.F. Ryabov124, M. Rybar166, G. Rybkin118, S. Ryu6 Ryzhov131, G.F. Rzehorz56, A.F. Saavedra151, G. Sabato108, S. Sacerdoti29, A. Ryzhov131, G.F. Rzehorz56, A.F. Saavedra151, G. Sabato108, S. Sacerdoti29, F-W. Sadrozinski138, R. Sadykov67, F. Safai Tehrani133a, P. Saha109, M. Sahinsoy60a, H.F-W. Sadrozinski138, R. Sadykov67, F. Safai Tehrani133a, P. Saha109, M. Sahinsoy60a, M. Saimpert137, T. Saito156, H. Sakamoto156, Y. Sakurai171, G. Salamanna135a,135b, M. Saimpert137, T. Saito156, H. Sakamoto156, Y. Sakurai171, G. Salamanna135a,135b, y g A. Salnikov144, J. Salt167, D. Salvatore39a,39b, F. Salvatore150, A. Salvucci62a, A. Salzburger32, A. Salnikov144, J. Salt167, D. Salvatore39a,39b, F. Salvatore150, A. Salvucci62a, A. Salzburger32, F. Salvatore150, A. Salvucci62a, A. Salzburger32, Sammel50, D. Sampsonidis155, A. Sanchez105a,105b, J. S´anchez167, V. Sanchez Martinez167, D. Sammel50, D. Sampsonidis155, A. Sanchez105a,105b, J. S´anchez167, V. Sanchez Martinez167 H. Sandaker120, R.L. Sandbach78, H.G. Sander85, M. Sandhoff175, C. Sandoval21, H. Sandaker120, R.L. Sandbach78, H.G. Sander85, M. Sandhoff175, C. Sandoval21, Sandstroem102, D.P.C. Sankey132, M. Sannino52a,52b, A. Sansoni49, C. Santoni36, R. Sandstroem102, D.P.C. Sankey132, M. Sannino52a,52b, A. Sansoni49, C. Santoni36, , A. Sansoni49, C. Santoni36, Santonico134a,134b, H. Santos127a, I. Santoyo Castillo150, K. Sapp126, A. Sapronov67, R. Santonico134a,134b, H. Santos127a, I. Santoyo Castillo150, K. Sapp126, A. Sapronov67, Saraiva127a,127d, B. Sarrazin23, O. Sasaki68, Y. Sasaki156, K. Sato161, G. Sauvage5,∗, J.G. Saraiva127a,127d, B. Sarrazin23, O. Sasaki68, Y. Sasaki156, K. Sato161, G. Sauvage5,∗, E. Sauvan5, G. Savage79, P. Savard159,d, C. Sawyer132, L. Sawyer81,q, J. Saxon33, C. Sbarra22a, E. Sauvan5, G. Savage79, P. Savard159,d, C. Sawyer132, L. Sawyer81,q, J. Saxon33, C. Sbarra22a, – 27 – A. Sbrizzi22a,22b, T. Scanlon80, D.A. Scannicchio163, M. Scarcella151, V. Scarfone39a,39b, , , , , J. The ATLAS collaboration Tsirintanis9, S. Tsiskaridze13, V. Tsiskaridze50, E.G. Tskhadadze53a, K.M. Tsui62a, I.I. Tsukerman98, V. Tsulaia16, S. Tsuno68, D. Tsybychev149, A. Tudorache28b, V. Tud . Tsukerman98, V. Tsulaia16, S. Tsuno68, D. Tsybychev149, A. Tudorache28b, V. Tudorache28b, N. Tuna59, S.A. Tupputi22a,22b, S. Turchikhin100,al, D. Turecek129, D. Turgeman172, A.N. Tuna59, S.A. Tupputi22a,22b, S. Turchikhin100,al, D. Turecek129, D. Turgeman172, R. Turra93a,93b, A.J. Turvey42, P.M. Tuts37, M. Tyndel132, G. Ucchielli22a,22b, I. Ueda156, R. Turra93a,93b, A.J. Turvey42, P.M. Tuts37, M. Tyndel132, G. Ucchielli22a,22b, I. Ueda156 R. Ueno31, M. Ughetto147a,147b, F. Ukegawa161, G. Unal32, A. Undrus27, G. Unel163, R. Ueno31, M. Ughetto147a,147b, F. Ukegawa161, G. Unal32, A. Undrus27, G. Unel163, F.C. Ungaro90, Y. Unno68, C. Unverdorben101, J. Urban145b, P. Urquijo90, P. Urrejola85, F.C. Ungaro90, Y. Unno68, C. Unverdorben101, J. Urban145b, P. Urquijo90, P. Urrejola85, G. Usai8, A. Usanova64, L. Vacavant87, V. Vacek129, B. Vachon89, C. Valderanis101, G. Usai8, A. Usanova64, L. Vacavant87, V. Vacek129, B. Vachon89, C. Valderanis101, E. Valdes Santurio147a,147b, N. Valencic108, S. Valentinetti22a,22b, A. Valero167, L. Valery13, E. Valdes Santurio147a,147b, N. Valencic108, S. Valentinetti22a,22b, A. Valero167, L. Valery1 S. Valkar130, S. Vallecorsa51, J.A. Valls Ferrer167, W. Van Den Wollenberg108, S. Valkar130, S. Vallecorsa51, J.A. Valls Ferrer167, W. Van Den Wollenberg108, P.C. Van Der Deijl108, R. van der Geer108, H. van der Graaf108, N. van Eldik153, P.C. Van Der Deijl108, R. van der Geer108, H. van der Graaf108, N. van Eldik153, P. van Gemmeren6, J. Van Nieuwkoop143, I. van Vulpen108, M.C. van Woerden32, van Gemmeren6, J. Van Nieuwkoop143, I. van Vulpen108, M.C. van Woerden32, M. Vanadia133a,133b, W. Vandelli32, R. Vanguri123, A. Vaniachine6, P. Vankov108, M. Vanadia133a,133b, W. Vandelli32, R. Vanguri123, A. Vaniachine6, P. Vankov108, A. Vartapetian8, K.E. Varvell151, J.G. Vasquez176, F. Vazeille36, T. Vazquez Schroeder A. Vartapetian8, K.E. Varvell151, J.G. Vasquez176, F. Vazeille36, T. Vazquez Schroeder J. Veatch56, L.M. Veloce159, F. Veloso127a,127c, S. Veneziano133a, A. Ventura75a,75b, M Veatch56, L.M. Veloce159, F. Veloso127a,127c, S. Veneziano133a, A. Ventura75a,75b, M. Venturi169 N. Venturi159, A. Venturini25, V. Vercesi122a, M. Verducci133a,133b, W. Verkerke108, N. Venturi159, A. Venturini25, V. Vercesi122a, M. Verducci133a,133b, W. Verkerke108, C. Vermeulen108, A. Vest46,aq, M.C. Vetterli143,d, O. Viazlo83, I. Vichou166, T. Vickey140, J.C. Vermeulen108, A. Vest46,aq, M.C. Vetterli143,d, O. Viazlo83, I. Vichou166, T. Vic O.E. Vickey Boeriu140, G.H.A. Viehhauser121, S. Viel16, L. Vigani121, R. Vigne64, M. Villa22a,22b, 93 93b 49 31 67 22 22b O.E. Vickey Boeriu140, G.H.A. Viehhauser121, S. Viel16, L. Vigani121, R. Vigne64, M. Villa22a,22b, key Boeriu140, G.H.A. Viehhauser121, S. Viel16, L. Vigani121, R. Vigne64, M. The ATLAS collaboration Subramaniam81, S. Suchek60a, Y. Sugaya119, M. Suk129, V.V. Sulin97, S. Sultansoy4c, T. Sumida70, S. Sun59, X. Sun35a, J.E. Sundermann50, K. Suruliz150, G. Susinno39a,39b, , , , , , , M.R. Sutton150, S. Suzuki68, M. Svatos128, M. Swiatlowski33, I. Sykora145a, T. Sykora130, D. Ta50, C. Taccini135a,135b, K. Tackmann44, J. Taenzer159, A. Taffard163, R. Tafirout160a, N. Taiblum154, H. Takai27, R. Takashima71, T. Takeshita141, Y. Takubo68, M. Talby87, A.A. Talyshev110,c, , , , , , , M.R. Sutton150, S. Suzuki68, M. Svatos128, M. Swiatlowski33, I. Sykora145a, T. Sykora130, D. Ta50, , , , , , , M.R. Sutton150, S. Suzuki68, M. Svatos128, M. Swiatlowski33, I. Sykora145a, T. Sykora130, D. Ta50, C. Taccini135a,135b, K. Tackmann44, J. Taenzer159, A. Taffard163, R. Tafirout160a, N. Taiblum154, H. Takai27, R. Takashima71, T. Takeshita141, Y. Takubo68, M. Talby87, A.A. Talyshev110,c, K.G. Tan90, J. Tanaka156, R. Tanaka118, S. Tanaka68, B.B. Tannenwald112, S. Tapia Araya34b, S. Tapprogge85, S. Tarem153, G.F. Tartarelli93a, P. Tas130, M. Tasevsky128, T. Tashiro70, H. Takai27, R. Takashima71, T. Takeshita141, Y. Takubo68, M. Talby87, A.A. Talyshev110,c, S. Tapprogge85, S. Tarem153, G.F. Tartarelli93a, P. Tas130, M. Tasevsky128, T. Tashiro70, – 28 – E. Tassi39a,39b, A. Tavares Delgado127a,127b, Y. Tayalati136d, A.C. Taylor106, G.N. Taylor90, P.T.E. Taylor90, W. Taylor160b, F.A. Teischinger32, P. Teixeira-Dias79, K.K. Temming50, y , y , g , , g , D. Temple143, H. Ten Kate32, P.K. Teng152, J.J. Teoh119, F. Tepel175, S. Terada68, K. Terashi15 p , , g , , p , , J. Terron84, S. Terzo102, M. Testa49, R.J. Teuscher159,l, T. Theveneaux-Pelzer87, J.P. Thomas19 J. Thomas-Wilsker79, E.N. Thompson37, P.D. Thompson19, A.S. Thompson55, L.A. Thomsen176 E. Thomson123, M. Thomson30, M.J. Tibbetts16, R.E. Ticse Torres87, V.O. Tikhomirov97,an Yu.A. Tikhonov110,c, S. Timoshenko99, P. Tipton176, S. Tisserant87, K. Todome158, T. Todorov5,∗, S. Todorova-Nova130, J. Tojo72, S. Tok´ar145a, K. Tokushuku68, E. Tol T. Todorov5,∗, S. Todorova-Nova130, J. Tojo72, S. Tok´ar145a, K. Tokushuku68, E. Tolley5 omlinson86, M. Tomoto104, L. Tompkins144,ao, K H. Torres143, E. Torr´o Pastor139, J. Toth87,ap, F. Touchard87, D.R. Tovey140, T. Trefzger174, H. Torres143, E. Torr´o Pastor139, J. Toth87,ap, F. Touchard87, D.R. Tovey140, T. Trefzger174, A. Tricoli27, I.M. Trigger160a, S. Trincaz-Duvoid82, M.F. Tripiana13, W. Trischuk159, B. Trocm´e JHEP11(2016)112 A. Trofymov44, C. Troncon93a, M. Trottier-McDonald16, M. Trovatelli169, L. Truong164a A. Trofymov44, C. Troncon93a, M. Trottier-McDonald16, M. Trovatelli169, L. Truong164a,164c, M. Trzebinski41, A. Trzupek41, J.C-L. Tseng121, P.V. Tsiareshka94, G. Tsipolitis10, Trzebinski41, A. Trzupek41, J.C-L. Tseng121, P.V. Tsiareshka94, G. Tsipolitis10, N. Tsirintanis9, S. Tsiskaridze13, V. Tsiskaridze50, E.G. Tskhadadze53a, K.M. Tsui62a, N. The ATLAS collaboration Villa22a,22b, Villaplana Perez93a,93b, E. Vilucchi49, M.G. Vincter31, V.B. Vinogradov67, C. Vittori22a,22b, Vivarelli150, S. Vlachos10, M. Vlasak129, M. Vogel175, P. Vokac129, G. Volpi125a,125b, M. Volpi90, I. Vivarelli150, S. Vlachos10, M. Vlasak129, M. Vogel175, P. Vokac129, G. Volpi125a,125b, M. von der Schmitt102, E. von Toerne23, V. Vorobel130, K. Vorobev99, M. Vos167, R. Voss32, H. von der Schmitt102, E. von Toerne23, V. Vorobel130, K. Vorobev99, M. Vos167, R. Voss32, H. Vossebeld76, N. Vranjes14, M. Vranjes Milosavljevic14, V. Vrba128, M. Vreeswijk108, J.H. Vossebeld76, N. Vranjes14, M. Vranjes Milosavljevic14, V. Vrba128, M. Vreeswijk108, R. Vuillermet32, I. Vukotic33, Z. Vykydal129, P. Wagner23, W. Wagner175, H. Wahlberg73, R. Vuillermet32, I. Vukotic33, Z. Vykydal129, P. Wagner23, W. Wagner175, H. Wahlberg73 S. Wahrmund46, J. Wakabayashi104, J. Walder74, R. Walker101, W. Walkowiak142, S. Wahrmund46, J. Wakabayashi104, J. Walder74, R. Walker101, W. Walkowiak142, V. Wallangen147a,147b, C. Wang35c, C. Wang35d,87, F. Wang173, H. Wang16, H. Wang42, V. Wallangen147a,147b, C. Wang35c, C. Wang35d,87, F. Wang173, H. Wang16, H. Wang42, J. Wang44, J. Wang151, K. Wang89, R. Wang6, S.M. Wang152, T. Wang23, T. Wang37, W. Wang35b, X. Wang176, C. Wanotayaroj117, A. Warburton89, C.P. Ward30, D.R. Wardrope80, W. Wang35b, X. Wang176, C. Wanotayaroj117, A. Warburton89, C.P. Ward30, D.R. Wardrope80, B.M. Waugh80, S. Webb85, M.S. Weber18, S.W. Weber174, J.S. Webster6, A.R. Weidberg121, L.A.M. Wiik-Fuchs23, A. Wildauer102, F. Wilk86, H.G. Wilkens32, H.H. Williams123, S. Williams108, C. Willis92, S. Willocq88, J.A. Wilson19, I. Wingerter-Seez5, F. Winklmeier11 – 29 – O.J. Winston150, B.T. Winter23, M. Wittgen144, J. Wittkowski101, S.J. Wollstadt85, J. Winston150, B.T. Winter23, M. Wittgen144, J. Wittkowski101, S.J. Wollstadt85, g M.W. Wolter41, H. Wolters127a,127c, B.K. Wosiek41, J. Wotschack32, M.J. Woudstra86, W. Wolter41, H. Wolters127a,127c, B.K. Wosiek41, J. Wotschack32, M.J. Woudstra86, , , , , , K.W. Wozniak41, M. Wu57, M. Wu33, S.L. Wu173, X. Wu51, Y. Wu91, T.R. Wyatt86, , , , , , W. Wozniak41, M. Wu57, M. Wu33, S.L. Wu173, X. Wu51, Y. Wu91, T.R. Wyatt86, B.M. Wynne48, S. Xella38, D. Xu35a, L. Xu27, B. Yabsley151, S. Yacoob146a, R. Yakabe69, B.M. Wynne48, S. Xella38, D. Xu35a, L. Xu27, B. Yabsley151, S. Yacoob146a, R. Yakabe69 D. Yamaguchi158, Y. Yamaguchi119, A. Yamamoto68, S. Yamamoto156, T. Yamanaka156, D. Yamaguchi158, Y. Yamaguchi119, A. Yamamoto68, S. Yamamoto156, T. Yamanaka156, K. Yamauchi104, Y. Yamazaki69, Z. Yan24, H. Yang35e, H. Yang173, Y. Yang152, Z. Yang15, K. Yamauchi104, Y. Yamazaki69, Z. Yan24, H. Yang35e, H. Yang173, Y. Yang152, Z. Yang1 W-M. Yao16, Y.C. Yap82, Y. Yasu68, E. Yatsenko5, K.H. Yau Wong23, J. Ye42, S. The ATLAS collaboration Ye27, I. Yeletskikh67, A.L. Yen59, E. Yildirim85, K. Yorita171, R. Yoshida6, K. Yosh S.P.Y. Yuen23, I. Yusuff30,ar, B. Zabinski41, R. Zaidan35d, A.M. Zaitsev131,ae, N JHEP11(2016)112 M. Zeman129, A. Zemla40a, J.C. Zeng166, Q. Zeng144, K. Zengel25, O. Zenin131, T. ˇZeniˇs145a, D. Zerwas118, D. Zhang91, F. Zhang173, G. Zhang35b,am, H. Zhang35c, J. Zhang6, L. Zhang50, R. Zhang23, R. Zhang35b,as, X. Zhang35d, Z. Zhang118, X. Zhao42, Y. Zhao35d, Z. Zhao35b, R. Zhang23, R. Zhang35b,as, X. Zhang35d, Z. Zhang118, X. Zhao42, Y. Zhao35d, Z. Zhao35b, A Zh h 67 J Zh 121 B Zh 91 C Zh 47 L Zh 37 L Zh 42 M Zh 149 A. Zhemchugov67, J. Zhong121, B. Zhou91, C. Zhou47, L. Zhou37, L. Zhou42, M. Zhou149, N. Zhou35f, C.G. Zhu35d, H. Zhu35a, J. Zhu91, Y. Zhu35b, X. Zhuang35a, K. Zhukov97, A. Zibell174, D. Zieminska63, N.I. Zimine67, C. Zimmermann85, S. Zimmermann50, Z. Zinonos56 85 142 ˇ 14 173 22 22b 17 A. Zibell174, D. Zieminska63, N.I. Zimine67, C. Zimmermann85, S. Zimmermann50, Z. Zinonos56, M. Zinser85, M. Ziolkowski142, L. ˇZivkovi´c14, G. Zobernig173, A. Zoccoli22a,22b, M. zur Nedden17, G. Zurzolo105a,105b and L. Zwalinski32 Zinser85, M. Ziolkowski142, L. ˇZivkovi´c14, G. Zobernig173, A. Zoccoli22a,22b, M. zur Nedden17, Zurzolo105a,105b and L. Zwalinski32 G. Zurzolo105a,105b and L. The ATLAS collaboration Zwalinski32 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany NY, United States of America 3 Department of Physics, University of Alberta, Edmonton AB, Canada Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5 LAPP, CNRS/IN2P3 and Universit´e Savoie Mont Blanc, Annecy-le-Vieux, France 5 LAPP, CNRS/IN2P3 and Universit´e Savoie Mont Blanc, Annecy-le-Vieux, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United Sta 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of Amer 7 Department of Physics, University of Arizona, Tucson AZ, United States of Ameri 8 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 8 Department of Physics, The University of Texas at Arlington, Arlington TX, United States of America sics Department, University of Athens, Athens, Gree 10 Physics Department, National Technical University of Athens, Zografou, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Department of Physics, The University of Texas at Austin, Austin TX, United States of America 11 Department of Physics, The University of Texas at Austin, Austin TX, United 12 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 12 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13 Institut de F´ısica d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain, Spain 14 Institute of Physics, University of Belgrade, Belgrade, Serbia 14 Institute of Physics, University of Belgrade, Belgrade, Serbia 16 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 16 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 17 Department of Physics, Humboldt University, Berlin, Germany 17 Department of Physics, Humboldt University, Berlin, Germany 18 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 18 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, stein Center for Fundamental Physics and Laboratory 20 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics Engineering, Gaziantep University, Gaziantep; (d) Istanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul,Turkey; (e) Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey, Turkey 21 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica e Astronomia, Universit`a di Bologna, Bologna, Italy – 30 – 23 Physikalisches Institut, University of Bonn, Bonn, Germany 24 Department of Physics, Boston University, Boston MA, United States of America 25 Department of Physics, Brandeis University, Waltham MA, United States of America Department of Physics, Brandeis University, Waltham MA, United States of America 26 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 27 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America ( ) ( ) 28 (a) Transilvania University of Brasov, Brasov, Romania; (b) National Institute of Physics and Nuclear Engineering, Bucharest; (c) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (d) University Politehnica Bucharest, Bucharest; (e) West University in Timisoara, Timisoara, Romania JHEP11(2016)112 29 Departamento de F´ısica, Universidad de Buenos Aires, Buenos Aires, Argentina 30 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 31 Department of Physics, Carleton University, Ottawa ON, Canada 32 CERN, Geneva, Switzerland 32 CERN, Geneva, Switzerland 33 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 34 (a) Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b) Departamento de F´ısica, Universidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Chile 35 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Department of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and Cosmology, Shanghai Jiao Tong University, Shanghai; (also affiliated with PKU-CHEP); (f) Physics Department, Tsinghua University, Beijing 100084, China Tsinghua University, Beijing 100084, China 36 Laboratoire de Physique Corpusculaire, Clermont Universit´e and Universit´e Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 37 Nevis Laboratory, Columbia University, Irvington NY, United States of America 37 Nevis Laboratory, Columbia University, Irv 38 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark ( ) 39 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Fisica, Universit`a della Calabria, Rende, Italy 39 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di 39 (a) INFN Gruppo Collegato di Cosenza, Laborat Fisica, Universit`a della Calabria, Rende, Italy 39 (a) INFN Gruppo Collegato di Cosenza, Laborat Fisica, Universit`a della Calabria, Rende, Italy 40 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 40 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Scien K k (b) M i S l h ki I i f Ph i J i ll i U i i K k P l d ; f y , g y, , 41 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 41 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland Physics Polish Academy of Sciences, Krakow, Polan 42 Physics Department, Southern Methodist University, Dallas TX, United States of America 42 Physics Department, Southern Methodist University, Dallas TX, United States of America 43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 44 DESY, Hamburg and Zeuthen, Germany 43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 44 DESY Hamburg and Zeuthen Germany 43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 44 DESY, Hamburg and Zeuthen, Germany y p , y f , , f 44 DESY, Hamburg and Zeuthen, Germany 44 DESY, Hamburg and Zeuthen, Germany 45 Institut f¨ur Experimentelle Physik IV, Technische Universit¨at Dortmund, Dortmund, Germany 46 Institut f¨ur Kern- und Teilchenphysik, Technische Universit¨at Dresden, Dresden, Germany 48 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 48 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy UPA - School of Physics and Astronomy, University 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy 50 Fakult¨at f¨ur Mathematik und Physik, Albert-Ludwigs-Universit¨at, Freiburg, Germany 51 Section de Physique, Universit´e de Gen`eve, Geneva, Switzerland 52 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit`a di Genova, Genova, Italy 53 (a) E. The ATLAS collaboration Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 53 (a) E. Andronikashvili Institute of Physics, Iv. The ATLAS collaboration Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 54 II Physikalisches Institut, Justus-Liebig-Universit¨at Giessen, Giessen, Germany hes Institut, Justus-Liebig-Universit¨at Giessen, Giess 55 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56 II Physikalisches Institut, Georg-August-Universit¨at, G¨ottingen, Germany 57 Laboratoire de Physique Subatomique et de Cosmologie, Universit´e Grenoble-Alpes, CNRS/IN2P3, Grenoble, France – 31 – 58 Department of Physics, Hampton University, Hampton VA, United States of America 59 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge MA, United States of America f 60 (a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (c) ZITI Insti 60 (a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (c) ZITI Institut f¨ur technische Informatik, Ruprecht-Karls-Universit¨at Heidelberg, Mannheim, Germany technische Informatik, Ruprecht-Karls-Universit¨at Heidelberg, Mannheim, Germany 61 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 62 (a) Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b) Department of Physics, The University of Hong Kong, Hong Kong; (c) Department of Physics, The H K U i it f S i d T h l Cl W t B K l H K Chi (a) Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b) Department of Physics The University of Hong Kong Hong Kong; (c) Department of Physics The Physics, The University of Hong Kong, Hong Kong Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 63 Department of Physics, Indiana University, Bloomington IN, United States of America Institut f¨ur Astro- und Teilchenphysik, Leopold-Franz JHEP11(2016)112 65 University of Iowa, Iowa City IA, United States of America 65 University of Iowa, Iowa City IA, United States of America 66 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of Ameri 66 Department of Physics and Astronomy, Iowa State University, 67 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 68 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan K, High Energy Accelerator Research Organization, T 69 Graduate School of Science, Kobe University, Kobe, Japan 70 Faculty of Science, Kyoto University, Kyoto, Japan 71 Kyoto University of Education, Kyoto, Japan 72 Department of Physics, Kyushu University, Fukuoka, Japan 72 Department of Physics, Kyushu University, Fukuoka, Japan 73 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argenti 73 Instituto de F´ısica La Plata, Universidad Nacional de La Plata 74 Physics Department, Lancaster University, Lancaster, United Kingdom 75 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Universit`a del Salento, Lecc Italy 76 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 78 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom nd Astronomy, Queen Mary University of London, L 79 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom p f y , y y y f , y, g 80 Department of Physics and Astronomy, University College London, London, United Kingdom 81 80 Department of Physics and Astronomy, University College London, London, United Kingdom 81 Louisiana Tech University, Ruston LA, United States of America 82 Laboratoire de Physique Nucl´eaire et de Hautes Energies, UPMC and Universit´e Paris-Diderot and CNRS/IN2P3, Paris, France Fysiska institutionen, Lunds universitet, Lund, Swed 84 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spa Departamento de Fisica Teorica C-15, Universidad A 85 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 86 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 86 School of Physics and Astronomy, University of Manchester, Ma 87 CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France CPPM, Aix-Marseille Universit´e and CNRS/IN2P 88 Department of Physics, University of Massachusetts, Amherst MA, United States of America 89 Department of Physics, McGill University, Montreal QC, Canada of Physics, University of Melbourne, Victoria, Austr 90 School of Physics, University of Melbourne, Victoria, Australia 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of America 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States 92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of America 93 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Universit`a di Milano, Milano, Italy 93 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Universit`a di Milano, Milano, Italy 94 B.I. The ATLAS collaboration Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America 115 Department of Physics, Oklahoma State University, Stillwater OK, United States of America Department of Physics, Oklahoma State University, Stillwater OK, United States of America Palack´y University, RCPTM, Olomouc, Czech Republic 116 Palack´y University, RCPTM, Olomouc, Czech Republic 116 Palack´y University, RCPTM, Olomouc, Czech Republic alacky University, RC , Olomouc, Czech Republic 117 C t f Hi h E Ph i U i it f O E OR U it d St t f A i y U y, RC , O , C R p 117 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America 118 y y, , , p 117 Center for High Energy Physics University of Oregon Eugene OR United States of America 117 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America enter for High Energy Physics, University of Oregon, 118 LAL, Univ. Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France 119 Graduate School of Science, Osaka University, Osaka, Japan epartment of Physics, University of Oslo, Oslo, Norw 120 Department of Physics, University of Oslo, Oslo, Norway 121 Department of Physics, Oxford University, Oxford, United Kingdom ( ) ( ) partment of Physics, Oxford University, Oxford, Unit ( ) 122 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 123 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 123 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 124 National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics 124 National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia 125 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. The ATLAS collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus 94 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus 95 National Scientific and Educational Centre for Particle and High Energy Physics, Minsk, Republic of Belarus 96 Group of Particle Physics, University of Montreal, Montreal QC, Canada 97 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russ 98 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 99 National Research Nuclear University MEPhI, Moscow, Russia – 32 – 100 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 101 Fakult¨at f¨ur Physik, Ludwig-Maximilians-Universit¨at M¨unchen, M¨unchen, Germany 102 Max-Planck-Institut f¨ur Physik (Werner-Heisenberg-Institut), M¨unchen, German agasaki Institute of Applied Science, Nagasaki, Japan 104 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan ( ) ( ) 104 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 105 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica Universit`a di Napoli Napoli Italy Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 105 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 105 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Universit`a di Napoli 106 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 107 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 108 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands JHEP11(2016)112 109 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 110 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 111 Department of Physics, New York University, New York NY, United States of America ent of Physics, New York University, New York NY, 112 Ohio State University, Columbus OH, United States of America 113 Faculty of Science, Okayama University, Okayama, Japan 114 Homer L. The ATLAS collaboration Fermi, Universit`a di Pisa, Pisa, Italy 126 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America 127 (a) Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas - LIP, Lisboa; (b) Faculdade de Ciˆencias, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra, Coimbra; (d) Centro de F´ısica Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Braga; (f) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal gia, Universidade Nova de Lisboa, Caparica, Portuga 128 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Re 129 Czech Technical University in Prague, Praha, Czech Republic 130 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 130 Faculty of Mathematics and Physics, Charles University in Pra Research Center Institute for High Energy Physics (P 131 State Research Center Institute for High Energy Physics (Protvino), NRC KI, Russia 132 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 133 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Universit`a Roma Tre, Roma, Italy 136 (a) Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques – 33 – Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech; Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech; (d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des sciences Universit´e Mohammed V Rabat Morocco Nucleaires, Rabat; Faculte des Sciences Semlalia, Universite Cadi Ayyad, LPHEA-Marrakech; (d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des i U i it´ M h d V R b t M (d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des sciences, Universit´e Mohammed V, Rabat, Morocco 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat `a l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of America 139 Department of Physics, University of Washington, Seattle WA, United States of America 140 Department of Physics and Astronomy, University of Sheffield, S 140 Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 141 Department of Physics, Shinshu University, Nagano, Japan 142 Fachbereich Physik, Universit¨at Siegen, Siegen, Germany epartment of Physics, Simon Fraser University, Burn JHEP11(2016)112 144 SLAC National Accelerator Laboratory, Stanford CA, United States of America 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics Institute of Experimental Physics of the Slovak Acade 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic Sciences, Kosice, Slovak Republic Sciences, Kosice, Slovak Republic 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand Johannesburg, South Africa p f y , y f p , p ; p f y , University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa 147 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 148 Physics Department, Royal Institute of Technology, Stockholm, Sweden 149 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 150 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 151 School of Physics, University of Sydney, Sydney, Australia 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 153 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 153 Department of Physics Technion: Israel Institute of Technology Haifa Israel 153 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 154 154 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 155 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 156 International Center for Elementary Particle Physics and Department of Physics, The Universit of Tokyo, Tokyo, Japan 157 Graduate School of Science and Technology, Tokyo Metropolitan Univer 158 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan nt of Physics, Tokyo Institute of Technology, Tokyo, J 159 Department of Physics, University of Toronto, Toronto ON, Canada 160 (a) TRIUMF, Vancouver BC; (b) Department of Physics and Astronomy, York University, Toronto ON, Canada 161 Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba, Tsukuba, Japan 162 Department of Physics and Astronomy, Tufts University, Medford MA, United States of America 163 partment of Physics and Astronomy, Tufts University 163 Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of America 164 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) 164 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di Chimica Fisica e Ambiente Universit`a di Udine Udine Italy ( ) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; ( ) ICTP, Trieste Dipartimento di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy Dipartimento di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy 165 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 166 166 Department of Physics, University of Illinois, Urbana IL, United States of America 167 Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingenier´ıa Electr´onica and Instituto de Microelectr´onica de Barcelona (IMB-CNM) University of Valencia and CSIC Valencia Spain Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingenier´ıa Electr´onica and Instituto de Microelectr´onica de Barcelona and Departamento de Ingenier´ıa Electr´onica and Instituto de Microelectr´onica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 168 Department of Physics, University of British Columbia, Vancouver BC, Canada 169 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 170 Department of Physics, University of Warwick, Coventry, United Kingdom – 34 – 171 Waseda University, Tokyo, Japan 172 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 173 Department of Physics, University of Wisconsin, Madison WI, United States of America 174 Fakult¨at f¨ur Physik und Astronomie, Julius-Maximilians-Universit¨at, W¨urzburg, Germany 175 Fakult¨at f¨ur Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universit¨at Wuppertal, Wuppertal, Germany Wuppertal, Wuppertal, Germany 176 Department of Physics, Yale University, New Haven CT, United States of America 177 Yerevan Physics Institute, Yerevan, Armenia 178 177 Yerevan Physics Institute, Yerevan, Armenia 178 Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3), Villeurbanne, France JHEP11(2016)112 b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan b Also at Institute of Physics, Azerbaijan Academy of Sciences c Also at Novosibirsk State University, Novosibirsk, Russia d Also at TRIUMF, Vancouver BC, Canada e Also at Department of Physics & Astronomy, University of Louisville, Louisville, KY, United States of America States of America f Also at Department of Physics, California State University, Fresno CA, United States of America partment of Physics, University of Fribourg, Fribourg h Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain i Also at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto, Portugal j Also at Tomsk State University, Tomsk, Russia k Also at Universita di Napoli Parthenope, Napoli, Italy l Also at Institute of Particle Physics (IPP), Canada m Also at National Institute of Physics and Nuclear Engineering, Bucharest, Romania n Also at Department of Physics, St. Petersburg State Polytechnical University, St. States of America Petersburg, Russia o Also at Department of Physics, The University of Michigan, Ann Arbor MI, United States of America p Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa r Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barc s Also at Graduate School of Science, Osaka University, Osaka, Japan s Also at Graduate School of Science, Osaka University, Osaka, Japan t Also at Department of Physics, National Tsing Hua University, Taiwan t Also at Department of Physics, National Tsing Hua University, Taiwan u Also at Institute for Mathematics, Astrophysics and Particle Physics, Radbou Nijmegen/Nikhef, Nijmegen, Netherlands u Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University u Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands v Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of America v Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of America w Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia w Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia lso at Institute of Theoretical Physics, Ilia State Univ x Also at CERN, Geneva, Switzerland y Also at Georgian Technical University (GTU),Tbilisi, Georgia y Also at Georgian Technical University (GTU),Tbilisi, Georgia y Also at Georgian Technical University z Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan z Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan aa Also at Manhattan College, New York NY, United States of America aa Also at Manhattan College, New York NY, United States of America Also at Manhattan College, New York NY, United S ab Also at Hellenic Open University, Patras, Greece ac Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan ac Also at Academia Sinica Grid Computing, Institute of Physics, Acad ad Also at School of Physics, Shandong University, Shandong, China o at School of Physics, Shandong University, Shando ae Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia ae Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, R af Also at Section de Physique, Universit´e de Gen`eve, Geneva, Switzerland af Also at Section de Physique, Universit´e de Gen`eve, Geneva, Switzerland ag Also at Eotvos Lorand University, Budapest, Hungary ag Also at Eotvos Lorand University, Budapest, Hungary ah Also at International School for Advanced Studies (SISSA), Trieste, Italy ah Also at International School for Advanced Studies (SISSA), Trieste, Italy – 35 – ai Also at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United States of America aj Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China ak Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Sofia, Bulgaria al Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia am Also at Institute of Physics, Academia Sinica, Taipei, Taiwan an Also at National Research Nuclear University MEPhI, Moscow, Russia ao Also at Department of Physics, Stanford University, Stanford CA, United States of America ap Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapes Hungary JHEP11(2016)112 ar Also at University of Malaya, Department of Physics, Kuala Lumpur, Mala as Also at CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France as Also at CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France ∗Deceased , / , , ∗Deceased – 36 –
https://openalex.org/W2100959617
https://ro-journal.biomedcentral.com/counter/pdf/10.1186/1748-717X-6-7
English
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Enhancement of radiosensitivity in human glioblastoma cells by the DNA N-mustard alkylating agent BO-1051 through augmented and sustained DNA damage response
Radiation oncology
2,011
cc-by
12,228
* Correspondence: uf004693@mail2000.com.tw † Contributed equally 1Graduate Institutes of Life Sciences, National Defense Medical Center & Department of Neurological Surgery, Tri-Service General Hospital, Taipei, Taiwan Full list of author information is available at the end of the article RESEARCH Open Access Open Access © 2011 Chu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin-9-ylamino)phenyl]urea (BO-1051) is an N-mustard DNA alkylating agent reported to exhibit antitumor activity. Here we further investigate the effects of this compound on radiation responses of human gliomas, which are notorious for the high resistance to radiotherapy. Methods: The clonogenic assay was used to determine the IC50 and radiosensitivity of human glioma cell lines (U87MG, U251MG and GBM-3) following BO-1051. DNA histogram and propidium iodide-Annexin V staining were used to determine the cell cycle distribution and the apoptosis, respectively. DNA damage and repair state were determined by g-H2AX foci, and mitotic catastrophe was measure using nuclear fragmentation. Xenograft tumors were measured with a caliper, and the survival rate was determined using Kaplan-Meier method. Results: BO-1051 inhibited growth of human gliomas in a dose- and time-dependent manner. Using the dosage at IC50, BO-1051 significantly enhanced radiosensitivity to different extents [The sensitizer enhancement ratio was between 1.24 and 1.50 at 10% of survival fraction]. The radiosensitive G2/M population was raised by BO-1051, whereas apoptosis and mitotic catastrophe were not affected. g-H2AX foci was greatly increased and sustained by combined BO-1051 and g-rays, suggested that DNA damage or repair capacity was impaired during treatment. In vivo studies further demonstrated that BO-1051 enhanced the radiotherapeutic effects on GBM-3-beared xenograft tumors, by which the sensitizer enhancement ratio was 1.97. The survival rate of treated mice was also increased accordingly. Conclusions: These results indicate that BO-1051 can effectively enhance glioma cell radiosensitivity in vitro and in vivo. It suggests that BO-1051 is a potent radiosensitizer for treating human glioma cells. Enhancement of radiosensitivity in human glioblastoma cells by the DNA N-mustard alkylating agent BO-1051 through augmented and sustained DNA damage response Pei-Ming Chu1, Shih-Hwa Chiou2,3,4†, Tsann-Long Su5†, Yi-Jang Lee6†, Li-Hsin Chen3, Yi-Wei Chen4,7, Sang-Hue Yen7, Ming-Teh Chen8, Ming-Hsiung Chen8, Yang-Hsin Shih8, Pang-Hsien Tu5, Hsin-I Ma1* Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Materials and Methods Cell lines and treatment This research followed the tenets of the Declaration of Helsinki. All samples were obtained after patients pro- vided informed consent. The study was approved by the Institutional Ethics Committee/Institutional Review Board of Tri-Service General Hospital. The commercial available U87MG, and U251MG glioma cell lines as well as primary GBM cell line (GBM-3), which was isolated from tumor sample obtained from patient undergoing surgery for a GBM (World Health Organizing Grade 4 astrocytoma), were grown as attached monolayers in 75-cm2 flasks in DMEM media (Invitrogen) supplemen- ted with glutamate (5 mmol/L) and 10% fetal bovine serum. Cells were incubated at the exponential growth phase in humidified 5% CO2/95% air atmosphere at 37℃. The GBM-3 cells used for the experiments had already undergone > 100 passages. 1-{4-[bis(2-chlor- oethyl)amino]phenyl}-3-[2-methyl-5- (4-methylacridin-9- ylamino)phenyl]urea (BO-0151, Figure 1A) was dissolved in DMSO to a stock concentration of 5 mM and stored at -20℃. Gamma radiation (ionizing irradiation) was delivered with a T-1000 Theratronic cobalt unit (Thera- tronic International, Inc., Ottawa, Canada) at a dose rate of 1.1 Gy/min (SSD = 57.5 cm). BO-0742 was found to have a potent therapeutic effi- cacy against human leukemia and solid tumor cell growth in vitro. Also, it has a good therapeutic index with leuke- mia being 10-40 times more sensitive than hematopoietic progenitors. Administration of BO-0742 at an optimal dose schedule, based on its pharmacokinetics, signifi- cantly suppressed the growth of xenograft tumors in mice bearing human breast and ovarian cancers. How- ever, BO-0742’s bioavailability is low because it has a nar- row therapeutic window and is chemically unstable in mice (half-life < 25 min) [12]. To improve the poor phar- macokinetics of BO-0742, we have recently synthesized a series of phenyl N-mustard-9-anilinoacridine conjugates via a urea linker [13,14]. Of these agents, BO-1051 was found to be more chemically stable than BO-0742 in rat plasma (54.2 vs. 0.4 h). BO-1051, an agent capable of inducing marked dose-dependent levels of DNA inter- strand cross-linking (ICLs), revealed a broad spectrum of anti-cancer activities in vitro without cross-resistance to taxol or vinblastine. Due to BO-1051’s hydrophobic abil- ity, it can penetrate through the blood-brain barrier to brain cortex. BO-1051 has been shown to possess thera- peutic efficacy in nude mice bearing human breast MX-1 tumors and human glioma in vivo [14]. Interestingly, we found that obvious tumor suppression was observed in mice and sustained over 70 days without relapse [14]. Background growth and recurrence rate [1]. A multicenter study has shown that addition of concurrent temozolomide (TMZ) to radical radiation therapy improves the survival in patients who suffered from GBM [2,3]. These studies have demonstrated an improvement for patients who received TMZ, compared to those who did not, in the median survival time and in the 2-year survival rate (14.6 vs. 12 months, 27% vs. 10%, respectively). Unfortunately, the survival rate remains low using TMZ, and it prompts investigators to seek new and more effective chemothera- peutic agents for the treatment of malignant gliomas. Malignant gliomas account for approximately 30% of all intracranial tumors, and of them, glioblastoma multi- forme (GBM) is considered as the most frequent and aggressive type. Removal of GBM by surgical resection is usually not feasible due to the highly diffuse infiltrative Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 2 of 13 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 DNA alkylating agents are used widely for treatment of a variety of pediatric and adult cancers because the cyto- toxic effects of these agents can directly modify DNA and cause DNA lesions [4]. However, the development of new alkylating N-mustard agents is slow due to their low tumor specificity, high chemical reactivity and an induc- tion of bone marrow toxicity [5,6]. To overcome these drawbacks, one strategy has been to design DNA- directed alkylating agents by linking the alkylating pharmacophore to the DNA-affinity molecules (e.g., DNA intercalating agents, DNA minor groove binder) [7,8]. In most cases, the DNA-directed alkylating agents have more selective, cytotoxic and potential than the cor- responding untargeted derivatives [8-10]. Among these agents, the compound BO-0742 exhibited significant cytotoxicity (107-fold higher) on human lymphoblastic leukemic cells than its parent analogue 3-(9-acridinyla- mino)-5-hydroxymethylaniline [9,11]. treatment with BO-1051 at nanomolar concentrations sensitizes the glioma cells to radiation-induced cellular lethality. These data indicate that BO-1051 enhances tumor radiosensitivity in vitro and in vivo. Moreover, this sensitization correlates with its enhancement arrest in the radiosensitive cell cycle phase and the delayed dispersion of phosphorylated histone H2AX (g-H2AX) foci, which suggests an inhibition of the repair to the DNA double-strand breaks (DBSs). SF PE PE xnM,thr 0nM,thr xnM thr , = Materials and Methods Cell lines and treatment The results indicated that BO-1051 was more potent than cyclophosphamide with low toxicity to the host (15% body-weight drop) suggesting that this agent is a promising candidate for preclinical studies. SF PE PE xnM,thr 0nM,thr xnM thr , = Assay of BO-1051 cytotoxicity  KͲϭϬϱϭ 0.1 1 6 hours 12 hours 24 hours Surviving fraction  Dose of BO-1051 (nM) – U87MG 400 50 100 200   Surviving fraction 0.1 1 6 hours 12 hours 24 hours  Surviving fraction Dose of BO-1051 (nM) – U251MG 400 50 100 200 0.1 1 6 hours 12 hours 24 hours 0.1 1.0 6 hours 12 hours 24 hours 24 hours  Surviving fraction Surviving fraction Dose of BO-1051 (nM Dose of BO-1051 (nM) – U251MG  50 100 400 50 100 200    Surviving fraction Surviving fraction Figure 1 Clonogenic survival of human glioma cells treated with BO-1051. (A) Chemical structure of 1-{4-[bis(2-chloroethyl)amino]phenyl}-3- [2-methyl-5-(4- methylacridin-9-ylamino)phenyl]urea (BO-1051). (B) U87MG, (C) U251MG and (D) GBM-3 cells were exposed to escalating doses (50-400 nM) of BO-1051 or vehicle (DMSO). At 6, 12 and 24 h after the addition of BO-1051, the BO-1051- containing medium was removed, rinsed, and then fed with fresh growth media. Colony- forming efficiency was determined 10-14 days later, and the survival fractions of BO-1051- treated cells were calculated after normalizing for the plating efficiencies of untreated cells. Points: mean for at least 3 independent experiments; bars, SD. generated after normalizing for the amount of BO-1051-induced cell death. The radiation SF of cells pretreated with × nM BO-1051 was calculated as [15]: This protocol was used in an attempt to eliminate any effects of trypsinization on post-treatment or post-irradia- tion signaling/recovery processes [16-20]. Moreover, this protocol allows for the irradiation of single cells but not microcolonies, which eliminates the confounding para- meter of multiplicity and its effects on the radiosensitivity. SF PE PE xnM,DGy xnM,DGy xnM,0Gy = Assay of BO-1051 cytotoxicity y y y For these studies, a specified number of single cells were seeded into a 25-T flask, and after 6 h, to allow for cell attachment (but no division), the cells were treated with 0, 50, 100, 200 or 400 nM BO-1051. At 0, 6, 12 and 24 h after the BO-1051 addition, the BO-1051-contain- ing medium was removed; the cells were washed with sterile PBS, and fresh media was added. After 10 to 14 days of incubation, colonies were fixed with methanol and stained with Giemsa. The number of colonies con- taining at least 50 cells was determined, and the plating efficiency (PE) and surviving fractions (SF) were calcu- lated. The SF of cells exposed to × nM BO-1051 for t h was calculated as [15]: Given that radiotherapy is considered to be the most effective adjuvant treatment with surgery, we tested if the therapeutic ability of BO-1051 could be translated into antitumor activity. In this study, we investigated the effects of BO-1051 on the radiosensitivity of a panel of three human glioma cell lines, and we found that SF PE PE xnM,thr 0nM,thr xnM thr , = SF PE PE xnM,thr 0nM,thr xnM thr , = Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 3 of 13 0.1 1 6 hours 12 hours 24 hours 0.1 1.0 6 hours 12 hours 24 hours 0.1 1 6 hours 12 hours 24 hours Surviving fraction    KͲϭϬϱϭ Surviving fraction Surviving fraction Dose of BO-1051 (nM) – GBM-3 Dose of BO-1051 (nM) – U87MG Dose of BO-1051 (nM) – U251MG  400 50 100 200 400 50 100 200 400 50 100 200 Figure 1 Clonogenic survival of human glioma cells treated with BO-1051. (A) Chemical structure of 1-{4-[bis(2-chloroethyl)amino]phenyl}-3- [2-methyl-5-(4- methylacridin-9-ylamino)phenyl]urea (BO-1051). (B) U87MG, (C) U251MG and (D) GBM-3 cells were exposed to escalating doses (50-400 nM) of BO-1051 or vehicle (DMSO). At 6, 12 and 24 h after the addition of BO-1051, the BO-1051- containing medium was removed, rinsed, and then fed with fresh growth media. Colony- forming efficiency was determined 10-14 days later, and the survival fractions of BO-1051- treated cells were calculated after normalizing for the plating efficiencies of untreated cells. Points: mean for at least 3 independent experiments; bars, SD. Annexin V-PI apoptosis assay g y y The tumor re-growth delay assay measures the time required for a tumor to reach a given size post-treatment. When tumors grew to a mean volume of ~150 mm3, mice were randomly assigned to one of four treatment groups: vehicle control (14 animals), BO-1051 (12 animals), 4 Gy irradiation (9 animals), or combined BO-1051 and radia- tion (8 animals). BO-1051 treatment was performed, which consisted of an intraperitoneal (i.p.) injection proto- col of 50 mg/kg administered at 3-day intervals over a 6-day period (3 injections on days 0, 3, 6; Q3D × 3). For irradiation, unanesthetized animals were immobilized in a lead jig that allowed for the localized irradiation of the implanted tumors. Gamma radiation was delivered by a T-1000 Theratronic cobalt unit (Theratronic International, Inc.; Ottawa, Canada) at a dose rate of 1.1 Gy/min (SSD = 57.5 cm). For the BO-1051-plus-radiation group, BO-1051 (50 mg/kg) was delivered via i.p. injection on days 0, 3, and 6, with day 0 being the day of randomization. Radia- tion (4 Gy) was delivered to animals restrained in a cus- tom lead jig 24 h after the first injection of BO-1051 (day 1 after randomization). Tumor volume is a critical para- meter in determining radiation-induced growth delay with smaller tumors appearing more radiosensitive. To ensure BO-1051-induced growth delay did not bias the results of the combination treatment (BO-1051 plus 4 Gy), it was important that the two irradiated groups (4 Gy and BO- 1051 plus 4 Gy) received radiation when the tumors were approximately the same size. To obtain tumor growth curves, perpendicular diameter measurements of each tumor were made every day with digital calipers, and the volumes were calculated using the formula for volume of an ellipsoid: 4Π/3 × L/2 × W/2 × H/2, where L = length, W = width, and H = height. The time for the tumor to grow again to ten times the initial volume (about 1500 mm3) was calculated for each animal. Absolute To evaluate apoptosis as a mechanism of cell death, approximately 2 × 106 cells were plated in 100-mm petri dishes. Cells were exposed to 200 nM or higher concentration (1.2 μM) of BO-1051 prior to irradiation and were stained at 24 and 48 h postirradiation (2 Gy). Both adherent and detached cells were collected, centri- fuged, and double stained with Annexin V-FITC and propidium iodide (PI). Combination of BO-1051 and irradiation The combined therapeutic effects based on drug and ionizing irradiation was obtained by the survival frac- tions measured by separate treatment as reported pre- viously [21]. The expected effect by two separate treatments was determined by the formula SF(Drug) × SF (Rad), which was compared to the observed survival fraction. After allowing the cells time to attach, the culture medium was then replaced with fresh medium that contained 200 nM BO-1051, and the flasks were irra- diated 24 h later. Immediately after irradiation, the growth media was aspirated, and fresh media was added. Colonies were stained with Giemsa 10 to 14 days after seeding. Survival curves were then Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 4 of 13 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 In vivo tumor model Six-week-old female nude mice were used in these stu- dies. Mice were caged in groups of five or less, and all animals were fed a diet of animal chow and water ad libitum. All procedures involving animals were per- formed in accordance with the institutional animal wel- fare guidelines of the Taipei Veterans General Hospital. Tumors were generated by injecting 5 × 106 GBM-3 cells subcutaneous (s.c.) into the right hind leg. Irradia- tion was performed using a T-1000 Theratronic cobalt unit (Theratronic International, Inc.; Ottawa, Canada) irradiator with animals restrained in a custom jig. Annexin V-PI apoptosis assay Apoptotic cells were quantified with flow cytometry using a FACSCalibur cytometer (BD FACS Caliber, Mountain View, CA). Cell-cycle analysis analysis software package (Scanalytics, Inc.; Fairfax, VA) running on a Macintosh G3 computer. For each treat- ment condition, g-H2AX foci were determined in at least 150 cells. After treatment, cells were prepared for fluorescence- activated cell sorting (FACS) to assess the relative distri- bution in the respective phases of the cell cycle. Cells were harvested 24 h after of treatment with BO-1051, pelleted by centrifugation, re-suspended in PBS, fixed in 70% ethanol and stored at -20℃. Immediately before flow cytometry, the cells were washed in cold PBS (4℃), incubated in Ribonuclease A (Sigma) for 20 min at room temperature, labeled by adding an equal volume of propidium iodide solution (100 μg/ml) and incubated in the dark for 20 min at 4℃. These samples were mea- sured (20,000 events collected from each) in a FACSCa- libur cytometer (BD FACS Caliber; Mountain View, CA). The data shown are for one experiment, but the results were reproduced and confirmed in at least three identical experiments. Immunofluorescent staining for g-H2AX g g Cells were treated with or without BO-1051 for 24 h prior to irradiation (2 Gy) and fed with BO-1051-free medium, and the average number of foci per cell was measured beginning at 1 h after irradiation and followed thereafter for 24 h. At specified times, the media were aspirated and cells were fixed in 1% paraformaldehyde for 10 min at room temperature. Paraformaldehyde was aspirated, and the cells were treated with a 0.2% NP40/ PBS solution for 15 min. Cells were then washed in PBS twice, and the anti-gH2AX antibody was added at a dilution of 1:500 in 1% BSA and incubated overnight at 4℃. Again, the cells were washed twice in PBS before incubating in the dark for 1 h with a FITC-labeled sec- ondary antibody at a dilution of 1:100 in 1% BSA. The secondary antibody solution was then aspirated, and the cells were washed twice in PBS. The cells were then incubated in the dark with PI (1 μg/ml) in PBS for 30 min, washed twice, and coverslips were mounted with an anti-fade solution (Dako Corp.; Carpinteria, CA). Slides were examined with a confocal fluorescent microscope (Wetzlar, Germany). Images were captured by a Photometrics Sensys CCD camera (Roper Scientific; Tucson, AZ) and imported into the IP Labs image Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 5 of 13 tumor growth delay was calculated as the number of days for the treated tumors to reach ten times the initial tumor volume minus the number of days for the control group to reach the same size. Enhancement of radiosensitivity in glioma cells by BO-1051 To investigate if BO-1051 enhances the cellular sensitiv- ity to ionizing radiation, the glioma cells were exposed to BO-1051 for 24 h before irradiation and subjected to the clonogenic assay. The results showed that the SFs at different radiation dosages were apparently reduced in U87MG, U251MG and GBM-3 cells after they were exposed to BO-1051 (Figure. 2A-C). SFs after 2 Gy of BO-1051-pretreated cells were significantly lower than those of untreated cells (Figure 2D). Besides, the SERs were 1.50 for U87MG, 1.24 for U251MG and 1.31 for GBM-3 at a 10% cell survival (0.1). At 50% cell survival (0.5), the SERs were 1.87 for U87MG, 1.83 for U251MG and 1.68 for GBM-3 (Figure 2A-C, and 2E). Determination of the cytotoxicity of BO-1051 on different human glioma cell lines Determination of the cytotoxicity of BO-1051 on different human glioma cell lines To determine the effects of BO-1051 on glioma cell cyto- toxicity by clonogenic survival, MTT assay was per- formed in a panel of 3 human malignant glioma cell lines (U87MG, U251MG and GBM-3). The IC50 (concentra- tion resulting in cell viability of 50% of control) values of BO-1051 for U87MG, U251MG and GBM-3 cells were 2.7, 2.5 and 1.5 μM, respectively. However, the clonogenic survival analysis showed little or no colony formation for 24 h post-exposure to the concentrations of BO-1051 > 400 nM. We found that the appropriate dosage range of BO-1051 for colony formation in these glioma cell lines was between 50 and 400 nM. The cyto- toxicity of U87MG, U251MG and GBM-3 cells were sig- nificantly influenced by BO-1051 in a time-dependent and dose-dependent manner. The 24-h treatment of 200 nM BO-1051 resulted in SFs of 0.470 ± 0.091, 0.485 ± 0.041 and 0.510 ± 0.042 for U87MG, U251MG, and GBM-3, respectively (Figure 1). Because approximately 50% of survival fractions were reached using 200 nM BO-1051 treatments on each glioma cells at 24 h, we chose this dose for the following experiments. Induction of a G2/M phase arrest in glioma cells exposed to BO-1051 Given that radiosensitivity is distinct in different phases of the cell cycle, we tested the cell cycle distribution in BO-1051 treated glioma cells [22,23]. Cells were treated with BO-1051 for 24 h and then subjected to flow cyto- metric analysis. As illustrated in the DNA histograms, BO-1051 treatment significantly disturbed the cell cycle progression and showed a dramatic increase in G2/M phase populations in U87MG cells compared with the untreated controls (Figure 3A). Quantitative analysis of the cell-cycle distribution at 24 h post-exposure to BO-1051 at different concentrations from 200 nM to 1200 nM is shown in Figure 3B-D, which shows that G2/M phase arrest was caused by pre-treatment with BO-1051 in a dose-dependent manner for all 3 glioma cells (Figure 3A-D). Because the G2/M phase is known as the cell cycle’s most radiosensitive phase [22,23], it may in part account for the effects of BO-1051 on the enhancement of radiosensitivity of glioma cell line. Immunofluorescent staining for g-H2AX As a result, the radiation survival curves obtained by the clonogenic assay showed that BO-1051 pretreatment sensitized human glioma cells to the ionizing radiation. Besides, Table 1 summarizes the relative reduction in SFs and compares them with a virtual value, expected for each of the combination of BO-1051 and irradiation dose. The actual SF measured for combinations is smaller than that expected on the basis of the treatment effects of each modality separately. It indicates a significant synergistic interaction in all three glioma cells. The mean size of tumors receiving the combination treatment was compared to the mean size of tumors in mice from each of the other groups (receiving vehicle control, radiation alone, or BO-1051 alone). The analysis was done on day 42 after the treatment started because this was the last day that all animals were still alive. Time to treatment failure (TTF) was defined as the time from the initiation of treatment (experimental or control) to the time a tumor was severely necrotic or had reached a volume > 1500 mm3. Normalized tumor growth delay is defined as the time in days for tumors to reach 10 times the initial volume in mice treated with the combination of BO-1051 and radiation minus the time in days for the tumors to reach 10 times the initial volume in mice trea- ted with BO-1051 only, which was 6.7 days (i.e., 16 minus 9.3 days). Statistical analysis The results are reported as mean ± SD. Statistical analy- sis was performed using a Student’s t-test, one-way ANOVA test or two-way ANOVA test followed by Tukey’s test, as appropriate. A P < 0.05 was considered to be statistically significant. Induction of a G2/M phase arrest in glioma cells exposed to BO-1051 Table 1 Relative reduction in surviving fraction of three glioma cells due to combination of irradiation and BO- 1051 treatment Table 1 Relative reduction in surviving fraction of three glioma cells due to combination of irradiation and BO- 1051 treatment Table 1 Relative reduction in surviving fraction of three glioma cells due to combination of irradiation and BO- 1051 treatment Irradiation dose (Gy) Relative reduction (%) U87MG U251MG GBM-3 2 41.7 42.6 40.6 4 70.4 45.6 47.0 6 74.2 47.9 64.3 8 76.3 50.0 73.9 Percentage relative reduction of the observed surviving fraction (SF) compared to the expected SF (calculated on the basis of combing individual treatment component, each with respective SF value). Irradiation dose (Gy) Relative reduction (%) U87MG U251MG GBM-3 2 41.7 42.6 40.6 4 70.4 45.6 47.0 6 74.2 47.9 64.3 8 76.3 50.0 73.9 Enhancement of radiosensitivity by BO-1051 treatment is not caused by apoptosis or mitotic catastrophes in glioma cells Figure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells were exposed to 200 nM of BO-1051 or DMSO (IR only) for 24 h and irradiated with graded doses of g-rays, rinsed, and fed with fresh growth media. Colony-forming efficiency was determined 10-14 days later, and survival curves were generated after normalizing for cell killing by BO-1051 alone. Points: mean survival fraction from at least 3 independent experiments; bars: SD. (D) The survival fraction after 2 Gy (SF2), corrected for independent cytotoxic effect of BO-1051, of human glioma cells treated with 200 nM of BO-1051 or control (DMSO) for 24 h pre-radiation was measured. Values are the mean survival fraction ± SD of at least 3 independent experiments. * p < 0.05. (E) Sensitizer enhancement ratios (SER) of human glioma cells. SERs were calculated at 10% or 50% cell survival (0.1 or 0.5) by dividing the dose of radiation from the radiation-only surviving curve with the corresponding dose from the BO-1051 plus radiation curve. 5% of apoptosis (Figure 4). Moreover, treatment with 1200 nM BO-1051 significantly induced approximately 20% of apoptosis in all 3 cell lines, but the combined protocol did not show obvious enhancement on the pro- portion of apoptotic cell deaths (Figure 4). An increase in radiosensitivity may be caused by radiation-induced mitotic catastrophes. Nevertheless, no significant mitotic catastrophes were detected in glioma cells treated with both BO-1051 and irradiation up to 72 h (unpublished data). These data indicate that the BO-1051-mediated increase in radiosensitivity is not due to the apoptosis and mitotic catastrophes. BO-1051 concentrations (from 200 to 1200 nM) for 24 h, and then were irradiated with 2 Gy of g-rays. The Annexin V/PI staining was then determined with FACS analysis. Cells treated with either 200 nM of BO-1051 alone or combined with irradiation exhibited less than Percentage relative reduction of the observed surviving fraction (SF) compared to the expected SF (calculated on the basis of combing individual treatment component, each with respective SF value). Enhancement of radiosensitivity by BO-1051 treatment is not caused by apoptosis or mitotic catastrophes in glioma cells 0 2 4 6 8 0.001 0.01 0.1 1 IR only 200 nM BO + IR 0 2 4 6 8 0.001 0.01 0.1 1 IR only 200 nM BO + IR 0 2 4 6 8 0.001 0.01 0.1 1 IR only 200 nM BO + IR Surviving fraction Radiation dose (Gy) Surviving fraction Radiation dose (Gy) Surviving fraction Radiation dose (Gy)    U87MG U251MG GBM-3 0 2 4 6 8 0.001 0.01 0.1 1 IR only 200 nM BO + IR Surviving fraction Radiation dose (Gy)  GBM-3    Surviving fraction Survival fraction at 2 Gy Sensitizer enhancement ratio 1.0 1.2 1.4 1.6 1.8 2.0 0.0 0.2 0.4 0.6 0.8 1.0 Control 200 nM BO U87MG U251MG GBM-3 Ύ Ύ Ύ   U87MG Survival fraction at 2 Gy Sensitizer enhancement ratio 1.0 1.2 1.4 1.6 1.8 2.0 SF0.1 SF0.5 0.0 0.2 0.4 0.6 0.8 1.0 Control 200 nM BO U87MG U251MG GBM-3 Ύ Ύ Ύ   U87MG U251MG GBM-3 Survival fraction at 2 Gy 0.0 0.2 0.4 0.6 0.8 1.0 Control 200 nM BO U87MG U251MG GBM-3 Ύ Ύ Ύ  Sensitizer enhancement ratio 1.0 1.2 1.4 1.6 1.8 2.0 SF0.1 SF0.5  U87MG U251MG GBM-3  igure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells we Figure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells were exposed to 200 nM of BO-1051 or DMSO (IR only) for 24 h and irradiated with graded doses of g-rays, rinsed, and fed with fresh growth media. Colony-forming efficiency was determined 10-14 days later, and survival curves were generated after normalizing for cell killing by BO-1051 alone. Points: mean survival fraction from at least 3 independent experiments; bars: SD. (D) The survival fraction after 2 Gy (SF2), corrected for independent cytotoxic effect of BO-1051, of human glioma cells treated with 200 nM of BO-1051 or control (DMSO) for 24 h pre-radiation was measured. Values are the mean survival fraction ± SD of at least 3 independent experiments. * p < 0.05. (E) Sensitizer enhancement ratios (SER) of human glioma cells. SERs were calculated at 10% or 50% cell survival (0.1 or 0.5) by dividing the dose of radiation from the radiation-only surviving curve with the corresponding dose from the BO-1051 plus radiation curve. Enhancement of radiosensitivity by BO-1051 treatment is not caused by apoptosis or mitotic catastrophes in glioma cells We next investigated whether BO-1051 enhanced radia- tion sensitivity of glioma cells was associated with increase of apoptosis. Cells were exposed to a range of Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 6 of 13 0 2 4 6 8 0.001 0.01 0.1 1 IR only 200 nM BO + IR 0 2 4 6 8 0.001 0.01 0.1 1 IR only 200 nM BO + IR 0 2 4 6 8 0.001 0.01 0.1 1 IR only 200 nM BO + IR Surviving fraction Radiation dose (Gy) Surviving fraction Radiation dose (Gy) Surviving fraction Radiation dose (Gy) Survival fraction at 2 Gy Sensitizer enhancement ratio    1.0 1.2 1.4 1.6 1.8 2.0 SF0.1 SF0.5 0.0 0.2 0.4 0.6 0.8 1.0 Control 200 nM BO U87MG U251MG GBM-3 Ύ Ύ Ύ   U87MG U251MG GBM-3 U87MG U251MG GBM-3 Figure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells were exposed to 200 nM of BO-1051 or DMSO (IR only) for 24 h and irradiated with graded doses of g-rays, rinsed, and fed with fresh growth media. Colony-forming efficiency was determined 10-14 days later, and survival curves were generated after normalizing for cell killing by BO-1051 alone. Points: mean survival fraction from at least 3 independent experiments; bars: SD. (D) The survival fraction after 2 Gy (SF2), corrected for independent cytotoxic effect of BO-1051, of human glioma cells treated with 200 nM of BO-1051 or control (DMSO) for 24 h pre-radiation was measured. Values are the mean survival fraction ± SD of at least 3 independent experiments. * p < 0.05. (E) Sensitizer enhancement ratios (SER) of human glioma cells. SERs were calculated at 10% or 50% cell survival (0.1 or 0.5) by dividing the dose of radiation from the radiation-only surviving curve with the corresponding dose from the BO-1051 plus radiation curve. BO-1051 combined with g-rays causes prolonged DNA damage response in glioma cells Cells in exponential growth were sham treated (left panel), treated with BO-1051 (200 nM, right panel) and then harvested 24 h later. (B-D) Cell cycle distributions of a panel of 3 human glioma cell lines (U87MG, U251MG and GBM-3) were exposed to the designated concentrations of BO- 1051 for 24 h. Data displayed by the DNA content profiles were analyzed, and the cell cycle phase information is represented graphically. markers for evaluating the level of DNA damage [24]. To investigate if BO-1051 can affect the extent of DNA damage by g-rays, the formation of g-H2AX foci in cell nuclei was determined. Cells were treated with or with- out BO-1051 for 24 h prior to irradiation (2 Gy) and fed with BO-1051-free medium, and the average number of foci per cell was measured beginning at 1 h after irradia- tion and followed thereafter for 24 h. The results showed that exposure of glioma cells to either BO-1051 or irradiation (2 Gy) resulted in a significant increase of g-H2AX foci at 1 h that was sustained for 6 h, and then the g-H2AX foci declined to almost basal level at least 24 h after irradiation or drug removal (Figure 5A and 5B). The combined protocol resulted in a greater num- ber of g-H2AX foci than either of the individual treat- ments at 1 or 6 h. However, the number of residual g-H2AX foci per cell 24 h post-irradiation was greater in BO-1051 plus irradiation (19.9 ± 2.5 per cell) compared with the number of foci in cells treated with either irradiation or BO-1051 alone (7.9 ± 2.8 and 11.2 ± 1.9 per cell, respectively) (Figure 5A and 5B). Further- more, the frequency of g-H2AX foci distribution at 24 h post-irradiation showed that the percentage of > 30 foci of g-H2AX was higher than additive in BO-1051 plus irradiation (24.9%) compared with the percentage of foci in cells treated with either irradiation or BO-1051 alone (0.3% and 12.0%, respectively). These results suggest that BO-1051 produces supra-additive and prolonged effects of irradiation on glioma cells. BO-1051 combined with g-rays causes prolonged DNA damage response in glioma cells DNA damage is the most important biological effects caused by ionizing radiation. It has been reported that the nuclear foci of g-H2AX is one of the canonical Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 7 of 13 0 20 40 60 80 100 G1 S G2/M Control  0 20 40 60 80 100 G1 S G2/M 0 20 40 60 80 100 G1 S G2/M 200 400 600 0 1200 BO-1051 (nM) 200 nM BO 200 400 600 0 1200 BO-1051 (nM) B U87MG C U251MG D GBM-3 200 400 600 0 1200 BO-1051 (nM) Cell Cycle Distribution (%) Cell Cycle Distribution (%) Cell Cycle Distribution (%) DNA content Cell count 2n 4n 2n 4n Figure 3 Effect of BO-1051 on cell cycle profile in human glioma cells. Cultures were exposed to BO-1051 for 24 h before collection and FACS analysis of the propidium iodide-stained cells. (A) The DNA histograms depict cell cycle phase distributions of U87MG 24 h post-treatment. Cells in exponential growth were sham treated (left panel), treated with BO-1051 (200 nM, right panel) and then harvested 24 h later. (B-D) Cell cycle distributions of a panel of 3 human glioma cell lines (U87MG, U251MG and GBM-3) were exposed to the designated concentrations of BO- 1051 for 24 h. Data displayed by the DNA content profiles were analyzed, and the cell cycle phase information is represented graphically. Control  0 20 40 60 80 100 G1 S G2/M 200 400 600 0 1200 BO-1051 (nM) 200 nM BO B U87MG Cell Cycle Distribution (%) DNA content Cell count 2n 4n 2n 4n  0 20 40 60 80 100 G1 S G2/M 0 20 40 60 80 100 G1 S G2/M BO 1051 (nM) 200 400 600 0 1200 BO-1051 (nM) C U251MG D GBM-3 200 400 600 0 1200 BO-1051 (nM) Cell Cycle Distribution (%) Cell Cycle Distribution (%) 0 20 40 60 80 100 G1 S G2/M ( ) D GBM-3 200 400 600 0 1200 BO-1051 (nM) Cell Cycle Distribution (%) Figure 3 Effect of BO-1051 on cell cycle profile in human glioma cells. Cultures were exposed to BO-1051 for 24 h before collection and FACS analysis of the propidium iodide-stained cells. (A) The DNA histograms depict cell cycle phase distributions of U87MG 24 h post-treatment. BO-1051 delays the growth of xenograft gliomas exposed to irradiation For each group, the time for tumors to grow from 150 to 1500 mm3 (i.e., a 10-fold increase in tumor size) was calculated using tumor volumes from the individual mice in each group (mean + SD). The time required for tumors to reach 10-times the starting volume increased from 20.2 days for control mice to 29.5 days for BO- 1051-treated mice. Irradiation treatment alone increased the time to reach 10-times the initial volume to 23.6 days. However, in mice that received the combination therapy, the time for tumors to reach 10-times the initial volume increased to 36.2 days, which is signifi- cantly greater than the individual treatment groups (Figure 6A; Table 2, p > 0.05). Thus, the growth delay after the combined treatment was more than the sum of the growth delays caused by either BO-1051 or radiation alone. To calculate an SER comparing the tumor radia- tion responses in mice with and without the BO-1051 treatment, the normalized tumor growth delay was was used. Mice bearing s.c. xenografts (~150 mm3) were stratified by size and randomized into 4 groups: control, BO-1051 alone, irradiation alone (4 Gy), or combined BO-1051 plus radiation. For the BO-1051 treatments, mice were i.p. injected with dosage at 50 mg/kg on days 0, 3 and 6. The growth rates for the GBM-3 tumors exposed to each treatment are shown in Figure 6A. For each group, the time for tumors to grow from 150 to 1500 mm3 (i.e., a 10-fold increase in tumor size) was calculated using tumor volumes from the individual mice in each group (mean + SD). The time required for tumors to reach 10-times the starting volume increased from 20.2 days for control mice to 29.5 days for BO- 1051-treated mice. Irradiation treatment alone increased the time to reach 10-times the initial volume to 23.6 days. However, in mice that received the combination therapy, the time for tumors to reach 10-times the initial volume increased to 36.2 days, which is signifi- cantly greater than the individual treatment groups (Figure 6A; Table 2, p > 0.05). Thus, the growth delay after the combined treatment was more than the sum of the growth delays caused by either BO-1051 or radiation alone. BO-1051 delays the growth of xenograft gliomas exposed to irradiation To determine if the enhanced radiosensitivity of BO-1051 treated glioma cells could be translated into an in vivo tumor model, a tumor growth delay assay using GBM-3 cells grown s.c. in the hind leg of nude mice Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 8 of 13 Figure 4 Apoptotic effects of BO-1051 in combination with irradiation in glioma cells. U87MG, U251MG and GBM-3 were exposed to 200 nM or higher concentration (1200 nM) of BO-1051 for 24 h and irradiated with 2 Gy, followed by FACS analysis of Annexin V-FITC and PI staining 24 h later. Control: no treatment; IR: ionizing radiation at 2 Gy; BO: BO-1051; BO+IR: cells exposed to BO-1051 for 24 h and then irradiation with 2 Gy of g-ray. Values are the means ± SD of 3 independent experiments. * p < 0.05. Figure 4 Apoptotic effects of BO-1051 in combination with irradiation in glioma cells. U87MG, U251MG and GBM-3 were exposed to 200 nM or higher concentration (1200 nM) of BO-1051 for 24 h and irradiated with 2 Gy, followed by FACS analysis of Annexin V-FITC and PI staining 24 h later. Control: no treatment; IR: ionizing radiation at 2 Gy; BO: BO-1051; BO+IR: cells exposed to BO-1051 for 24 h and then irradiation with 2 Gy of g-ray. Values are the means ± SD of 3 independent experiments. * p < 0.05. Figure 4 Apoptotic effects of BO-1051 in combination with irradiation in glioma cells. U87MG, U251MG and GBM-3 were exposed to 200 nM or higher concentration (1200 nM) of BO-1051 for 24 h and irradiated with 2 Gy, followed by FACS analysis of Annexin V-FITC and PI staining 24 h later. Control: no treatment; IR: ionizing radiation at 2 Gy; BO: BO-1051; BO+IR: cells exposed to BO-1051 for 24 h and then irradiation with 2 Gy of g-ray. Values are the means ± SD of 3 independent experiments. * p < 0.05. was used. Mice bearing s.c. xenografts (~150 mm3) were stratified by size and randomized into 4 groups: control, BO-1051 alone, irradiation alone (4 Gy), or combined BO-1051 plus radiation. For the BO-1051 treatments, mice were i.p. injected with dosage at 50 mg/kg on days 0, 3 and 6. The growth rates for the GBM-3 tumors exposed to each treatment are shown in Figure 6A. BO-1051 delays the growth of xenograft gliomas exposed to irradiation To calculate an SER comparing the tumor radia- tion responses in mice with and without the BO-1051 treatment, the normalized tumor growth delay was measured to determine the role of BO-1051 on tumor growth delay induced by the combination treatment. The SER of the xenograft gliomas was 1.97 with versus without the combined treatment of BO-1051 and irra- diation (Table 2). Thus, BO-1051 alone slows tumor growth and enhances the effect of radiation, which is similar to the results obtained in vitro. Finally, the Kaplan-Meier survival curves of the combined treated mice revealed a trend toward longer survival in mice (Figure. 6B). We also noticed that the maximal toxicity of these agents decreased with body weight, and there was no more than a 15% weight reduction compared to the pretreatment body weight. However after cession of treatment, the body weight recovered (data not shown). Discussion Control 2Gy-1h 2Gy-6h 2Gy-24h BO-1051 200nM, 24h BO-1051 2Gy-1h BO-1051 2Gy-6h BO-1051 2Gy-24h unirradiated unirradiated  0 20 40 60 80 100 0-10 11-30 >30 0 5 10 15 20 25 30 35 vehicle BO ȖH2AX foci per cell 1h 6h 24h 0 Gy 1h 6h 24h 2 Gy Control 2Gy-1h 2Gy-6h 2Gy-24h BO-1051 200nM, 24h BO-1051 2Gy-1h BO-1051 2Gy-6h BO-1051 2Gy-24h Ctrl BO IR BO/IR unirradiated unirradiated Percent of cells with ȖH2AX foci Ύ    Figure 5 Influence of BO-1051 on the repair of radiation-induced DSBs. GBM-3 cells growing on slides in 35-mm dishes were exposed to 200 nM of BO-1051 for 24 h, irradiated (2Gy), and then fixed at the specified times for immunofluorescent analysis of nuclear g-H2AX foci using a confocal microscope. (A) Immunofluorescent microscopy images of GBM-3 cells untreated or treated with 200 nM BO-1051 24 h before irradiation, fixed after 0, 1, 6, 24 h and then stained for g-H2AX foci. (B) Quantitative analysis of g-H2AX foci presented in irradiated cells following the above treatments. Filled columns: data from vehicle-treated cells; open columns: data from cells exposed to BO-1051. Values are the means ± SD of 3 independent experiments. * p < 0.05. (C) Distribution of g-H2AX foci numbers per cell for one representative experiment at 24 h after irradiation. Ctrl: no treatment; IR: ironing radiation at 2 Gy; BO: cells exposed to 200 nM BO-1051; BO/IR: cells exposed to 200 nM BO-1051 for 24  2Gy-1h 0 20 40 60 80 100 0-10 11-30 >30 Ctrl BO IR BO/IR Percent of cells with ȖH2AX foci    Figure 5 Influence of BO-1051 on the repair of radiation-induced DSBs. GBM-3 cells growing on slides in 35-mm dishes were exposed to 200 nM of BO-1051 for 24 h, irradiated (2Gy), and then fixed at the specified times for immunofluorescent analysis of nuclear g-H2AX foci using a confocal microscope. (A) Immunofluorescent microscopy images of GBM-3 cells untreated or treated with 200 nM BO-1051 24 h before irradiation, fixed after 0, 1, 6, 24 h and then stained for g-H2AX foci. (B) Quantitative analysis of g-H2AX foci presented in irradiated cells following the above treatments. Filled columns: data from vehicle-treated cells; open columns: data from cells exposed to BO-1051. Values are the means ± SD of 3 independent experiments. * p < 0.05. Discussion Although human GBM is one of the most radio-resis- tant tumors, radiotherapy remains routinely applied for patient treatment. Lots of efforts are made to develop methods for enhancing the radiosensitivity of GBM for promising therapy. Previous studies have shown that temozolomide (TMZ) combined with radiation exposure results in an increase of survival rate in a subset of human tumors [3,25,26]. Clinical studies also indicate that delivery of TMZ during radiotherapy increases Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 9 of 13 0 20 40 60 80 100 0-10 11-30 >30 0 5 10 15 20 25 30 35 vehicle BO ȖH2AX foci per cell 1h 6h 24h 0 Gy 1h 6h 24h 2 Gy Control 2Gy-1h 2Gy-6h 2Gy-24h BO-1051 200nM, 24h BO-1051 2Gy-1h BO-1051 2Gy-6h BO-1051 2Gy-24h Ctrl BO IR BO/IR unirradiated unirradiated Percent of cells with ȖH2AX foci Ύ    Figure 5 Influence of BO-1051 on the repair of radiation-induced DSBs. GBM-3 cells growing on slides in 35-mm dishes were exposed to 200 nM of BO-1051 for 24 h, irradiated (2Gy), and then fixed at the specified times for immunofluorescent analysis of nuclear g-H2AX foci using a confocal microscope. (A) Immunofluorescent microscopy images of GBM-3 cells untreated or treated with 200 nM BO-1051 24 h before irradiation, fixed after 0, 1, 6, 24 h and then stained for g-H2AX foci. (B) Quantitative analysis of g-H2AX foci presented in irradiated cells following the above treatments. Filled columns: data from vehicle-treated cells; open columns: data from cells exposed to BO-1051. Values are the means ± SD of 3 independent experiments. * p < 0.05. (C) Distribution of g-H2AX foci numbers per cell for one representative experiment at 24 h after irradiation. Ctrl: no treatment; IR: ironing radiation at 2 Gy; BO: cells exposed to 200 nM BO-1051; BO/IR: cells exposed to 200 nM BO-1051 for 24 h and then irradiated with 2 Gy of g-rays. Foci were evaluated in 100 nuclei per treatment for each cell type. Values are the means at least of 3 independent experiments. Discussion (C) Distribution of g-H2AX foci numbers per cell for one representative experiment at 24 h after irradiation. Ctrl: no treatment; IR: ironing radiation at 2 Gy; BO: cells exposed to 200 nM BO-1051; BO/IR: cells exposed to 200 nM BO-1051 for 24 h and then irradiated with 2 Gy of g-rays. Foci were evaluated in 100 nuclei per treatment for each cell type. Values are the means at least of 3 independent experiments. survival rates of GBM patients, which suggests that this DNA alkylating agent can enhance the radiosensitivity of GBM [2,27,28]. Based on these previous studies, more efficient and safe DNA alkylating agents should be developed to increase the radiosensitivity in human GBM. Use of BO-1051 for cancer treatment has been supported by in vitro and in vivo preclinical studies [3,26]. The data presented here showed that the treat- ment of primary glioma cells and established cell lines with BO-1051 resulted in a dose-dependent induction of clonogenic cell death. It is supposed that BO-1051 can enhance the radiosensitivity via a synergistic effect since the survival fractions of combined treatment are lower than that of each individual treatment on glioma cell. However, additional studies are required to confirm that BO-1051 plays a synergistic or additive role on radiotherapy of gliomas. The anti-tumor and radiosensi- tizing effects of BO-1051 are encouraging because drugs showing efficacy against malignant glioma are still uncommon. Bifunctional N-mustard alkylating agents, such as BO- 1051, exhibits anticancer activity due to its ability to produce DNA interstrand and/or intrastrand cross-links [29,30]. As has been known, bifunctional alkylating agents induce collapsed replication forks that can lead to either cell cycle arrest, DNA repair, or apoptosis [31]. For example, the new synthesized alkylating agent BO-1012 shows anticancer activity on xenograft tumors Page 10 of 13 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Figure 6 The effects of BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in nude mice bearing GBM-3 xenografts. When tumors reached 150 mm3, the nude mice with established GBM-3 flank xenografts were randomized into 4 groups: control (black circle), radiation (white circle), BO-1051 (black triangle) or BO-1051 plus radiation (white triangle). BO-1051 (50 mg/kg) was delivered via i.p. injection on days 0, 3 and 6, where day 0 begins on the day of randomization. Discussion Radiation (4 Gy) was delivered 24 h after the first injection of BO-1051 (day 1 after randomization), which corresponded to the same tumor size. Each group contained at least 8 mice. (A) Tumor growth rates for each treatment group were plotted as the mean relative tumor volume ± SD. Arrows indicate the time of BO-1051 and irradiation treatment. (B) Kaplan-Meier survival rates of nude mice with GBM-3 flank xenografts for each of the four treatments is depicted. Survival analysis was monitored daily. Treatment failure was defined as tumor size greater than 1500 mm3 or the development of severe necrosis requiring euthanasia. f BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in 3 Figure 6 The effects of BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in nude mice bearing GBM-3 xenografts. When tumors reached 150 mm3, the nude mice with established GBM-3 flank xenografts were randomized into 4 groups: control (black circle), radiation (white circle), BO-1051 (black triangle) or BO-1051 plus radiation (white triangle). BO-1051 (50 mg/kg) was delivered via i.p. injection on days 0, 3 and 6, where day 0 begins on the day of randomization. Radiation (4 Gy) was delivered 24 h after the first injection of BO-1051 (day 1 after randomization), which corresponded to the same tumor size. Each group contained at least 8 mice. (A) Tumor growth rates for each treatment group were plotted as the mean relative tumor volume ± SD. Arrows indicate the time of BO-1051 and irradiation treatment. (B) Kaplan-Meier survival rates of nude mice with GBM-3 flank xenografts for each of the four treatments is depicted. Survival analysis was monitored daily. Treatment failure was defined as tumor size greater than 1500 mm3 or the development of severe necrosis requiring euthanasia. Figure 6 The effects of BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in nude mice bearing GBM-3 xenografts. When tumors reached 150 mm3, the nude mice with established GBM-3 flank xenografts were randomized into 4 groups: control (black circle), radiation (white circle), BO-1051 (black triangle) or BO-1051 plus radiation (white triangle). BO-1051 (50 mg/kg) was delivered via i.p. injection on days 0, 3 and 6, where day 0 begins on the day of randomization. only group to reach the same size. # Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. * Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text). Discussion Radiation (4 Gy) was delivered 24 h after the first injection of BO-1051 (day 1 after randomization), which corresponded to the same tumor size. Each group contained at least 8 mice. (A) Tumor growth rates for each treatment group were plotted as the mean relative tumor volume ± SD. Arrows indicate the time of BO-1051 and irradiation treatment. (B) Kaplan-Meier survival rates of nude mice with GBM-3 flank xenografts for each of the four treatments is depicted. Survival analysis was monitored daily. Treatment failure was defined as tumor size greater than 1500 mm3 or the development of severe necrosis requiring euthanasia. that are formed by various human lung and bladder cancer cells [32]. BO-1051 and its analog(s) also exhibit similar behavior, and several related synthetic bifunc- tional N-mustards are under development [33]. Because BO-1051 contains the inherent lipophilicity for penetra- tion through blood-brain barrier, it has efficiently demonstrated the ability to inhibit the growth of xeno- graft glioma in nude mice. Compared to other clinically used alkylating agents, such as melphalan and cisplatin, BO-1051 induced a higher level of ICLs [14]. BO-1051 also enhances the radiosensitivity of human glioma cell lines. Although repair mechanisms such as homologous recombination and nonhomologous end-joining are important mammalian responses to double-strand DNA damage, cell cycle regulation is perhaps the most impor- tant determinant of irradiation sensitivity [22,34]. The cell cycle is strongly affected by DNA damage, and a cell’s radiosensitivity depends on cell cycle position and progression [22]. Conventionally, the G2/M phase is the Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts Treatment group Tumor growth period, days* Absolute growth delay† Normalized growth delay‡ Enhancement ratio# Control 20.2 BO-1051 29.5 9.3 IR 23.6 3.4 BO-1051+IR 36.2 16 6.7 1.97 * Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text). † The number of days for the treated tumors to reach 10 times the initial tumor volume minus the number of days for the control group to reach the same size. ‡ The number of days for the tumors in the BO-1051+IR group to reach 10 times the initial tumor volume minus the number of days for tumors in the BO-1051- only group to reach the same size. # Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text). † The number of days for the treated tumors to reach 10 times the initial tumor volume minus the number of days for the control group to reach the same size. ‡ The number of days for the tumors in the BO-1051+IR group to reach 10 times the initial tumor volume minus the number of days for tumors in the BO-1051- only group to reach the same size. # Normalized growth delay for the BO 1051+IR group divided by absolute growth delay for the radiation only group y g p # Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Discussion Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts Treatment group Tumor growth period, days* Absolute growth delay† Normalized growth delay‡ Enhancement ratio# Control 20.2 BO-1051 29.5 9.3 IR 23.6 3.4 BO-1051+IR 36.2 16 6.7 1.97 * Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text). † The number of days for the treated tumors to reach 10 times the initial tumor volume minus the number of days for the control group to reach the same size. ‡ The number of days for the tumors in the BO-1051+IR group to reach 10 times the initial tumor volume minus the number of days for tumors in the BO-1051- only group to reach the same size. # Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts Treatment group Tumor growth period, days* Absolute growth delay† Normalized growth delay‡ Enhancement ratio# Control 20.2 BO-1051 29.5 9.3 IR 23.6 3.4 BO-1051+IR 36.2 16 6.7 1.97 * Ti f b f h i i i l l 0 i ( ) Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts y g p # Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Page 11 of 13 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 11 of 13 Page 11 of 13 However, theses phenomenon were not detected in glioma cells exposed to BO-1051 following irradiation. Recently, we found that BO-1051 can induce autophagy in glioma cell lines (unpublished observation), and sev- eral lines of evidence have supported that autophagy is one of the causes of radiosensitization instead of apopto- sis [51-53]. Therefore, the correlation between autophagy and radiosensitivity needs to be further investigated. most radiosensitive phase compared to others. Several chemotherapeutic agents have been reported to enhance the radiosensitivity of cancer cells by accumulating the G2/M population, such as paclitaxel, indomethacin, 2-methoxyestradiol and TMZ [3,22,25,35-37]. From the results in this study, BO-1051 works by partially synchro- nized glioma cells in the most radiosensitive phase of the cell cycle, and it is suggested that BO-1051 may be a use- ful agent for adjuvant therapy on the glioma. Given that human GBM usually exhibits high radiore- sistance, it is necessary to search for a specific radiosen- sitizer to enhance the radiosensitivity of GBM during radiotherapy. Discussion Kil et al. have demonstrated that TMZ may be used as a radiosensitizer because it can enhance the radiosensitivity of U251MG cells formed xenograft tumors [26]. Nevertheless, we found that TMZ was neither able to increase the radiosensitivity of xenograft tumors derived from GBM-3 cells nor able to delay tumor growth and improve animal survival after treat- ment (unpublished observation). Therefore, TMZ may exhibit cell specific effects for the treatment of different sources of human GBM. However, BO-1051 enhances the radiosensitivity of various glioma cell lines, as well as that of the corresponding xenograft tumors formed by GBM-3 cells. These results suggest that BO-1051 is a radiosensitizer with broader effects on different human GBM, and it may possess a clinical potential in the ther- apeutic strategy for treating malignant gliomas. The phosphatidyl-inositol kinase-related protein ATM (ataxia-telangiectasia mutated), the most proximal signal transducer initiating cell cycle changes after the DNA damage/genomic stress [38], can be activated by BO-1051 in a dose-dependent manner in SAS cell line. It also acti- vates the checkpoint kinase 2 (Chk2) in squamous cell car- cinoma cell line after exposure to BO-1051 (unpublished observation). Chk2 activity is necessary for the phosphory- lation of the dual-specificity phosphatases Cdc25A/C, which inactivates the enzymes, blocks CDK1 activation and causes a G2 arrest [39]. Furthermore, ATM’s essential role in DNA damage and repair is highlighted by the extreme sensitivity to ionizing radiation of cells with defec- tive ATM [40,41]. It, together with DNA-dependent pro- tein kinase, phosphorylate the histone g-H2AX foci, which can be visualized by immunofluorescence microscope as a discrete nuclear foci reflecting sites of DNA DSBs [42,43]. Although the specific relationship between the appearance of g-H2AX foci and the repair of DSBs has not been com- pletely defined, the reduction in the number of g-H2AX foci in irradiated cells correlates with DNA repair, which is associated with the radiosensitivity [44-46]. It is also known that g-H2AX is present in focal aggregates at sites of double-strand DNA damage and complex with other important repair molecules. g-H2AX is required for foci formation for numerous factors including p53, MRN com- plex (MRE-11, RAD50, and NBS1), and BRCA1 [47]. MRN complex has also been implicated in the repair of small fraction of DSBs detectable as g-H2AX foci that remain 24 h post-irradiation [48] Therefore, the observa- tion that combined BO-1051 plus radiation significantly increased the levels of g-H2AX foci. BO-1051: 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin- 9-ylamino)phenyl]urea; DBSs: Double-strand breaks; GBM: Glioblastoma multiforme; ICLs: Interstrand cross-linking; PE: Plating efficacy; SER: Sensitizer enhancement ratio; SF: Surviving fraction; TMZ: Temozolomide; TTF: Time to treatment failure. Abbreviations BO 0 { [B BO-1051: 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin- 9-ylamino)phenyl]urea; DBSs: Double-strand breaks; GBM: Glioblastoma multiforme; ICLs: Interstrand cross-linking; PE: Plating efficacy; SER: Sensitizer enhancement ratio; SF: Surviving fraction; TMZ: Temozolomide; TTF: Time to treatment failure. Radiation sensitization could occur through any one of multiple modes of cell death. Zou et al. observed radio- sensitization through the promotion of apoptosis [35], while another research group reported radiosensitization through a mitotic catastrophe [26,49] or senescence [50]. Conclusions GBM is the most malignant primary brain tumor in adults, but the effective therapeutic strategies remain under investigation. BO-1051 has been shown to inhibit the growth of gliomas. Here we further demonstrate that BO-1051 can significantly enhance the radiosensi- tivity. The enhanced radiosensitivity was found to be associated with G2/M phase arrest as well as the sus- tained DNA damage. In vivo studies further demon- strated that BO-1051 enhanced the radiotherapeutic effects on GBM-3-beared xenograft tumors. In this model, the combination of BO-1051 plus radiation pro- duced the best response in terms of both local control and survival. These data suggest that BO-1051 provides a new strategy to improve therapeutic gain for radiation therapy. Discussion Because the pro- longed expression of radiation-induced g-H2AX foci may reflect the end result of disparate processes and events leading to maintenance of unrepaired DSBs, a distinctly different mechanism may be involved. Whereas the mechanism of this repair inhibition is not revealed in this investigation, additional investigations are required to define the molecular processes responsible for BO-1051- mediated radiosensitization. References Wadhwa PD, Zielske SP, Roth JC, Ballas CB, Bowman JE, Gerson SL: Cancer gene therapy: scientific basis. Annu Rev Med 2002, 53:437-452. 24. Pilch DR, Sedelnikova OA, Redon C, Celeste A, Nussenzweig A, Bonner WM: Characteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem Cell Biol 2003, 81(3):123-129. 5. Grando SA, Kawashima K, Wessler I: Introduction: the non-neuronal cholinergic system in humans. Life Sci 2003, 72(18-19):2009-2012. 25. Chakravarti A, Erkkinen MG, Nestler U, Stupp R, Mehta M, Aldape K, Gilbert MR, Black PM, Loeffler JS: Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms. Clin Cancer Res 2006, 12(15):4738-4746. 6. Maze R, Carney JP, Kelley MR, Glassner BJ, Williams DA, Samson L: Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2- chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent. 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Gourdie TA, Valu KK, Gravatt GL, Boritzki TJ, Baguley BC, Wakelin LP, Wilson WR, Woodgate PD, Denny WA: DNA-directed alkylating agents. 1. Structure-activity relationships for acridine-linked aniline mustards: consequences of varying the reactivity of the mustard. J Med Chem 1990, 33(4):1177-1186. 28. Yung WK, Albright RE, Olson J, Fredericks R, Fink K, Prados MD, Brada M, Spence A, Hohl RJ, Shapiro W, et al: A phase II study of temozolomide vs. procarbazine in patients with glioblastoma multiforme at first relapse. Br J Cancer 2000, 83(5):588-593. 9. Author details 1 12. Lee CH, Chou TC, Su TL, Yu J, Shao LE, Yu AL: BO-0742, a derivative of AHMA and N-mustard, has selective toxicity to drug sensitive and drug resistant leukemia cells and solid tumors. Cancer Lett 2009, 276(2):204-211 1Graduate Institutes of Life Sciences, National Defense Medical Center & Department of Neurological Surgery, Tri-Service General Hospital, Taipei, Taiwan. 2Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan. 3Institute of Pharmacology, National Yang- Ming University, Taipei, Taiwan. 4Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. 5Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. 6Department of Biomedical Image and Radiological Sciences, School of Biomedical Science and Engineering, National Yang-Ming University, Taipei, Taiwan. 7Cancer Center, Taipei Veterans General Hospital, Taipei, Taiwan. 8Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan. 13. Kapuriya N, Kapuriya K, Dong H, Zhang X, Chou TC, Chen YT, Lee TC, Lee WC, Tsai TH, Naliapara Y, et al: Novel DNA-directed alkylating agents: design, synthesis and potent antitumor effect of phenyl N-mustard-9- anilinoacridine conjugates via a carbamate or carbonate linker. Bioorg Med Chem 2009, 17(3):1264-1275. 14. Kapuriya N, Kapuriya K, Zhang X, Chou TC, Kakadiya R, Wu YT, Tsai TH, Chen YT, Lee TC, Shah A, et al: Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage. Bioorg Med Chem 2008, 16(10):5413-5423. 15. Kim JH, Shin JH, Kim IH: Susceptibility and radiosensitization of human glioblastoma cells to trichostatin A, a histone deacetylase inhibitor. Int J Radiat Oncol Biol Phys 2004, 59(4):1174-1180. Competing interests The authors declare that they have no competing interest. 18. Dote H, Cerna D, Burgan WE, Carter DJ, Cerra MA, Hollingshead MG, Camphausen K, Tofilon PJ: Enhancement of in vitro and in vivo tumor cell radiosensitivity by the DNA methylation inhibitor zebularine. Clin Cancer Res 2005, 11(12):4571-4579. Received: 1 September 2010 Accepted: 19 January 2011 Received: 1 September 2010 Accepted: 19 January 2011 Received: 1 September 2010 Accepted: 19 January 2011 Published: 19 January 2011 Received: 1 September 2010 Accepted: 19 January 2011 Published: 19 January 2011 Published: 19 January 2011 Published: 19 January 2011 19. Barker CA, Burgan WE, Carter DJ, Cerna D, Gius D, Hollingshead MG, Camphausen K, Tofilon PJ: In vitro and in vivo radiosensitization induced by the ribonucleotide reductase inhibitor Triapine (3-aminopyridine-2- carboxaldehyde-thiosemicarbazone). Clin Cancer Res 2006, 12(9):2912-2918. Acknowledgements This study was supported by research grants from National Science Council- (NSC 97-3111-B-075-001-MY3, NSC99-2811-B-016-007-MY3, 98-2320-B-075-003- MY3), Taipei Veterans General Hospital (V97B1-006, E1-008, F-001), Tri-Service Page 12 of 13 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 General Hospital (TSGH-C100-047), the Joint Projects of UTVGH (VGHUST 98-p1- 01), Yen-Tjing-Ling Medical Foundation (96/97/98), National Yang-Ming University (Ministry of Education, Aim for the Top University Plan) & Genomic Center Project, Institute of Biological medicine, Academia Sinica (IBMS-CRC99- p01), and Center of Excellence for Cancer Research at Taipei Veterans General Hospital (DOH99-TD-C-111-007), Taiwan. 10. Su TL, Lin YW, Chou TC, Zhang X, Bacherikov VA, Chen CH, Liu LF, Tsai TJ: Potent antitumor 9-anilinoacridines and acridines bearing an alkylating N-mustard residue on the acridine chromophore: synthesis and biological activity. J Med Chem 2006, 49(12):3710-3718. 11. Su TL: Development of DNA topoisomerase II-mediated anticancer agents, 3-(9-acridinylamino)-5-hydroxymethylanilines (AHMAs) and related compounds. Curr Med Chem 2002, 9(18):1677-1688. Authors’ contributions PMC carried out most of the study, participated in its design, and drafted the manuscript. LHC, MTC, MHC, and YHS did parts of the statistical analysis and helped in discussion of data. YWC, SHY, PHT involved in drafting the manuscript in the section of radiotherapy techniques. HIM, SHC, TLS and YJL jointly conceived of the study, and coordination, participated in its design and drafted the manuscript. All authors read and approved the manuscript. 16. Camphausen K, Brady KJ, Burgan WE, Cerra MA, Russell JS, Bull EE, Tofilon PJ: Flavopiridol enhances human tumor cell radiosensitivity and prolongs expression of gammaH2AX foci. Mol Cancer Ther 2004, 3(4):409-416. 17. McCord AM, Jamal M, Williams ES, Camphausen K, Tofilon PJ: CD133+ glioblastoma stem-like cells are radiosensitive with a defective DNA damage response compared with established cell lines. Clin Cancer Res 2009, 15(16):5145-5153. References 1. Walker MD, Green SB, Byar DP, Alexander E Jr, Batzdorf U, Brooks WH, Hunt WE, MacCarty CS, Mahaley MS Jr, Mealey J Jr, et al: Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 1980, 303(23):1323-1329 1. Walker MD, Green SB, Byar DP, Alexander E Jr, Batzdorf U, Brooks WH, Hunt WE, MacCarty CS, Mahaley MS Jr, Mealey J Jr, et al: Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 1980, 303(23):1323-1329. 20. Russell JS, Burgan W, Oswald KA, Camphausen K, Tofilon PJ: Enhanced cell killing induced by the combination of radiation and the heat shock protein 90 inhibitor 17-allylamino-17- demethoxygeldanamycin: a multitarget approach to radiosensitization. Clin Cancer Res 2003, 9(10 Pt 1):3749-3755. 2. Stupp R, Dietrich PY, Ostermann Kraljevic S, Pica A, Maillard I, Maeder P, Meuli R, Janzer R, Pizzolato G, Miralbell R, et al: Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide. J Clin Oncol 2002, 20(5):1375-1382. 2. Stupp R, Dietrich PY, Ostermann Kraljevic S, Pica A, Maillard I, Maeder P, Meuli R, Janzer R, Pizzolato G, Miralbell R, et al: Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide. J Clin Oncol 2002, 20(5):1375-1382. 21. Geldof AA, Slotman BJ: Radiosensitizing effect of cisplatin in prostate cancer cell lines. Cancer Lett 1996, 101(2):233-239. 3. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, et al: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352(10):987-996. 3. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, et al: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352(10):987-996. 22. Pawlik TM, Keyomarsi K: Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys 2004, 59(4):928-942. 23. Sinclair WK, Morton RA: X-ray sensitivity during the cell generation cycle of cultured Chinese hamster cells. Radiat Res 1966, 29(3):450-474. 4. Wadhwa PD, Zielske SP, Roth JC, Ballas CB, Bowman JE, Gerson SL: Cancer gene therapy: scientific basis. Annu Rev Med 2002, 53:437-452. 4. References Bacherikov VA, Chou TC, Dong HJ, Zhang X, Chen CH, Lin YW, Tsai TJ, Lee RZ, Liu LF, Su TL: Potent antitumor 9-anilinoacridines bearing an alkylating N-mustard residue on the anilino ring: synthesis and biological activity. Bioorg Med Chem 2005, 13(12):3993-4006. 29. Tomasz M, Palom Y: The mitomycin bioreductive antitumor agents: cross- linking and alkylation of DNA as the molecular basis of their activity. Pharmacol Ther 1997, 76(1-3):73-87. Page 13 of 13 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 Page 13 of 13 Page 13 of 13 Chu et al. Radiation Oncology 2011, 6:7 http://www.ro-journal.com/content/6/1/7 30. Panasci L, Xu ZY, Bello V, Aloyz R: The role of DNA repair in nitrogen mustard drug resistance. Anticancer Drugs 2002, 13(3):211-220. radiotherapy in a mouse model of lung cancer. Autophagy 2008, 4(5):659-668. 53. Tsuboi Y, Kurimoto M, Nagai S, Hayakawa Y, Kamiyama H, Hayashi N, Kitajima I, Endo S: Induction of autophagic cell death and radiosensitization by the pharmacological inhibition of nuclear factor- kappa B activation in human glioma cell lines. J Neurosurg 2009, 110(3):594-604. 31. Sorensen CS, Hansen LT, Dziegielewski J, Syljuasen RG, Lundin C, Bartek J, Helleday T: The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol 2005, 7(2):195-201. 32. Kakadiya R, Dong H, Lee PC, Kapuriya N, Zhang X, Chou TC, Lee TC, Kapuriya K, Shah A, Su TL: Potent antitumor bifunctional DNA alkylating agents, synthesis and biological activities of 3a-aza-cyclopenta[a] indenes. Bioorg Med Chem 2009, 17(15):5614-5626. doi:10.1186/1748-717X-6-7 Cite this article as: Chu et al.: Enhancement of radiosensitivity in human glioblastoma cells by the DNA N-mustard alkylating agent BO-1051 through augmented and sustained DNA damage response. Radiation Oncology 2011 6:7. 33. Kakadiya R, Dong H, Kumar A, Narsinh D, Zhang X, Chou TC, Lee TC, Shah A, Su TL: Potent DNA-directed alkylating agents: Synthesis and biological activity of phenyl N-mustard-quinoline conjugates having a urea or hydrazinecarboxamide linker. Bioorg Med Chem 2010, 18(6):2285-2299. 34. Shrivastav M, De Haro LP, Nickoloff JA: Regulation of DNA double-strand break repair pathway choice. Cell Res 2008, 18(1):134-147. 35. Zou H, Zhao S, Zhang J, Lv G, Zhang X, Yu H, Wang H, Wang L: Enhanced radiation-induced cytotoxic effect by 2-ME in glioma cells is mediated by induction of cell cycle arrest and DNA damage via activation of ATM pathways. Brain Res 2007, 1185:231-238. 36. References Jin C, Wu H, Liu J, Bai L, Guo G: The effect of paclitaxel-loaded nanoparticles with radiation on hypoxic MCF-7 cells. J Clin Pharm Ther 2007, 32(1):41-47. 37. Furuta Y, Hunter N, Barkley T Jr, Hall E, Milas L: Increase in radioresponse of murine tumors by treatment with indomethacin. Cancer Res 1988, 48(11):3008-3013. 38. Samuel T, Weber HO, Funk JO: Linking DNA damage to cell cycle checkpoints. Cell Cycle 2002, 1(3):162-168. 39. Herzinger T, Funk JO, Hillmer K, Eick D, Wolf DA, Kind P: Ultraviolet B irradiation-induced G2 cell cycle arrest in human keratinocytes by inhibitory phosphorylation of the cdc2 cell cycle kinase. Oncogene 1995, 11(10):2151-2156. 40. Kastan MB, Lim DS: The many substrates and functions of ATM. Nat Rev Mol Cell Biol 2000, 1(3):179-186. 41. Collis SJ, Swartz MJ, Nelson WG, DeWeese TL: Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res 2003, 63(7):1550-1554. 42. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM: DNA double- stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998, 273(10):5858-5868. 43. Sedelnikova OA, Rogakou EP, Panyutin IG, Bonner WM: Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma- H2AX antibody. Radiat Res 2002, 158(4):486-492. 44. Olive PL, Banath JP: Phosphorylation of histone H2AX as a measure of radiosensitivity. Int J Radiat Oncol Biol Phys 2004, 58(2):331-335. 45. Nazarov IB, Smirnova AN, Krutilina RI, Svetlova MP, Solovjeva LV, Nikiforov AA, Oei SL, Zalenskaya IA, Yau PM, Bradbury EM, et al: Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A. Radiat Res 2003, 160(3):309-317. 46. Banath JP, Macphail SH, Olive PL: Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines. Cancer Res 2004, 64(19):7144-7149. 47. Bilsland E, Downs JA: Tails of histones in DNA double-strand break repair. Mutagenesis 2005, 20(3):153-163. 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 48. Lobrich M, Jeggo PA: Harmonising the response to DSBs: a new string in the ATM bow. DNA Repair (Amst) 2005, 4(7):749-759. References Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 49. Mitchell JB, Choudhuri R, Fabre K, Sowers AL, Citrin D, Zabludoff SD, Cook JA: In vitro and in vivo radiation sensitization of human tumor cells by a novel checkpoint kinase inhibitor, AZD7762. Clin Cancer Res 2010, 16(7):2076-2084. 50. 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Trabalho paralelo e trabalho doméstico: um diálogo teórico possível?
Revista Estudos Feministas
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Trabalho fora do trabalho: uma etnografia das percepções. de me tornar etnógrafa, e este livro guarda tais vestígios [...]” (p. 272-273). O livro se divide em três partes: “O ofício de etnógrafo”, que trata do método e da teoria da pesquisa; “Formas e funções do trabalho paralelo”, que busca explorar a prática do trabalho paralelo em si e suas valorações por parte dos trabalhadores observados; e “Pertencimentos sociais, pertencimentos locais”, que explora as relações nas quais as valorações desse tipo de atividade se constroem. WEBER, Florence. Tradução de Roberta Ceva. Rio de Janeiro: Garamond, 2009. 384 p. Tradução de Roberta Ceva. Rio de Janeiro: Garamond, 2009. 384 p. Tradução de Roberta Ceva. Rio de Janeiro: Garamond, 2009. 384 p. A análise é cuidadosa. As pessoas em geral são observadas em diferentes aspectos, não só pelo pertencimento àquilo que a autora chama de operariado, mas a descendência, o sexo, a renda, as relações políticas e a faixa etária são aspectos levados em conta. Considerando que, para ter acesso a informações que dados estatísticos não resolveriam sobre o trabalho realizado fora do ambiente formal (que muitas vezes não era declarado por medo de uma fiscalização governamental sobre sua irregula- ridade ou até pelo caráter de lazer atribuído pelos pesquisados a esse trabalho), a pesquisadora se insere no ambiente estudado para obter as informações. Assim, em muitos momentos, ela coloca suas percepções encarando-as como sendo parte do meio, apesar de trabalhar com a ideia de distanciamento entre pesquisador e objeto de pesquisa. Apesar de os estudos que têm como objeto o operariado, nas Ciências Humanas, serem inquestionavelmente extensos, de forma geral esses estão ligados a uma identidade operária construída na relação com a fábrica – ou outro que seja seu ambiente de trabalho formal – ou então construída na resistência a ela. Nas pesquisas que se preocupam em observar essas questões mais de perto, o ideal de bom operário ou o de bom militante não aparece puro, estando circulando e se relacionando de forma não estática, não engessada. Florence Weber, em sua pesquisa realizada entre trabalhadores/as de uma área rural francesa entre 1983 e 1985, buscou observar exatamente o que não estava nem na fábrica nem no sindicato, questionando- se sobre o que faziam os operários fora de seu trabalho em uma fábrica metalúrgica. TTTTTrabalho paralelo e trabalho doméstico: rabalho paralelo e trabalho doméstico: rabalho paralelo e trabalho doméstico: rabalho paralelo e trabalho doméstico: rabalho paralelo e trabalho doméstico: um diálogo teórico possível? um diálogo teórico possível? um diálogo teórico possível? um diálogo teórico possível? um diálogo teórico possível? Trabalho fora do trabalho: uma etnografia das percepções. Trabalho fora do trabalho: uma etnografia das percepções. Quer dizer, é possível que na época da pesquisa ela intencionalmente não tenha se apropriado dessas questões feministas por razões políticas, pessoais ou até de legitimidade acadêmica. operárias? Mesmo que representando uma minoria faziam parte do quadro da fábrica. O que elas faziam com seu tempo livre? Estando convivendo na cidade por mais de um ano e tendo relações próximas com uma operária que inclusive a ajudou na pesquisa, essas perguntas poderiam ter sido feitas. Leituras de teóricas feministas sobre como o amor à família e a ideia de lazer2 se entrelaçam com o trabalho doméstico poderiam inclusive ajudar nas observações sobre a realização pessoal imbricada no “trabalho paralelo”. Os debates sobre os lugares de produção e lugares de reprodução continuam atuais,3 ainda que transformados em muitos aspectos. Talvez isso justifique a retomada desta obra (de 1989 e republicada vinte anos depois), a qual no século XXI pode oferecer subsídios para se pensarem as relações de trabalho diluídas na vida privada, a constituição de identidades construídas sobre práticas laborais e a não fixidez das relações que se dão nesses espaços; este último ponto inclusive ressaltado por Florence Weber em sua escrita. Para os estudos feministas e de gênero, talvez a maior contribuição da pesquisadora tenha sido, paradoxalmente, o que ela considerou o resultado não feminista de sua pesquisa: a conclusão de que não só as mulheres produzem fora dos circuitos de mercado. Essa conclusão tanto pode contribuir para estudos de masculinidades e identidades laborais, de trabalho reprodutivo e de relações de gênero quanto para dar mais consistência às conceituações sobre trabalho reprodutivo para se questionar a constituição hierárquica da noção de trabalho produtivo versus trabalho reprodutivo, entre outros debates afins. Outro ponto, que nem ao menos no posfácio é tratado, é sobre as mulheres operárias. A obra é problemática na medida em que utiliza o tempo todo “os operários” como sujeito universal, e nunca especifica com clareza que se escolheu trabalhar apenas com homens. De repente, aparece uma operária que a ajuda na pesquisa, uma açougueira é citada. Mas o trabalho paralelo analisado é apenas masculino. Trabalho fora do trabalho: uma etnografia das percepções. Socióloga e antropóloga francesa, Florence Weber se desloca na década de 1980 de Paris para Montbard, pequena cidade na qual residiu durante a infância, para, através da pesquisa etnográfica, buscar “descobrir, a partir da observação dos comportamentos operários fora da fábrica, valores e gostos positivos que permitem explicar certas práticas operárias sem reduzi-las a uma simples submissão à necessidade” (p. 254). O foco principal da pesquisadora é o “trabalho paralelo”, um termo que ela classifica como nativo (próprio dos pesquisados) e que designa uma série de atividades que produzem bens de uso que não são voltados, ou especificamente voltados, para a mercantilização. No posfácio de 2009, acrescido à edição original de 1989, a autora faz duas afirmações que podem nos ajudar na leitura de seu trabalho: “Sem dúvida nenhuma, eu era marxista e feminista (nesta ordem), antes Nota-se que ela se preocupa em marcar seu afastamento de abordagens que carregam a alcunha de “pós-modernas”, percepção que se completa com a ideia de que “uma interpreta- ção aprofundada destes acontecimentos microscópicos [atividades reconhecidas como trabalho paralelo] pode torná-los exemplares” (p. 15). Mas lembra, em seu posfácio datado de 2009, que seus resultados não foram marxistas porque os operários que descreve não são proletários inteiramente presos à ordem industrial capitalista. Tais afirmações podem gerar debates. De qualquer forma, em meio a essa discussão sobre ser ou não marxista ou pós-estruturalista, a ideia de “trabalho paralelo” como revanche ao trabalho na fábrica, como uma prática de resis- tência, uma das hipóteses iniciais da abordagem, foi abandonada no decorrer de sua pesquisa. Sua posição perante o feminismo chama especial atenção, principalmente, quando ela 335 Estudos Feministas, Florianópolis, 20(1): 313-337, janeiro-abril/2012 335 mesma reconhece, em 2009, e talvez após receber algumas críticas nesse sentido, que sua preocupação em descrever a vida cotidiana, a produção doméstica e trocas não mercantis aproximá-la-ia, nesses aspectos, aos trabalhos feministas que se interessam pela parte invisível da economia. Uma primeira questão que tais afirmações podem gerar é de que essas discussões sobre a economia invisível, por parte dos feminismos, já existiam e tinham razoável circulação quando ela escreveu seu livro, em meados para o final da década de 1980.1 Mas o trabalho paralelo das mulheres é sucintamente citado no livro, em duas ou três linhas, apenas no posfácio de 2009. Notas Notas Notas Notas Notas 1 Exemplos de trabalhos europeus neste sentido em Maria Angeles DURAN, 1983; e Andrée KARTCHEVSKY-BULPORT et al., 1986. 1 Exemplos de trabalhos europeus neste sentido em Maria Angeles DURAN, 1983; e Andrée KARTCHEVSKY-BULPORT et al., 1986. 2 Referência em DURAN, 1983. 2 Referência em DURAN, 1983. E o trabalho paralelo feminino? Esse, ela qualifica em geral como trabalho doméstico, por seu caráter de obrigatoriedade. Mas a partir do momento em que ao trabalho paralelo que ela analisa e conceitua são agregados valores de satisfação pessoal e até lazer que os diferenciariam de uma dupla jornada de trabalho, com afirmações como “Caso se trabalhasse para um patrão, faria-se muito menos” (p. 67), não entendo a dificuldade em utilizar seu próprio conceito para uma análise voltada também às mulheres. É claro que as donas de casa, esposas de operários, não eram seu objeto, apesar de se terem feito incursões sobre elas. Mas e as mulheres 3 Exemplos em Cristina BRUSCHINI, 2007; e Cristina CARRASCO, 2008. 3 Exemplos em Cristina BRUSCHINI, 2007; e Cristina CARRASCO, 2008. Trabalho fora do trabalho: uma etnografia das percepções. Se ao ler a obra publicada em 1989 a crítica construir-se-ia no sentido de problematizar como uma pesquisa voltada ao trabalho invisível se esquece das mulheres que fazem parte de seu objeto (sendo o trabalho invisível de forma geral referido às mulheres), após ler o posfácio de 2009 e a identificação da pesquisadora como femi- nista (ainda que seja uma identificação secundá- ria em relação à de marxista), pode-se questionar como uma feminista, que tomou cuidado com tantas questões, invisibilizou as mulheres em sua obra, que quando aparecem é pelo caráter de “colaboração” – termo da autora – das mulheres nesses trabalhos paralelos masculinos. DURAN, Maria Angeles. A dona de casa: crítica política da economia doméstica. Rio de Janeiro: Edições Graal, 1983. Lucia da; TITO, Neuza. Trabalho doméstico e de cuidados: por outro paradigma de sustentabilidade da vida humana. São Paulo: SOF, 2008. p. 91-104. KARTCHEVSKY-BULPORT, Andrée et al. O sexo do trabalho. Rio de Janeiro: Paz e Terra, 1986. KARTCHEVSKY-BULPORT, Andrée et al. O sexo do trabalho. Rio de Janeiro: Paz e Terra, 1986. Soraia Carolina de Mello Universidade Federal de Santa Catarina Referências Referências Referências Referências Referências BRUSCHINI, Cristina. “Trabalho doméstico: inativi- dade econômica ou trabalho não remune- rado?”. In: ARAÚJO, Clara; PICANÇO, Felícia; SCALON, Celi (Org.). Novas conciliações e antigas tensões? Gênero, família e trabalho em perspectiva comparada. Bauru: EDUSC, 2007. p. 21-58. CARRASCO, Cristina. “Por uma economia não androcêntrica: debates e propostas a partir da economia feminista”. In: SILVEIRA, Maria CARRASCO, Cristina. “Por uma economia não androcêntrica: debates e propostas a partir da economia feminista”. In: SILVEIRA, Maria 336 36 Estudos Feministas, Florianópolis, 20(1): 313-337, janeiro-abril/2012 KARTCHEVSKY-BULPORT, Andrée et al. O sexo do trabalho. Rio de Janeiro: Paz e Terra, 1986. Soraia Carolina de Mello Universidade Federal de Santa Catarina DURAN, Maria Angeles. A dona de casa: crítica política da economia doméstica. Rio de Janeiro: Edições Graal, 1983. Estudos Feministas, Florianópolis, 20(1): 313-337, janeiro-abril/2012 337
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Clinical significance of peripheral blood-derived inflammation markers in advanced gastric cancer after radical resection
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Clinical significance of peripheral blood-derived inflammation markers in advanced gastric cancer after radical resection Lihu Gu  Ningbo No 2 Hospital Bangsheng Chen  Ningbo Yinzhou No 2 Hospital Dingcheng Zheng  Ningbo No 2 Hospital Jiahang Mo  Zhejiang Chinese Medical University Lingling Yuan  Ningbo No 2 Hospital Feiyan Mao  Ningbo No 2 Hospital Kang Zhang  Ningbo University Medical School Derry Minyao Ng  Ningbo University Medical School Ping Chen Feiyan Mao  Ningbo No 2 Hospital Research article Keywords: Gastric cancer, Systemic inflammatory marker, Platelet lymphocyte ratio, Prognosis, Ch th Keywords: Gastric cancer, Systemic inflammatory marker, Platelet lymphocyte ratio, Prognosis, Chemotherapy Posted Date: March 4th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-15911/v1 Version of Record: A version of this preprint was published at BMC Surgery on October 2nd, 2020. See the published version at https://doi.org/10.1186/s12893-020-00884-8. DOI: https://doi.org/10.21203/rs.3.rs-15911/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Page 1/20 Version of Record: A version of this preprint was published at BMC Surgery on October 2nd, 2020. See the published version at https://doi.org/10.1186/s12893-020-00884-8. Version of Record: A version of this preprint was published at BMC Surgery on October 2nd, 2020. See the published version at https://doi.org/10.1186/s12893-020-00884-8. Page 2/20 Abstract Background and objective The prognostic significance of peripheral blood-derived inflammation markers in patients with gastric cancer (GC) has not been elucidated. This study aimed to investigate the relationship between systemic inflammatory markers and GC prognosis. Methods A prospective observational cohort study involving 598 patients was conducted to analyze the prognosis of GC based on systemic inflammatory markers. The following peripheral blood-derived inflammation markers were evaluated: the neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), systemic immune- inflammation index (SII), C-reactive protein/albumin (CRP/Alb) ratio, Glasgow Prognostic Score (GPS), modified Glasgow Prognostic Score (mGPS), prognostic nutrition index (PNI), and prognostic index (PI). The receiver operating characteristics (ROC) curve and the Youden index were used to determine the optimal cutoff values. Univariate and multivariate analysis of prognostic factors was conducted accordingly. Results The optimal cutoff values of the PNI, fibrinogen, NLR, PLR, SII, and CRP/Alb were 49.5, 397ng/dl, 2.5, 154, 556, and 0.05, respectively. Multivariate analysis showed that age, PLR, TNM stage, and chemotherapy were the independent prognostic factors for advanced gastric cancer (AGC). Adjuvant chemotherapy improved the long-term prognosis of patients with PLR ≥154, but chemotherapy had no significant effect on the survival of patients with PLR <154. Conclusions Our findings show that higher PLR (≥154) is an independent risk factor for poor prognosis in GC patients. Besides, PLR can predict adjuvant chemotherapy (oxaliplatin/5-fluorouracil combination) response in patients with GC after surgery. Study population This was a prospective observational cohort study involving patients who underwent radical gastrectomy from January 2013 to December 2016 at HwaMei Hospital, University of Chinese Academy of Sciences. And was approved by the Ethics Committee of the HwaMei Hospital and the University of Chinese Academy of Sciences (approval NO. PJ-NBEY-KY-2019-153-01). Written consent was obtained from all patients before enrollment. The inclusion criteria were: (1) patients with histologically proven primary adenocarcinoma of the stomach; (2) patients without a history of gastrectomy or other malignant tumor; (3) patients without inflammatory or hematological diseases; (4) patients with pathologically negative resection margins (R0); (5) patients without neoadjuvant chemoradiotherapy; (6) follow-up of at least 36 months. Treatment with 5-fluorouracil (5-FU)-based or platinum-based adjuvant chemotherapy was recommended for all patients with stage II-III of the disease[13]. Introduction Gastric cancer (GC) is one of the most common malignant tumors and that poses a serious threat to human health, especially in Asia. Approximately 300,000 deaths and 400,000 new cases of GC occur in China every year[1]. Despite the advancement in diagnostic and treatment methods, the prognosis of advanced gastric cancer (AGC) patients has remained poor[2]. Tumor, Node, Metastasis (TNM) staging based on the International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) guidelines is currently the standard approach of determining the prognosis of GC patients[3]. However, several prognostic factors related to GC have been proposed, which include peripheral blood-derived inflammation markers, such as neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), systemic immune-inflammation index (SII), C-reactive protein/albumin (CRP/Alb) ratio, and Glasgow Prognostic Score (GPS)[4–6]. Some studies have combined the TNM staging system with GC related risk factors to improve the accuracy of the long-term prognosis of the disease[7]. Routine peripheral blood-derived inflammation markers are closely associated with the pathogenesis of GC[8]. Moreover, the use of these markers as prognostic factors is advantageous because most of the peripheral blood-derived inflammation markers belong to the routine test items; the test cost is cheap and does not require special equipment. Page 3/20 Page 3/20 Park indicated that preoperative body mass index (BMI) and prognostic nutritional index (PNI), as well as their postoperative changes, are related to the prognosis of stage II/III GC[9]. Also, Jagadesham reported that the combination of one or more markers of systemic inflammation could precisely predict the prognosis of advanced adenocarcinoma of the esophagus[10]. Studies have suggested that combining NLR and PLR could significantly improve the accuracy of predicting the first-line chemosensitivity in AGC[11]. Contrarily, Xu et al. indicated that CRP/Alb might be a promising predictor of early recurrence (recurrence within 12 months after radical gastrectomy) and postoperative chemotherapy in stage III GC[12]. Unfortunately, most of these findings are based on small sample retrospective studies with insufficient evidence, which could be the reason for the inconsistencies among the various reports. Herein, we designed a prospective observational cohort to examine the relationship between peripheral blood-derived inflammation markers and the prognosis of GC. Also, we hypothesized that derangements in one or more systemic inflammation markers may be associated with poor disease outcomes and the ineffectiveness of chemotherapy. Follow-up All patients were followed up every 3–6 months for the first two years and annually thereafter until death or at least five years after surgery. Disease-free survival (DFS) was defined as the time from surgery to death, loco-regional recurrence or distant recurrence. Disease-specific survival (DSS) was defined as the time from surgery to death as a result of GC. Patients for whom none of these events was recorded were censored at the date of their last known contact. The median follow-up time for the entire cohort was 50 months (range 4–83 months), and follow-up of all patients was stopped in December 2019. All patients were followed up every 3–6 months for the first two years and annually thereafter until death or at least five years after surgery. Disease-free survival (DFS) was defined as the time from surgery to death, loco-regional recurrence or distant recurrence. Disease-specific survival (DSS) was defined as the time from surgery to death as a result of GC. Patients for whom none of these events was recorded were censored at the date of their last known contact. The median follow-up time for the entire cohort was 50 months (range 4–83 months), and follow-up of all patients was stopped in December 2019. Statistical analysis Continuous variables were compared using the independent sample t-test or Wilcoxon rank-sum test, whereas categorical variables were compared using Pearson’s chi-squared test or Fisher’s exact test, as appropriate. The receiver operating characteristics (ROC) curve was calculated, and the Youden index (maximum = sensitivity + specificity − 1) was used to determine the optimal cutoff value for the number of lymph nodes retrieved, PNI, fibrinogen, NLR, PLR, SII, and CRP/Alb. The potentially relevant factors obtained from the univariate analysis were assessed in the multivariate model using Cox’s regression. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated. The DFS and DSS were calculated using the Kaplan-Meier method, and the log-rank test was employed to determine the significance. All statistical tests were performed 2-sided, and a p < 0.05 was considered statistically significant. All the analyses were performed using SPSS software (version 25.0, SPSS Inc. IL, USA). Systemic inflammatory markers and histological examination The whole blood and clinicopathological data were obtained one week before initial treatment. Blood samples were collected for routine laboratory tests, which included complete blood count, CRP, albumin, fibrinogen, and tumor markers, such as carcinoembryonic antigen (CEA). The following common peripheral blood-derived inflammation markers were included based on previous studies: NLR, PLR, SII, CRP/Alb, GPS, modified Glasgow Prognostic Score (mGPS), PNI, and prognostic index (PI). All surgical resection specimens were assessed according to the handling guideline of the third edition of the Japanese classification of gastric carcinoma. And the staging was conducted by pathologists using the 8th edition of the UICC/AJCC TNM staging system[3]. Page 4/20 Results A total of 598 patients were recruited from January 2013 to December 2016. The 5-year DFS and DSS rates of all patients were 72.6% and 75.4%, respectively. Male patients were about twice as many as female patients, and the tumors in the distal stomach accounted for 77% of all the tumors. Concerning the GC staging, patients with GC stage I, II, and III were 119, 113, and 366, respectively. The 5-year DFS and DSS rates after surgery for stage I patients were 97% and 98%, respectively, whereas, for stage II patients, the rates were 81.4% and 85.8%, respectively. For stage III patients, the rates were 52.1% and 55.6%, respectively (Supplementary Table 1). A total of 376 patients received adjuvant chemotherapy, of which 239 received SOX regimen [14] and 112 XELOX[15]. The remaining 25 patients received other chemotherapy treatments[16]. Optimal cutoff analysis The optimal cutoff value of the number of lymph nodes retrieved, PNI, fibrinogen, NLR, PLR, SII, and CRP/Alb were 30, 49.5, 397ng/dl, 2.5, 154, 556, and 0.05, respectively. Risk factors for advanced gastric cancer Considering the close correlation between the TNM stage and the 5-year DFS, the number of lymph nodes retrieved may be related to the prognosis. Lymph nodes less than 16 is considered inadequate lymph node dissection, especially for AGC. Therefore, only patients with the AGC lymph nodes ≥16 were included in the analysis. Multivariate analysis showed that age, PLR, and TNM stages were associated with 5-year DFS. Also, age, PLR, TNM stage, and chemotherapy were associated with 5-year DSS (Table 2). Clinicopathological factors and survival analysis Clinicopathological factors and survival analysis Page 5/20 Page 5/20 A total of 23 potential risk factors were selected. According to univariate analysis, the following 17 clinicopathological characteristics were found to be significantly associated with the 5-year DFS rate in all enrolled patients: age, tumor location, type of gastrectomy, tumor size, perineural invasion, lymphovascular invasion, T stage, N stage, chemotherapy, PNI, fibrinogen, NLR, PLR, SII, GPS, CRP/Alb, and CEA (Table 1). Multivariate Cox proportional hazards model analysis showed that age, T stage, N stage, number of lymph nodes retrieved, and PLR were independent prognostic factors for GC (Table 1). Risk factors associated with stage II and III gastric cancer Kaplan-Meier curves were used to determine the long-term disease outcomes of GC patients in stage II and stage III. In GC stage II, the 5-year DFS rates were similar among patients with age ≤60 and age >60 (p=0.213). However, in stage III, the 5-year DFS rate of GC patients aged >60 was worse than those aged ≤60 (p=0.016). Similarly, in GC stage III, the 5-year DFS rate of patients with PLR ≥154 was worse than those with PLR <154 (p<0.001). But this phenomenon was not observed in GC stage II (p=0.153). The effect of chemotherapy on the prognosis of patients with stage II GC was not statistically significant (p=0.260). In contrast, chemotherapy significantly improved the 5-year DFS in patients with stage III GC (p=0.017) [Figure 1]. With the 5-year DSS as a long-term prognostic index, age, PLR, and chemotherapy had the same effect on the prognosis of stage II and III GC patients as 5-year DFS, except that chemotherapy also improved the 5- year DSS of stage II GC patients (p=0.033) [Figure 2]. Combined with the above observations, PLR was found to be an independent risk factor for the prognosis of AGC. Patients with AGC were divided into the PLR <154 and PLR ≥154 subgroups. Further analysis of the clinicopathological factors between the two groups revealed that tumor lesions in the PLR <154 group were smaller than those in the PLR ≥154 group. But, there was no other difference between the groups (Table 3). Further analysis of the relationship between chemotherapy and PLR showed that chemotherapy improved the long-term prognosis of patients in the PLR ≥154 group, including 5-year DFS and DSS (p=0.026, p=0.014, respectively). Notably, chemotherapy had no significant effect on the long- term prognosis of patients in the PLR <154 group (Figure 3). Discussion Page 6/20 Many studies have been conducted to investigate the correlation between peripheral blood-derived inflammation markers and tumor prognosis[17, 18]. Liu showed that CRP/Alb was an independent prognostic marker for patients with ovarian cancer[19]. Also, the NLR and PLR are prognostic factors in patients with non-small cell lung cancer after stereotactic radiation therapy[20]. The independent risk factors for poor GC prognosis include NLR, PLR, fibrinogen, PNI, GPS, CRP/Alb, among others. Also, some studies have combined these systemic inflammatory markers with or without TNM stage to provide new prognostic tools[21–23]. However, most of these studies were retrospective, and reported inconsistent results, particularly on the significance of each inflammatory index and the threshold value. Therefore, in addition to the peripheral blood-derived inflammation markers reported in previous studies, this study further explored the prognostic value of some conventional systemic inflammatory marker in patients with GC. We prospectively analyzed 598 GC patients after radical surgery and found that independent risk factors for poor prognosis of GC included age, T stage, N stage, number of lymph nodes retrieved, and PLR. Currently, TNM staging is the standard prognostic tool for GC. Given the excellent prognosis of early gastric cancer, our focus was to analyze the prognosis of patients with AGC (stage II/III). Many studies have demonstrated that the number of lymph node dissection has an impact on the prognosis of GC; therefore, we further excluded patients with an inadequate amount of lymph node dissection (less than 16)[24–26]. Independent risk factors for AGC included age, PLR, TNM stages, and chemotherapy. Previous studies have investigated the effect of age on GC prognosis; however, most of these studies found no significant association between age and GC prognosis[27–29]. Takatsu analyzed 5000 GC cases and found that early-onset GC (age < 40 years) was likely to present lymph node metastases. But the survival rate of young GC patients was equivalent to that of older GC patients (age ≥ 60 years)[30]. In the present study, older GC patients (age > 60 years) had a worse prognosis, which was closely related to tumor recurrence. Nutritional status is associated with survival in patients with malignant tumors, including GC. Nutritional status is associated with survival in patients with malignant tumors, including GC. Preoperative underweight and low PNI are considered poor prognostic factors. Park suggested that careful nutritional intervention after surgery could improve the survival rate[9]. Discussion Besides, a meta-analysis concluded that a low PNI is significantly associated with poor overall survival except for stage IV GC[31]. However, consistent with the results of Li et al.[32], we observed that PNI was not associated with prognosis. Also, our results did not show a correlation between fibrinogen and the prognosis of GC. Recent studies have shown that fibrinogen is one of the risk factors for poor prognosis in upper gastric cancer[33]. Fibrinogen is the primary acute-phase protein, and as a critical component of the hemostatic system, it regulates the systemic inflammatory state and cancer progression. However, its clinical significance in the prognosis of GC has not been elucidated. The NLR and PLR are the most extensively studied markers of peripheral blood-derived inflammation, which are associated with the prognosis of GC. Accumulating evidence has shown that NLR and PLR are associated with distant metastases during GC progression[34, 35]. Kim reported that although both the PLR and NLR could reflect the prognosis, the NLR was more predictive of overall survival than the PLR in Page 7/20 Page 7/20 GC[36]. Also, they suggested that NLR and PLR might be associated with lymph node metastasis in early gastric cancer[37]. On the contrary, Zhu et al. indicated that NLR and PLR could not predict lymph node metastasis and prognosis in early gastric cancer[38]. In the present study, PLR was significantly correlated with the prognosis of GC, but there was no statistical difference between NLR and prognosis of GC. This observation seems to be inconsistent with previous studies, but the exact mechanism is still unclear. However, the inconsistencies could be because most previous studies focused on overall survival as the primary outcome, whereas herein, we used tumor recurrence and tumor-related mortality as observational indicators, which seem to be more accurate. In addition, the clinicopathological characteristics were similar between the PLR elevating group (PLR < 154) and the PLR decreasing group (PLR ≥ 154), except for tumor size, which further suggested that PLR might influence the prognosis of tumor through other mechanisms. A recent meta-analysis has revealed that PLR is associated with prognosis of GC[39]. Abnormal levels of CRP and Alb have been related to poor prognosis of tumor patients. It is noteworthy that the combinations of these two indicators can enhance the accuracy to predict the recurrence of multiple tumors. Among them, the most common evaluation criteria after combination include GPS and CRP/Alb. Discussion Besides, many studies have used GPS to predict the prognosis of various tumors, including GC. Hsueh recently recommended the use of GPS as a predictive and prognostic factor in patients with GC. A significant correlation was observed between the GPS, short-term postoperative complications, and long- term survival outcomes in patients with GC undergoing D2 gastrectomy[40]. Some studies have indicated that GPS and mGPS, used either alone or in combination, represent an independent prognostic factor for long-term outcome in resected GC[41, 42]. However, Walsh’s results did not show a correlation between prognosis of patients and mGPS levels, although mGPS was associated with advanced GC stage[17]. Liu et al. retrospectively analyzed 455 patients with resectable GC and showed that CRP/Alb, rather than GPS and mGPS, was associated with overall survival[43]. Similarly, Xu[12] and Lu[44] et al. also observed that CRP/Alb and CRP/prealbumin were associated with recurrence of GC based on the data from a phase III randomized clinical trial. On the contrary, our results showed that although CRP/Alb and GPS were associated with prognosis in the univariate analysis, the multivariate analysis showed that both were not related to long-term survival. Consistent with the results of previous clinical trials, our findings showed that chemotherapy could significantly improve the prognosis of AGC, especially for patients with stage III GC[45, 46]. Moreover, many studies have investigated the correlation between peripheral blood-derived inflammation markers and the effects of chemotherapy, to guide the selection of chemotherapy-sensitive patients. A study suggested that the sensitivity of chemotherapy (oxaliplatin/5-fluorouracil combination) might be closely related to NLR, PLR, and their changes in metastatic gastric cancer[47]. Hirahara believed that the combination of NLR and PLR might be more effective in predicting the chemotherapy response in patients with metastatic gastric cancer[48]. Besides, Tang[49] and Chen[50] concluded that PLR could predict the efficacy of neoadjuvant chemotherapy of GC patients treated with oxaliplatin and capecitabine regimens. In the current study, PLR effectively predicted adjuvant chemotherapy (oxaliplatin/5-fluorouracil combination) response in patients with AGC after surgery. For patients with Page 8/20 Page 8/20 PLR ≥ 154, chemotherapy significantly improved long-term survival, including DFS and DSS; however, patients with PLR < 154 did not benefit from adjuvant chemotherapy. Thus, we recommend that AGC patients with PLR ≥ 154 should actively receive adjuvant chemotherapy (oxaliplatin/5-fluorouracil combination) after surgery, whereas patients with PLR < 154 need to be cautious when choosing adjuvant chemotherapy. Conclusion Our findings show that PLR is significantly correlated with the prognosis of GC, especially for stage III. That is, higher PLR (≥154) is an independent risk factor for poor long-term survival rate of GC patients. Moreover, PLR can be used to predict adjuvant chemotherapy (oxaliplatin/5-fluorouracil combination) response in patients with GC after surgery. Discussion PLR ≥ 154, chemotherapy significantly improved long-term survival, including DFS and DSS; however, patients with PLR < 154 did not benefit from adjuvant chemotherapy. Thus, we recommend that AGC patients with PLR ≥ 154 should actively receive adjuvant chemotherapy (oxaliplatin/5-fluorouracil combination) after surgery, whereas patients with PLR < 154 need to be cautious when choosing adjuvant chemotherapy. This study systematically evaluated the relationship between peripheral blood-derived inflammation markers and the prognosis of GC. Notably, the potential ability of inflammatory markers to predict the effects of chemotherapy was further demonstrated. However, this study had a few limitations. Importantly, this was an observational study and was therefore influenced by other confounding factors. Also, the sample size was relatively small, and this may have reduced the reliability of the findings. Thus, these results need to be further validated by large multicenter randomized clinical trials. Conflict of interest Conflict of interest The authors declare no conflict of interest. Abbreviations GC, gastric cancer; AGC, advanced gastric cancer; UICC, International Union Against Cancer; AJCC, American Joint Committee on Cancer; NLR, neutrophil-lymphocyte ratio; PLR, platelet lymphocyte ratio; SII, systemic immune-inflammation index; CRP/Alb, C-reactive protein/albumin ratio; GPS, Glasgow Prognostic Score; mGPS, modified Glasgow Prognostic Score; BMI, body mass index; PNI, prognostic nutritional index; PI, prognostic index; DFS, disease-free survival; DSS, disease-specific survival; ROC, receiver operating characteristics; HR, hazard ratios; CI, confidence intervals Financial support This study was funded by the Key Laboratory of Diagnosis and Treatment of Digestive System Tumors of Zhejiang Province(2019E10020) Author contributions Author contributions Page 9/20 References 1. 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Page 9/20 Conceptualization: Dongjie Wang; Data curation: Lihu Gu, Bangsheng Chen, and Dingcheng Zheng; Formal analysis: Lihu Gu, Jiahang Mo; Investigation: Kang Zhang, Derry Minyao Ng, and Ping Chen; Writing original draft: Lingling Yuan, Feiyan Mao, and Lihu Gu. Conceptualization: Dongjie Wang; Data curation: Lihu Gu, Bangsheng Chen, and Dingcheng Zheng; Formal analysis: Lihu Gu, Jiahang Mo; Investigation: Kang Zhang, Derry Minyao Ng, and Ping Chen; Writing original draft: Lingling Yuan, Feiyan Mao, and Lihu Gu. Conceptualization: Dongjie Wang; Data curation: Lihu Gu, Bangsheng Chen, and Dingcheng Zheng; Formal analysis: Lihu Gu, Jiahang Mo; Investigation: Kang Zhang, Derry Minyao Ng, and Ping Chen; Writing original draft: Lingling Yuan, Feiyan Mao, and Lihu Gu. References Zheng L, Zou K, Yang C, Chen F, Guo T, Xiong B: Inflammation-based indexes and clinicopathologic features are strong predictive values of preoperative circulating tumor cell detection in gastric cancer patients. 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References Melling N, Gruning A, Tachezy M, Nentwich M, Reeh M, Uzunoglu FG, Vashist YK, Izbicki JR, Bogoevski D: Glasgow Prognostic Score may be a prognostic index for overall and perioperative survival in gastric cancer without perioperative treatment. Surgery 2016, 159(6):1548-1556. 42. Jiang X, Hiki N, Nunobe S, Kumagai K, Kubota T, Aikou S, Sano T, Yamaguchi T: Prognostic importance of the inflammation-based Glasgow prognostic score in patients with gastric cancer. British journal of cancer 2012, 107(2):275-279. 42. Jiang X, Hiki N, Nunobe S, Kumagai K, Kubota T, Aikou S, Sano T, Yamaguchi T: Prognostic importance of the inflammation-based Glasgow prognostic score in patients with gastric cancer. British journal of cancer 2012, 107(2):275-279. Page 13/20 Page 13/20 43. Liu X, Sun X, Liu J, Kong P, Chen S, Zhan Y, Xu D: Preoperative C-Reactive Protein/Albumin Ratio Predicts Prognosis of Patients after Curative Resection for Gastric Cancer. 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Table 1-2 Univariate and multivariate analysis of prognostic factors in patients with gastric cancer Tables Association between PLR and the patients’ characteristics Table 3. Association between PLR and the patients’ characteristics   PLR<154 PLR≥154 p value Age (year) 0.223 ≤60 78 55     >60 110 103   Gender 0.818   Male 128 105     Female 60 53   Tumor location 0.941 Upper third 26 47   Middle third 23 17   Lower third 133 116   Two-thirds or more 6 4   Tumor size <0.001 ≤5cm 135 73   >5cm 53 85   Histologic type 0.156 Differentiated 74 75   Undifferentiated 114 83   Perineural invasion 0.194 Absence 79 78     Presence 109 80   Lymphovascular invasion 0.509 Absence 77 59   Presence 111 99   T category 0.835 T1 7 4   T2 15 10   T3 7 6   T4a 153 135   T4b 6 3   N category 0.416 N0 35 28   N1 39 29   N2 58 41   N3a 45 43   N3b 11 17   CEA 0.275 ≤5 ng/mL 166 133   >5 ng/mL 22 25   PLR Platelet-lymphocyte ratio patients characteristics   PLR<154 PLR≥154 p value Age (year) 0.223 ≤60 78 55     >60 110 103   Gender 0.818   Male 128 105     Female 60 53   Tumor location 0.941 Upper third 26 47   Middle third 23 17   Lower third 133 116   Two-thirds or more 6 4   Tumor size <0.001 ≤5cm 135 73   >5cm 53 85   Histologic type 0.156 Differentiated 74 75   Undifferentiated 114 83   Perineural invasion 0.194 Absence 79 78     Presence 109 80   Lymphovascular invasion 0.509 Absence 77 59   Presence 111 99   T category 0.835 T1 7 4   T2 15 10   T3 7 6   T4a 153 135   T4b 6 3   N category 0.416 N0 35 28   N1 39 29   N2 58 41   N3a 45 43   N3b 11 17   CEA 0.275 ≤5 ng/mL 166 133   >5 ng/mL 22 25 PLR, Platelet-lymphocyte ratio PLR, Platelet-lymphocyte ratio Tables Table 1-1 Univariate and multivariate analysis of prognostic factors in patients with gastric cancer Page 14/20 Page 14/20 Page 14/20 Page 14/20 Clinicopathological Univariate analysis Multivariate analysis feature HR 95% CI p value HR 95% CI p value Age (years) ≤60 1     1     >60 1.62 1.16-2.25 0.005 1.63 1.15-2.31 0.007 Gender Male 1           Female 1.01 0.73-1.40 0.967       BMI ≤24 kg/m2 1           >24 kg/m2 0.77 0.52-1.13 0.175       Tumor location Upper third 1           Middle third 0.53 0.29-0.98 0.041       Lower third 0.47 0.31-0.71 <0.001       Two-thirds or more 0.76 0.27-2.15 0.600       Gastrectomy Distal 1           Total 1.57 1.10-2.23 0.013       Proximal NA           Tumor size ≤5cm 1           >5cm 2.60 1.90-3.54 <0.001       Histologic type Differentiated 1           Undifferentiated 1.29 0.95-1.76 0.106       Perineural invasion Absence 1           Presence 2.73 2.00-3.72 <0.001       Lymphovascular invasion Absence 1           Presence 2.78 2.02-3.83 <0.001       T category T1 1     1     T2 4.30 1.56-11.83 0.005 3.04 1.08-8.57 0.036 T3 2.04 0.25-16.92 0.510 1.69 0.20-14.50 0.633 T4a 15.79 6.97-35.76 <0.001 8.22 3.26-20.75 <0.001 T4b 28.21 9.78-81.36 <0.001 13.05 3.98-42.82 <0.001 N category N0 1     1     N1 3.22 1.76-5.90 <0.001 1.70 0.88-3.26 0.112 N2 4.71 2.75-8.05 <0.001 2.09 1.15-3.81 0.016 N3a 11.35 6.82-18.89 <0.001 5.38 3.00-9.65 <0.001 N3b 24.19 13.27-44.07 <0.001 11.01 5.50-22.04 <0.001 lti i t l i f ti f t i ti t ith t i Page 15/20 Clinicopathological  Univariate analysis Multivariate analysis feature HR 95% CI p value HR 95% CI p value Chemotherapy No 1     1     Yes 1.58 1.12-2.22 0.009 0.70 0.48-1.01 0.059 Number of lymph nodes retrieved ≤15 1     1     16-29 0.75 0.50-1.13 0.169 0.46 0.30-0.70 <0.001 ≥30 1.36 0.88-2.11 0.166 0.52 0.32-0.84 0.008 PI 0 1           1 1.54 0.96-2.49 0.075       PNI <49.5 1           ≥49.5 0.40 0.27-0.59 <0.001       Fibrinogen <397 ng/dl 1           ≥397 ng/dl 2.06 1.51-2.81 <0.001       NLR <2.5 1     1     ≥2.5 1.69 1.24-2.31 0.001 0.70 0.48-1.01 0.059 PLR <154 1     1     ≥154 2.21 1.62-3.01 <0.001 1.70 1.20-2.42 0.003 SII <556 1           ≥556 1.94 1.43-2.64 <0.001       GPS 0 1           1 1.76 1.24-2.49 0.002       2 1.89 1.04-3.43 0.037       mGPS 0 1           1 1.53 0.68-3.46 0.307       2 1.66 0.92-3.00 0.091       CRP/Alb <0.05 1           ≥0.05 1.91 1.40-2.60 <0.001       CEA ≤5 ng/mL 1           >5 ng/mL 1.76 1.16-2.68 0.008 HR, hazard ratios; CI, confidence interval; BMI, Body mass index; PNI, Prognostic nutritional index; PI, Prognostic index; GPS, Glasgow Prognostic Score; mGPS, Modified Glasgow Prognostic Score; NLR, Neutrophil-lymphocyte ratio; PLR, Platelet-lymphocyte ratio; CRP/Alb, C-reactive protein/albumin; SII, Systemic immune-inflammatory index Page 16/20 Table 2 Multivariate analysis of prognostic factors in patients with advanced gastric cancer Clinicopathological Multivariate analysis DFS Multivariate analysis DSS feature HR 95% CI p value HR 95% CI p value Age (years) ≤60 1     1     >60 1.61 1.10-2.35 0.014 1.61 1.06-2.42 0.024 PLR <154 1     1     ≥154 2.00 1.40-2.84 <0.001 2.09 1.43-3.04 <0.001 TNM II 1     1     III 4.46 2.51-7.91 <0.001 5.94 3.01-11.74 <0.001 Chemotherapy No 1     1     Yes 0.70 0.48-1.04 0.074 0.66 0.45-0.99 0.043 DFS, disease-free survival; DSS, disease-specific survival; HR, hazard ratios; CI, confidence interval; PLR, platelet-lymphocyte ratio Table 2 Multivariate analysis of prognostic factors in patients with advanced gastric cancer Page 16/20 DFS, disease-free survival; DSS, disease-specific survival; HR, hazard ratios; CI, confidence interval; PLR, platelet-lymphocyte ratio P 16/20 DFS, disease-free survival; DSS, disease-specific survival; HR, hazard ratios; CI, confidence interval; PLR, platelet-lymphocyte ratio Table 3. Figure 3 Comparison of survival curves between patients with or without chemotherapy in advanced gastric cancer. (A) PLR <154; (B) PLR ≥154. SupplementaryTable1.docx Figures Page 17/20 Page 17/20 Page 17/20 Figure 1 Disease-free survival (DFS) of patients with advanced gastric cancer according to the risk factors. (A) stage II; (B) stage III. Figure 1 Disease-free survival (DFS) of patients with advanced gastric cancer according to the risk factors. (A) stage II; (B) stage III. (A) Page 18/20 Figure 2 Disease-specific survival (DSS) of patients with advanced gastric cancer according to the risk factors. ( stage II; (B) stage III. Figure 2 Disease-specific survival (DSS) of patients with advanced gastric cancer according to the risk factors. (A) stage II; (B) stage III. (A) Page 19/20 Figure 3 Comparison of survival curves between patients with or without chemotherapy in advanced gastric cancer. (A) PLR <154; (B) PLR ≥154. Figure 3 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTable1.docx Page 20/20
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Inducible Ablation of Melanopsin-Expressing Retinal Ganglion Cells Reveals Their Central Role in Non-Image Forming Visual Responses
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Abstract * E-mail: satchin@salk.edu ¤ Current address: Kyorin University, Tokyo, Japan ¤ Current address: Kyorin University, Tokyo, Japan completely abolished in mice that lack both melanopsin and functional outer retina photoreceptors, thus establishing a dominant role of melanopsin photopigment in these processes [12,13]. Yet, the AOPs are attenuated, not eliminated, in melanopsin deficient (Opn42/2) mice, an observation that suggests a role for rod/cone photoreceptors in AOPs [14–16]. The mRGCs develop normally in Opn42/2 mice and make normal monosynaptic projections to the suprachiasmatic nucleus (SCN) and olivary pretectal nucleus (OPN) which regulate the circadian behavior and pupillary constriction, respectively [14]. Both the SCN and OPN also receive direct and indirect projections from RGCs that do not express melanopsin [17]. Therefore, it is unclear whether rod/cone-initiated light signal is transmitted primarily via the mRGCs or via other RGCs to brain regions that regulate AOPs. Additionally, the mRGCs have also been suggested to play a role in modulating rod/cone initiated image- forming functions [18,19]. Megumi Hatori1, Hiep Le1, Christopher Vollmers1, Sheena Racheal Keding1, Nobushige Tanaka1¤, Christian Schmedt2, Timothy Jegla3, Satchidananda Panda1* 1 The Salk Institute for Biological Studies, La Jolla, California, United States of America, 2 Genomics Institute of Novartis Research Foundation, San Diego, California, United States of America, 3 Department of Cell Biology and Institute for Childhood and Neglected Diseases, The Scripps Research Institute, La Jolla, California, United States of America PLoS ONE | www.plosone.org Abstract Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre- inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non- image forming photoresponses in mammals. Citation: Hatori M, Le H, Vollmers C, Keding SR, Tanaka N, et al. (2008) Inducible Ablation of Melanopsin-Expressing Retinal Ganglion Cells Reveals Their Central Role in Non-Image Forming Visual Responses. PLoS ONE 3(6): e2451. doi:10.1371/journal.pone.0002451 Editor: Michael Hendricks, Temasek Life Sciences Laboratory, Singapore Received April 25, 2008; Accepted May 14, 2008; Published June 11, 2008 ori et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. pyright:  2008 Hatori et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MH, HL, CV, SK, and SP were supported by Pew Scholars Program in Biomedical Sciences, Dana Foundation, Whitehall Foundation and NIH grant EY016807. CS and TJ were supported by funding from GNF. The funding agencies 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. Inducible Ablation of Melanopsin-Expressing Retinal Ganglion Cells Reveals Their Central Role in Non-Image Forming Visual Responses Megumi Hatori1, Hiep Le1, Christopher Vollmers1, Sheena Racheal Keding1, Nobushige Tanaka1¤, Christian Schmedt2, Timothy Jegla3, Satchidananda Panda1* Introduction The rod/cone photoreceptors of the outer retina signal via multisynaptic pathways to the retinal ganglion cells (RGCs) of the inner retina. The RGCs, in turn, transmit the visual information to the brain via their axonal projections. A small subset of RGCs exclusively expresses the functional photopigment melanopsin (OPN4) and is intrinsically photosensitive, but also receives rod/ cone inputs (Figure 1A) [1-5]. These mRGCs, along with the rod/ cone photoreceptors, mediate several non-image forming, or adaptive ocular photoresponses (AOPs), which help organisms optimize their physiological performance in variable ambient light conditions. These AOPs include rapid adjustment of pupil size, modulation of general activity and endocrine function, and tuning of the phase and period length of the circadian clock to adapt to the light environment (reviewed in [6]). Mouse genetics has established the complementary roles of both rod/cone and melanopsin in these AOPs. Mice with outer retinal degeneration (rd/rd) retain functional mRGCs and exhibit almost intact AOPs [7–11]. However, these adaptive responses are To understand the specific role of mRGCs in both image- forming and adaptive ocular photoresponses, we generated mice expressing Cre-inducible diphtheria toxin receptor exclusively in PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 1 June 2008 | Volume 3 | Issue 6 | e2451 Melanopsin RGC Ablation Figure 1. Strategy for fluorescent labeling or inducible ablation of mRGC lineage in the mouse retina. (A) The cellular circuitry underlying the rod/cone and mRGC contribution to visual responses. Thickness of the filled arrows roughly highlights the relative strength of information flow. (B) Strategy to fluorescently label mRGCs by breeding a mouse carrying a Cre recombinase ‘‘knocked-in’’ to the melanopsin promoter to Z/EG mouse that allows Cre-dependent expression of GFP from chicken beta-actin promoter. (C) Strategy to achieve inducible and specific ablation of mRGCs. Opn4Cre/+ mouse was bred to a mouse expressing Cre-dependent expression of simian diphtheria toxin receptor. The resulting progeny develop normal mRGCs expressing DTR, which allows specific ablation of these cells by DT administration. Schematic of two targeting vectors used to achieve inducible mRGC ablation are shown in (D). The Cre knock-in cassette for targeted insertion to melanopsin locus also carried coding sequences for CRE dependent expression of bTau-eYFP. However, fluorescence from bTau:eYFP was undetectable in retina from Opn4Cre/+ mice (data not shown). The targeting vector and generation of R26iDTR/+ mice are described in [20]. Introduction A schematic of the targeting vector is shown here. doi:10.1371/journal.pone.0002451.g001 Figure 1. Strategy for fluorescent labeling or inducible ablation of mRGC lineage in the mouse retina. (A) The cellular circuitry underlying the rod/cone and mRGC contribution to visual responses. Thickness of the filled arrows roughly highlights the relative strength of information flow. (B) Strategy to fluorescently label mRGCs by breeding a mouse carrying a Cre recombinase ‘‘knocked-in’’ to the melanopsin promoter to Z/EG mouse that allows Cre-dependent expression of GFP from chicken beta-actin promoter. (C) Strategy to achieve inducible and specific ablation of mRGCs. Opn4Cre/+ mouse was bred to a mouse expressing Cre-dependent expression of simian diphtheria toxin receptor. The resulting progeny develop normal mRGCs expressing DTR, which allows specific ablation of these cells by DT administration. Schematic of two targeting vectors used to achieve inducible mRGC ablation are shown in (D). The Cre knock-in cassette for targeted insertion to melanopsin locus also carried coding sequences for CRE dependent expression of bTau-eYFP. However, fluorescence from bTau:eYFP was undetectable in retina from Opn4Cre/+ mice (data not shown). The targeting vector and generation of R26iDTR/+ mice are described in [20]. A schematic of the targeting vector is shown here. doi:10 1371/journal pone 0002451 g001 doi:10.1371/journal.pone.0002451.g001 the mRGC lineage. Diphtheria toxin (DT) crosses the blood-brain barrier after systemic injection and has been shown to trigger cell death in neurons expressing a primate DTR without triggering a significant immune response [20,21]. This strategy allows normal embryonic and postnatal differentiation and development of the target cell type which can be verified in adult mice. Subsequent acute cell ablation with local or systemic DT administration circumvents any potential developmental compensation. Systemic DT injection in the adult mice with DTR expressing mRGCs triggers a profound loss of mRGCs. The mRGC-ablated mice lose non-image forming visual responses while maintaining largely intact image-forming functions. This demonstrates a central role of the mRGCs as the site of integration of melanopsin and rod/cone initiated photoresponses for generating adaptive light responses in mammals. (GFP) from a strong b-actin promoter, such that GFP is uniformly expressed in all mRGCs irrespective of the heterogeneity in the level of transcription from the native melanopsin locus or in the level of immunologically detectable melanopsin protein. In the retina of adult Opn4Cre/+;Z/EG mice, GFP expressing cells were mostly found in the RGC sub-layer, and these cells had extensive dendritic arborization characteristic of the mRGCs. Introduction An average of 131 GFP expressing cells/mm2 (625.4, SD, n = 3) were found in these retina, 42.6% of which also expressed immunologically detectable levels of melanopsin (Figures 2A–2C). These cells with detectable levels of melanopsin protein likely represent the M1 type mRGCs [23]. The second group of GFP positive mRGCs may express very low level of melanopsin representing the M2 type of mRGCs [23], or may represent cells where melanopsin promoter is almost silent in adulthood. A small number of RGCs stained positive for melanopsin, but showed no detectable level of GFP fluorescence. This may represent cells with insufficient Cre expression, Cre activity, or GFP level. In summary, GFP expression pattern in Opn4Cre/+;Z/EG mice established (a) restricted expression of melanopsin in RGC layer and (b) sufficient CRE activity in vast majority of both M1 and M2 type mRGCs. Fluorescent tagging of mRGCs To comprehensively tag mRGCs with transcriptionally active melanopsin locus, we generated a mouse line which carries a Cre recombinase and a Cre-dependent bTau-Yellow Fluorescent Protein (YFP) expression cassette knocked-in to the native melanopsin locus (Figure 1). However, no detectable YFP expression was found in the retina of Opn4Cre/+ mouse, which may be due to weak transcriptional activity from the native melanopsin promoter and/or low expression of the second transcript downstream of an internal ribosomal entry site (IRES) cassette. Next, we bred the Opn4Cre/+ mouse with Z/EG mouse (Figure 1B) [22]. This strategy allows Cre-dependent expression of green fluorescent protein Specific ablation of mRGCs in adult mice (F) Average melanopsin immunoreactive cell density in WT and Opn4Cre/+;R26iDTR/+ retina was comparable. Two weeks after DT administration, the number of cells in WT retina remained unchanged, while that in Opn4Cre/+;R26iDTR/+ retina reduced from 62.3 mm22 to 2.8 mm22. No melanopsin immunostaining was observed in Opn42/2 mice. It is important to note that retina from all DT-treated mice tested by immunostaining still retained a few melanopsin staining cells (arrows), implying incomplete expression of Cre in all mRGCs and/or insufficient level of bioavailable DT. Average cell counts (+SEM, n = 3 to 5 retinas) from each genotype/treatment group are shown. Significant difference in cell numbers (Student’s t test, p,0.05) between mRGCs without and with DT was highlighted with an asterisk. doi:10.1371/journal.pone.0002451.g002 Specific ablation of mRGCs in adult mice To achieve inducible ablation of mRGC lineage, we bred the Opn4Cre/+ mouse with a mouse strain in which the inducible diphtheria toxin receptor (simian Hbegf) (iDTR) is knocked-in to the ROSA26 locus (R26iDTR). The iDTR can only be expressed after Cre-mediated excision of a transcriptional STOP cassette [20] (Figures 1C and 1D). As melanopsin function is haplosuffi- PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 2 Melanopsin RGC Ablation Figure 2. Cre-dependent GFP labeling and inducible ablation of mRGCs. Retina of adult Opn4Cre/+;Z/EG mouse probed with (A) anti-OPN4 or (B) anti-GFP antibodies show staining of a small subset of cells. (C) Only a subset of GFP positive cells also stained with anti-OPN4 antibody. A section of the flat mount retina containing a cell (marked with an arrow) that stained with melanopsin antibody, but did not express detectable level of GFP is shown. (D) Retina of Opn4Cre/+;R26iDTR/+ showed normal melanopsin immunostaining in a small fraction of RGCs. (E) Two weeks after DT administration, the number of melanopsin-immunoreactive cells were significantly reduced. (F) Average melanopsin immunoreactive cell density in WT and Opn4Cre/+;R26iDTR/+ retina was comparable. Two weeks after DT administration, the number of cells in WT retina remained unchanged, while that in Opn4Cre/+;R26iDTR/+ retina reduced from 62.3 mm22 to 2.8 mm22. No melanopsin immunostaining was observed in Opn42/2 mice. It is important to note that retina from all DT-treated mice tested by immunostaining still retained a few melanopsin staining cells (arrows), implying incomplete expression of Cre in all mRGCs and/or insufficient level of bioavailable DT. Average cell counts (+SEM, n = 3 to 5 retinas) from each genotype/treatment group are shown. Significant difference in cell numbers (Student’s t test, p,0.05) between mRGCs without and with DT was highlighted with an asterisk. doi:10.1371/journal.pone.0002451.g002 Figure 2. Cre-dependent GFP labeling and inducible ablation of mRGCs. Retina of adult Opn4Cre/+;Z/EG mouse probed with (A) anti-OPN4 or (B) anti-GFP antibodies show staining of a small subset of cells. (C) Only a subset of GFP positive cells also stained with anti-OPN4 antibody. A section of the flat mount retina containing a cell (marked with an arrow) that stained with melanopsin antibody, but did not express detectable level of GFP is shown. (D) Retina of Opn4Cre/+;R26iDTR/+ showed normal melanopsin immunostaining in a small fraction of RGCs. (E) Two weeks after DT administration, the number of melanopsin-immunoreactive cells were significantly reduced. PLoS ONE | www.plosone.org Normal image-forming visual responses in mRGC ablated mice cient in mice [14], this approach allows normal differentiation and function of the melanopsin cell lineage in the double heterozygote mice (Opn4Cre/+;R26iDTR/+) and their specific ablation upon DT administration. Image-forming vision requires normal retina architecture, light- induced electrical activities in the retina and RGC-mediated transmission of the light information to the image processing brain centers. As shown in Figure 3A, DT-induced mRGC ablation had minimal impact on the overall retina architecture. These mice also exhibited normal electroretinogram (ERG) (Figure 3B). Rod- mediated responses to scotopic illumination, cone-mediated responses to photopic illumination after photobleaching of rods and maximal rod/cone combined responses were comparable in WT and Opn4Cre/+;R26iDTR/+ mice injected with DT. Finally, to evaluate pattern forming visual responses, we tested the perfor- mance of mice in a visual cliff test (Figure 3C). The test evaluates the ability of the mice to visually discriminate between a ‘‘safe’’ and an ‘‘unsafe’’ landing space and accordingly step down from a slightly raised platform to the safe area. Typically, mice with intact The retina of 8 week old adult Opn4Cre/+;R26iDTR/+ mice showed normal stratification and density of melanopsin-immuno- staining (Figures 2D and 3A). Two weeks after DT administration (intraperitoneal injections of DT at 50 mg/kg body weight, 2–3 times at 3 d apart) there was a dramatic reduction of mRGCs by over 90% in Opn4Cre/+;R26iDTR/+(Figure 2E), but not in wild type (WT) mice carrying only one or no copy of either of the two transgenes (Figure 2F). The DT administration did not cause any widespread cell death in the retina as the normal stratification of the major cell layers remained intact (Figure 3A). This demonstrated the accessibility of DT across the blood-retina barrier and the success of the iDTR system in specific ablation of differentiated RGC subtypes in live mice. PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 June 2008 | Volume 3 | Issue 6 | e2451 3 Melanopsin RGC Ablation Figure 3. mRGC ablation does not alter the normal retina architecture and image-forming responses. (A) Hematoxylin and Eosin staining of 5 mm thick paraffin embedded sections of retina from Opn4Cre/+;R26iDTR/+ mice without and with DT injection. DT application had no detectable adverse effect on the normal stratification of the retina (outer segment (OS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL)). Normal image-forming visual responses in mRGC ablated mice (B) Representative full-field ERG of WT and DT-treated Opn4Cre/+;R26iDTR/+ mice showing rod, cone and maximal combined responses. Responses from both eyes were simultaneously measured and plotted. Quantitative analysis of magnitude and timing of a-wave, b-wave and oscillatory potentials of these two genotype (3 i h) h d i ifi diff (d induced damage to the retina and thereby affect image-forming responses. Loss of circadian photoentrainment in mRGC ablated (A) Hematoxylin and Eosin staining of 5 mm thick paraffin embedded sections of retina from Opn4Cre/+;R26iDTR/+ mice without and with DT injection. DT application had no detectable adverse effect on the normal stratification of the retina (outer segment (OS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL)). (B) Representative full-field ERG of WT and DT-treated Opn4Cre/+;R26iDTR/+ mice showing rod, cone and maximal combined responses. Responses from both eyes were simultaneously measured and plotted. Quantitative analysis of magnitude and timing of a-wave, b-wave and oscillatory potentials of these two genotype groups (3 mice each) showed no significant difference (data not shown). (C) Image forming visual function as assessed by the visual cliff test was unaffected by mRGC ablation. Average percentage (+SEM, n = 5 to 13 mice) of positive choice in 10 trials for each mouse are shown. Mice with outer retina degeneration (rd/rd) made random choices while stepping down from the platform and were significantly different (Student’s t test, p,0.05; red asterisk) from the other four groups. No significant difference in test performance was found among native or DT-treated WT or Opn4Cre/+;R26iDTR/+ mice. doi:10.1371/journal.pone.0002451.g003 To clearly establish the roles of retina photopigments and photoreceptor cells in circadian entrainment, we compared the temporal activity rhythms of DT-treated WT and DT-treated Opn4Cre/+;R26iDTR/+ with those of rd/rd, Opn42/2 and Opn42/2;rd/rd mice under three different lighting conditions: LD, DD and LL (constant light). Mice with complete loss of photopigments or acute loss of mRGC lineage in adulthood showed a DD period length that was indistinguishable from that of WT mice, thus implying normal function of the SCN oscillator sustains without tonic input from the mRGCs. As shown before [8,15,16] under LD and LL conditions, the circadian clock in rd/rd and in Opn42/2 mice showed signs of light input. Both genotypes entrained normally to LD cycle; i.e. they exhibited 24 h LD period length. As reported earlier [13,15,16], under constant light, both rd/rd and Opn42/2 mice showed period lengthening–although the effect was less pronounced in the latter genotype. A parsimonious interpretation of circadian photoentrain- ment in melanopsin photopigment deficient (Opn42/2) mice, but not pattern forming vision make correct decisions and step on the safe side in .70% of trials, while mice lacking image-forming vision make random choices and choose the safe side in ,50% of trials. Loss of PLR in mRGC ablated mice Both rod/cone photoreceptors and melanopsin play complemen- tary roles in dynamically adapting the pupil size to ambient light. Specifically, rod/cone photoreceptors mediate response to low light intensity, while melanopsin partly regulates pupil constriction under high intensity light conditions [12–14,24]. The OPN, which mediates PLR, receives a significant number of mRGC projections [4,17]. To test the role of mRGCs in the non-image forming visual responses, we measured PLR at 20 mW of monochromatic blue light (470 nm, 10 nm half-peak width). This intensity of light triggers ,80% pupil constriction in WT mice. Both WT and the Opn4Cre/+;R26iDTR/+ mice showed comparable constriction to this light intensity (data not shown). After two doses of DT injection (50 mg/kg, 3 d apart), WT mice maintain normal pupil constriction. However, during the course of a week following the first DT injection, PLR in the Opn4Cre/+;R26iDTR/+ mice gradually lost sensitivity (Figures 4A and 4B). The rate of loss in PLR was highly variable among this group. Almost 90% sensitivity was lost in the first week, and complete loss of PLR as in Opn42/2;rd/rd mice was observed after the second week following DT injection (Figures 4C and 4D). The loss in PLR, coupled with normal ERG in DT-treated Opn4Cre/+;R26iDTR/+ mice implies a necessary role of mRGCs in dynamic adaptation of pupil size to ambient light. Loss of circadian photoentrainment in mRGC ablated We next tested the effect of mRGC ablation on circadian photoentrainment. The circadian wheel running activity of mice has an intrinsic periodicity of less than 24 h. Photic input to the SCN makes daily phase adjustments to the clock so that the animal’s activity rhythm maintains a constant phase relation with the ambient photoperiod (reviewed in [25]). The daily wheel running activity of Opn4Cre/+;R26iDTR/+ and of WT mice entrained normally to an imposed 12 h light:12 h dark (LD) cycle (Figures 5A and 5B). The mice consolidated their activity with activity commencement juxtaposed to the dark onset. After 10 days of entrainment all mice were administered DT. The WT mice continued to exhibit 24 h rhythm in activity-rest cycle with the activity onset coincident with the dark onset. However, after one week following DT injection, the Opn4Cre/+;R26iDTR/+ mice phenocopied the Opn42/2;rd/rd mice showing no sign of functional light input to the circadian clock. Their activity rhythm under LD conditions exhibited a period length of ,23.5 h which is similar to that under constant dark (DD) conditions (Figure 5C). We next tested the effect of mRGC ablation on circadian photoentrainment. The circadian wheel running activity of mice has an intrinsic periodicity of less than 24 h. Photic input to the SCN makes daily phase adjustments to the clock so that the animal’s activity rhythm maintains a constant phase relation with the ambient photoperiod (reviewed in [25]). The daily wheel running activity of Opn4Cre/+;R26iDTR/+ and of WT mice entrained normally to an imposed 12 h light:12 h dark (LD) cycle (Figures 5A and 5B). The mice consolidated their activity with activity commencement juxtaposed to the dark onset. After 10 days of entrainment all mice were administered DT. The WT mice continued to exhibit 24 h rhythm in activity-rest cycle with the activity onset coincident with the dark onset. However, after one week following DT injection, the Opn4Cre/+;R26iDTR/+ mice phenocopied the Opn42/2;rd/rd mice showing no sign of functional light input to the circadian clock. Their activity rhythm under LD conditions exhibited a period length of ,23.5 h which is similar to that under constant dark (DD) conditions (Figure 5C). Loss of circadian photoentrainment in mRGC ablated To clearly establish the roles of retina photopigments and photoreceptor cells in circadian entrainment, we compared the temporal activity rhythms of DT-treated WT and DT-treated Opn4Cre/+;R26iDTR/+ with those of rd/rd, Opn42/2 and Opn42/2;rd/rd mice under three different lighting conditions: LD, DD and LL (constant light). Mice with complete loss of photopigments or acute loss of mRGC lineage in adulthood showed a DD period length that was indistinguishable from that of WT mice, thus implying normal function of the SCN oscillator sustains without tonic input from the mRGCs. As shown before [8,15,16] under LD and LL conditions, the circadian clock in rd/rd and in Opn42/2 mice showed signs of light input. Both genotypes entrained normally to LD cycle; i.e. they exhibited 24 h LD period length. As reported earlier [13,15,16], under constant light, both rd/rd and Opn42/2 mice showed period lengthening–although the effect was less pronounced in the latter genotype. A parsimonious interpretation of circadian photoentrain- ment in melanopsin photopigment deficient (Opn42/2) mice, but not Figure 3. mRGC ablation does not alter the normal retina architecture and image-forming responses. (A) Hematoxylin and Eosin staining of 5 mm thick paraffin embedded sections of retina from Opn4Cre/+;R26iDTR/+ mice without and with DT injection. DT application had no detectable adverse effect on the normal stratification of the retina (outer segment (OS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL)). (B) Representative full-field ERG of WT and DT-treated Opn4Cre/+;R26iDTR/+ mice showing rod, cone and maximal combined responses. Responses from both eyes were simultaneously measured and plotted. Quantitative analysis of magnitude and timing of a-wave, b-wave and oscillatory potentials of these two genotype groups (3 mice each) showed no significant difference (data not shown). (C) Image forming visual function as assessed by the visual cliff test was unaffected by mRGC ablation. Average percentage (+SEM, n = 5 to 13 mice) of positive choice in 10 trials for each mouse are shown. Mice with outer retina degeneration (rd/rd) made random choices while stepping down from the platform and were significantly different (Student’s t test, p,0.05; red asterisk) from the other four groups. No significant difference in test performance was found among native or DT-treated WT or Opn4Cre/+;R26iDTR/+ mice. doi:10.1371/journal.pone.0002451.g003 Figure 3. mRGC ablation does not alter the normal retina architecture and image-forming responses. Loss of circadian photoentrainment in mRGC ablated Both WT and Opn4Cre/+;R26iDTR/+ mice treated with or without DT performed equally and made the safe choice in .70% of 10 repeated trials, while mice with outer retina degeneration (rd/rd) made random choices. These results indicate that the mRGCs do not play a major role in the overall image-forming and visual responses in mammals, which is consistent with the limited monosynaptic projections of the mRGCs to image processing brain regions. Nonetheless, mRGCs may still play some roles in visual responses. For instance, the loss of PLR may cause light- PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 4 Melanopsin RGC Ablation Figure 4. Necessity of mRGCs for PLR. DT injection severely attenuates pupil constriction in response to 20 mW of monochromatic blue light (470 nm) in Opn4Cre/+;R26iDTR/+ mice (A), but does not affect in such PLR response in WT mice (B). Normalized pupil constriction (-SD; n = 3 mice) measured one day prior to or every day following DT injection for up to 8 days are shown. There was variability in the rate of loss in PLR response among Opn4Cre/+;R26iDTR/+ as reflected in the larger error bars. (C) Pupil constriction in response to varying irradiance levels over 5 log units shows the necessity of mRGC for PLR. Average (+SEM, n = 5 to 6 mice) and fitted sigmoid curves for WT, Opn4Cre/+;R26iDTR/+, Opn42/2;rd/rd and DT-treated Opn4Cre/+;R26iDTR/+ mice are shown. (D) Representative frozen video images showing dark adapted pupil and pupil under low (1011 photons.cm22.s21) or high intensity light (1015 photons.cm22.s21) of 470 nm are shown. Notice the complete lack of pupil constriction in Opn42/2;rd/rd and in DT- treated Opn4Cre/+;R26iDTR/+ mice. For each genotype representative images of the same eye under three different conditions are shown. doi:10.1371/journal.pone.0002451.g004 Figure 4. Necessity of mRGCs for PLR. DT injection severely attenuates pupil constriction in response to 20 mW of monochromatic blue light (470 nm) in Opn4Cre/+;R26iDTR/+ mice (A), but does not affect in such PLR response in WT mice (B). Normalized pupil constriction (-SD; n = 3 mice) measured one day prior to or every day following DT injection for up to 8 days are shown. There was variability in the rate of loss in PLR response among Opn4Cre/+;R26iDTR/+ as reflected in the larger error bars. Loss of circadian photoentrainment in mRGC ablated (C) Pupil constriction in response to varying irradiance levels over 5 log units shows the necessity of mRGC for PLR. Average (+SEM, n = 5 to 6 mice) and fitted sigmoid curves for WT, Opn4Cre/+;R26iDTR/+, Opn42/2;rd/rd and DT-treated Opn4Cre/+;R26iDTR/+ mice are shown. (D) Representative frozen video images showing dark adapted pupil and pupil under low (1011 photons.cm22.s21) or high intensity light (1015 photons.cm22.s21) of 470 nm are shown. Notice the complete lack of pupil constriction in Opn42/2;rd/rd and in DT- treated Opn4Cre/+;R26iDTR/+ mice. For each genotype representative images of the same eye under three different conditions are shown. doi:10.1371/journal.pone.0002451.g004 Opn4Cre/+;R26iDTR/+mice firmly suggests specific ablation of mRGCs in these mice. in mRGC-ablated (DT-treated Opn4Cre/+;R26iDTR/+) mice is the photic input to the circadian clock in Opn42/2 mice must initiate from the outer-retina rod/cone photoreceptors and transmit through the mRGCs (which are intact in these mice). Degeneration or dysfunction of the outer retina photoreceptors along with lack of melanopsin photopigment or acute ablation of mRGCs severs light input to the SCN. mRGCs mediate light suppression of activity The wheel running activity of the WT mouse remained entrained to the LD cycle even after DT injection and was lengthened under constant light. Daily wheel running activity profile of mice were binned in 6 min and double plotted such that the activity from consecutive days are plotted to the right and beneath the data from previous day. Periods of darkness are shown in shaded area. (C) Genotypes of mice and their respective average (6SEM, n = 3 to 6 mice) period length of wheel running activity rhythm under conditions of LD, DD and LL as determined by periodogram analysis in Clocklab software [41] are shown. Within each lighting or treatment group (separated by solid box) significant difference (Student’s t test, p,0.05) from WT group is shown by red asterisk. (D) Anterograde CTB-Alexa Fluor 488 tracing in the optic tract (OT) is intact, but is completely abolished in the SCN of DT treated Opn4Cre/+;R26iDTR/+ mice. (E) Staining in both regions are left intact in WT mice treated with DT. doi:10.1371/journal.pone.0002451.g005 Figure 5. mRGCs are necessary for light adaptation of circadian wheel running activity rhythm. Representative daily wheel running activity profile of an (A) Opn4Cre/+;R26iDTR/+ and (B) WT mouse under 12 h light:12 h dark (LD), constant dark (DD) or constant light (LL) are shown. A week after DT injection (red arrows) the wheel running activity of Opn4Cre/+;R26iDTR/+ began to ‘‘free run’’ with a periodicity similar to that under no light. Constant light had no effect on the daily drift in activity onset in this mouse. The wheel running activity of the WT mouse remained entrained to the LD cycle even after DT injection and was lengthened under constant light. Daily wheel running activity profile of mice were binned in 6 min and double plotted such that the activity from consecutive days are plotted to the right and beneath the data from previous day. Periods of darkness are shown in shaded area. (C) Genotypes of mice and their respective average (6SEM, n = 3 to 6 mice) period length of wheel running activity rhythm under conditions of LD, DD and LL as determined by periodogram analysis in Clocklab software [41] are shown. Within each lighting or treatment group (separated by solid box) significant difference (Student’s t test, p,0.05) from WT group is shown by red asterisk. mRGCs mediate light suppression of activity The general locomotor activity of nocturnal rodents is acutely suppressed upon exposure to photopic light levels [26]. Such activity masking by light is left almost intact in rd/rd mice [27], mildly attenuated in Opn42/2 mice [28], and is abolished in Opn42/2;rd/rd mice [13]. Multiple brain centers are involved in this response, which requires photic input from the retina. To assess the role of mRGCs in negative masking, we measured the wheel running activity of mice subjected to a short photocycle of 4 h light and 4 h dark over several days (Figure 6). Such a short photocycle suppresses the normal circadian nocturnal activity and has been useful in uncovering the complementary roles of rod/ cone and melanopsin photopigments in negative masking [28]. Typically, activity during the light phase of the day is suppressed, so that the total activity is largely distributed over the three 4 h dark periods. The rd/rd, Opn42/2 and WT mice showed strong At the end of the circadian wheel running experiments, we intravitreally injected Alexa Fluor 488 conjugated Cholera Toxin subunit B (CTB) to the eyes of DT treated WT and Opn4Cre/ +;R26iDTR/+mice and monitored staining of the RGC axons and terminals in the brain. Brain sections of both genotypes showed normal staining in the optic tract (OT), which is known to consist of projections from both mRGCs and other RGCs. However, the SCN of Opn4Cre/+;R26iDTR/+mice showed very little staining, while the SCN of WT mice showed significant amount of fluorescence (Figures 5D and 5E). Therefore, the loss of melanopsin immunostaining in the retina along with the loss of a majority of projection to the SCN, but not in the OT of DT treated PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 5 Melanopsin RGC Ablation Figure 5. mRGCs are necessary for light adaptation of circadian wheel running activity rhythm. Representative daily wheel running activity profile of an (A) Opn4Cre/+;R26iDTR/+ and (B) WT mouse under 12 h light:12 h dark (LD), constant dark (DD) or constant light (LL) are shown. A week after DT injection (red arrows) the wheel running activity of Opn4Cre/+;R26iDTR/+ began to ‘‘free run’’ with a periodicity similar to that under no light. Constant light had no effect on the daily drift in activity onset in this mouse. mRGCs mediate light suppression of activity (D) Anterograde CTB-Alexa Fluor 488 tracing in the optic tract (OT) is intact, but is completely abolished in the SCN of DT treated Opn4Cre/+;R26iDTR/+ mice. (E) Staining in both regions are left intact in WT mice treated with DT. doi:10.1371/journal.pone.0002451.g005 negative masking, such that .80% of the daily activity was partitioned into the three dark phases, while both Opn42/2;rd/rd and DT-treated Opn4Cre/+;R26iDTR/+ mice continued to exhibit strong circadian activity consolidation that was unperturbed by periods of illumination (Figure 6C). They showed equal activity during light and dark phases. of the mammalian circadian system, multiple cell types in the SCN constitute a network of oscillators which is entrained to the ambient light via ocular photoreceptors (reviewed in [30]). The mRGCs have emerged as the predominant RGC sub-type making monosynaptic connection to SCN oscillators. In this study we generated a mouse line that expresses functional CRE recombinase from the native melanopsin locus. We used this mouse line to (a) comprehensively tag mRGC lineage with GFP and to (b) ablate the mRGCs after their normal differentiation. This approach revealed that the melanopsin promoter is active in a large number of RGCs, of which ,40% produce immunologically detectable level of melanopsin protein. Comparable heterogeneity Discussions Our approach of transgene tagging from a non-native promoter now allows comprehensive identification of all mRGCs and their classification into two distinct groups based on the level of OPN4 expression. Such differences in melanopsin protein levels along with possible heterogeneity in the relative expression levels of genes defining morphological and functional properties may further contribute to the functional diversity in mRGC population. in mRGC morphology (Provencio, personal communication), dendritic arborization [31], light induced calcium responses [11,32], electrical activity [33] and immunostaining [23] has already been reported. Our approach of transgene tagging from a non-native promoter now allows comprehensive identification of all mRGCs and their classification into two distinct groups based on the level of OPN4 expression. Such differences in melanopsin protein levels along with possible heterogeneity in the relative expression levels of genes defining morphological and functional properties may further contribute to the functional diversity in mRGC population. We have used the acute cell ablation to understand the cellular bases for light input to the circadian clock. Our results show a central non-redundant role for mRGCs in the relay of photic input to two brain regions that generate adaptive photoresponses. Rod/ cone and melanopsin initiated photo signals converge at the level of the mRGCs before relaying to oscillator neurons in the SCN. Similarly, mRGCs appear to serve as the sole functional relay for photic input to the OPN. These results highlight an important cellular design feature of the mammalian circadian system. The mammalian clock, like that of plants, invertebrates and lower vertebrates, uses multiple, mutually compensating photopigments to adapt to the ambient light [36]. However, in other organisms at least some circadian photoreceptors and the clock are cell autonomous and there appear to be multiple molecular nodes for integration of light signal to the molecular clock [25]. The mammalian circadian time keeping system recruits non-cell autonomous multiple photoreceptors, including rod, cones and melanopsin. The complete loss of light entrainment in mRGC The strategy to acutely ablate target cell types in mice with DT- DTR specificity has been successful for many peripheral and intracranial neuronal populations [20,21,34,35], but its efficacy for acute ablation of retina cell types has never been tested. From immunostaining, parallel loss of circadian photoentrainment and other adaptive ocular photoresponses, it is clear that DT can successfully cross the blood-retina barrier in sufficient quantity to trigger ablation of DTR expressing cells. Discussions Comprehensive identification and perturbation of genes and circuits are necessary to gain a systems level understanding of circadian regulation of behavior [29]. In the current cellular model PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 June 2008 | Volume 3 | Issue 6 | e2451 6 Melanopsin RGC Ablation Figure 6. Lack of negative masking in mRGC ablated mice. Representative wheel running activity profile of DT-treated (A) Opn4Cre/+;R26iDTR/+ and (B) WT mice held under ultradian cycle of 4 h light and 4 h darkness are shown. The sessions of darkness are shown by shaded area. Activity profile is plotted as in Figure 5A. (C) Average percentage (6SEM, n = 3 to 6 mice) of daily activity during the bouts of light sessions (on left) or during darkness (on right) for five different groups of mice are shown. Groups with percent activity during light phase significantly different (Student’s t test, p,0.05) from that of the DT-treated WT mice are shown with asterisk. doi:10.1371/journal.pone.0002451.g006 Figure 6. Lack of negative masking in mRGC ablated mice. Representative wheel running activity profile of DT-treated (A) Opn4Cre/+;R26iDTR/+ and (B) WT mice held under ultradian cycle of 4 h light and 4 h darkness are shown. The sessions of darkness are shown by shaded area. Activity profile is plotted as in Figure 5A. (C) Average percentage (6SEM, n = 3 to 6 mice) of daily activity during the bouts of light sessions (on left) or during darkness (on right) for five different groups of mice are shown. Groups with percent activity during light phase significantly different (Student’s t test, p,0.05) from that of the DT-treated WT mice are shown with asterisk. doi:10.1371/journal.pone.0002451.g006 ganglion cells. Although the tests employed here did not find any profound loss of image forming function of the retina in acute tests, we cannot rule out the possibility that mRGCs play a subtle direct or indirect role in sculpting the image-forming function of the retina. For instance, in the retina mRGCs may influence function of other RGCs via intercellular coupling [11], they may participate in relaying luminance information to the image processing [18], or by playing an indispensible role in the pupil constriction response, they may reduce direct photodamage to the retina. in mRGC morphology (Provencio, personal communication), dendritic arborization [31], light induced calcium responses [11,32], electrical activity [33] and immunostaining [23] has already been reported. PLoS ONE | www.plosone.org Discussions However, we found up to 10% of melanopsin expressing cells escaped cell death due to potential stochastic noise in CRE or DTR expression. It is interesting to note that these surviving cells are unable to generate AOPs. This implies that the SCN and OPN require a strong photic input to reset intrinsic activity. Alternatively, the surviving cells could represent a distinct mRGC population that does not play a role in relaying light information to the SCN or OPN. Our finding that mRGCs do not overtly disrupt image-forming processes implies a divergence of signals at the level of the retinal June 2008 | Volume 3 | Issue 6 | e2451 June 2008 | Volume 3 | Issue 6 | e2451 7 Melanopsin RGC Ablation Melanopsin RGC Ablation integration of Cre leading to loss of melanopsin protein in Opn4Cre/ Cre mice was verified by lack of anti-OPN4 immunostaining (data not shown). Opn4Cre/+;Z/EG mice were used for assessment of CRE function and fluorescent tagging of mRGCs in the retina. Littermate Opn4Cre/+;R26iDTR/+ and WT mice were used in all subsequent experiments for acute ablation of mRGCs. / ablated mice firmly establishes a unique mammalian-specific cellular network design to integrate light information from these photopigments to the circadian clock. The mRGCs in this design serve as the principal site of signal integration and therefore, have now emerged as a unique cellular target for therapeutic intervention in circadian clock related disorders. The complete loss of non-image forming photoresponses also highlights the role of mRGCs as the principal cellular node where photic information for image-forming and non-image forming responses diverge. The Opn42/2 mice used in this study were generated in 129S background and characterized earlier [15]. These mice were bred to a line of C57BL/6 carrying the Pde6brd/rd mutation. Progeny from this breeding, which were also in a 129S;C57BL/6 background were genotyped and mice of Opn42/2, rd/rd and Opn42/2;rd/rd genotypes were used for behavioral studies. During the course of this study, another manuscript [37] describing specific ablation of mRGCs was published. That study employed direct expression of an attenuated diphtheria toxin from the melanopsin locus, which caused slow degeneration of the mRGCs over several months, as assessed by the loss of a Tau:LacZ transgene expression or melanopsin immunostaining. Both these techniques detect only a subset of mRGCs ([23] and Figure 2). Therefore, the slow degeneration of non-image forming photo- sensitivity in the study by Guler et al. DT injection DT (D0564, Sigma-Aldrich, St. Louis, MO) was dissolved in sterile PBS (1 mg/ml) and stored at 280uC till use. Freshly thawed DT stock solution was diluted in sterile PBS and injected intraperitoneally (50 mg/kg body weight) [21] to 8–12 weeks old Opn4Cre/+;R26iDTR/+ and WT littermate mice. The dose was repeated once or twice at 3 d interval. Generation of mice All animal care and procedures were approved by the Institutional Animal Care and Use Committee of the Genomics Institute of the Novartis Research Foundation and The Salk Institute for Biological Studies. R26iDTR/+ mouse strain was a kind gift of Dr. Ari Waisman [20] and Z/EG mouse strain [22] was purchased from Jackson Laboratory. A schematic diagram of the targeting construct for generating the R26iDTR/+ mouse is shown in Figure 1D. The Opn4Cre/+ mouse was generated by replacing the first seven exons of the mouse melanopsin gene with a gene cassette containing Cre (Figure 1D). For generation of the targeting construct, genomic DNA of 129S1SvImJ inbred mouse strain was used as a template and 3.5 kb of genomic sequence immediately upstream of the translation start site and 3.4 kb of sequence 39 distal to the 7th exon of the of Opn4 gene were PCR amplified and cloned into two multicloning sites flanking the Cre:IRES:bTau:loxP:Neo:lox- P:eYFP cassette. The left arm of the targeting vector also carried a HSV-TK resistance gene. The construct was linearized by NotI digestion and microinjected into an embryonic stem (ES) cell line from 129S mice. ES cell clones with integration of the targeting construct were selected on G418, and 288 antibiotic resistant clones were screened by PCR for homologous recombination of the targeting construct to the melanopsin locus. Genomic DNA from seven PCR positive clones was digested with NheI and subjected to Southern blot hybridization with a probe that lies within the right arm. Sequences of the genotyping PCR primers are; wild type (Primer a = CACTTCAGAGACAGCCAGAAGCAGG, Primer b = GACTGACACTGAAGCCTGGCAAACG) and mutant (Primer a and Primer c = CCATTTCCGGTTATTCAACTTG- CACC). One clone with appropriately recombined DNA was injected into C57BL/6J blastocysts and introduced into C57BL/6J pseudopregnant females. Chimeric male progeny were bred to C57BL/6J females and the resulting heterozygous agouti coat- colored progeny were mated with C57BL/6J. Heterozygous mice were bred among each other and with Z/EG or Rosa26iDTR/+ mice which were also in 129S;C57BL/6 mixed background. Targeted Retina staining For flat mount, adult mice were sacrificed, the eyes quickly removed and placed into aerated Ames medium (Sigma-Aldrich, St. Louis, MO). After removal of the corneas and lenses, eyecups were fixed in 4% paraformaldehyde for 15 min. Following three washes in PBS, retinas were dissected from eyecups, stretched onto filter paper, and processed in 24-well plates. The retinas were incubated in a blocking solution (0.3% Triton X-100, 5% normal donkey serum, and 0.5% glycine in PBS) for 1 h at room temperature. After three washes in PBS, the retinas were incubated in a 1:5,000 dilution of rabbit anti-OPN4 antiserum (against a peptide consisting of the 15 N-terminal amino acids of mouse melanopsin [38]) or in 1:500 dilution of rabbit anti-GFP antibody (Cat# A11122, Invitrogen, Carlsbad, CA) in the blocking solution for overnight at 4uC and rinsed with PBS. Melanopsin immunoreactivity was visualized with Cy3-conjugated donkey anti-rabbit IgG (1:500, Cat# 711-165-152, Jackson ImmunoResearch Laboratories, West Grove, PA) or FITC- conjugated anti-rabbit IgG (1:20, Cat# 401314, Calbiochem, San Diego, CA) in blocking solution. Finally, retinas were washed with PBS and mounted with PermaFluor (Cat# IM0752, Beckman Coulter, Fullerton, CA). Fluorescent images were captured using an Olympus Fluoview500 confocal microscope or Leica TCS SP2 AOBS confocal microscope. For Hematoxylin and Eosin (H&E) staining, eyes were removed and eyecups were fixed in 4% paraformaldehyde for 2 h, washed three times with PBS, and embedded in paraffin. Five micron thick paraffin sections were used for H&E staining. Stained slides were visualized under a Leica microscope. Discussions [37] may have resulted from likely heterogeneity in toxin production from the native promoter, homeostatic synaptic strengthening and/or early developmental compensation. Despite the differences in the pace of cell death, and the approach, both manuscripts arrived at similar conclusion that the mRGCs are the primary node of convergence of melanopsin and rod/cone initiated photoresponses. Anterograde tracing with cholera toxin g g Mice were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg) and one drop each of 1% tropicamide and 0.5% proparacaine (Bausch & Lomb, Tampa, FL) were applied to their eyes. An incision was made with a 30 gauge needle below the limbus region and 1 ml of 1% Cholera Toxin-B subunit (CTB) conjugated to Alexa Fluor 488 (Cat# C34775, Invitrogen, CA) was injected into the vitreous. After 48 h, the mice were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg), perfused with 4% paraformaldehyde in 16PBS. The brain was extracted, further fixed at 4uC overnight, embedded in OCT and 40 mm thick sections cut in a microtome were imaged using an Olympus Fluoview500 confocal microscope. PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 PLoS ONE | www.plosone.org 8 Melanopsin RGC Ablation camera to improve the image quality. After collecting images of dark adapted pupils, the contralateral eye was exposed to light from the integrating sphere. Light from a 300 Watt Xenon Arc lamp light source (Sutter Instrument, Novato, CA, USA) was filtered, collimated and delivered to the integrating sphere through a liquid light guide. An inline 470 nm filter, a filter wheel with a series of neutral density filters, and a Lambda 10-3 optical filter changer with SmartShutterTM were used to control the spectral quality, intensity and duration of light. Light intensity was measured with a Melles Griot power meter. Video images of pupil constriction were analyzed by ViewPoint EyeTrackerTM software (Arrington Research Inc., Scottsdale, AZ). Dark adapted pupil area prior to light pulse and pupil area after 30 sec of light were measured and pupil constriction was defined as 1-(dark adapted pupil width)ˆ2/(pupil width after 30 sec of light)ˆ2. The squares come in to account for the calculation of pupil area. Pupil area constriction was plotted against the log of irradiance. Data was fitted to a sigmoidal dose response curve with the lower asymptote fixed to y = 0 using Origin lab 8 software. ERG Mice were dark adapted for 1 h, anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg). The eyes were applied with 1% tropicamide, 0.5% proparacaine and Genteal lubricant eye gel (CIBA vision, Duluth, GA, USA). Electrodes were connected under dim red light. ERGs were recorded from both eyes with an ERG system (Model LE-2000, Tomey, Nagoya, Japan). y 13. Panda S, Provencio I, Tu DC, Pires SS, Rollag MD, et al. (2003) Melanopsin is required for non-image-forming photic responses in blind mice. Science 301: 525–527. 15. Panda S, Sato TK, Castrucci AM, Rollag MD, DeGrip WJ, et al. (2002) Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting. Science 298: 2213–2216. 14. Lucas RJ, Hattar S, Takao M, Berson DM, Foster RG, et al. (2003) Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice. Science 299: 245–247. Locomotor activity measurement Daily locomotor activity of mice individually housed in wheel running cages was measured as described in [41]. Typically, 6– 10 week old mice were individually housed in wheel running cages placed inside light tight boxes with independent illumination. During the light phase, the mice received ,150 lux of white light from fluorescent light source. Wheel running activity in 1 min bins was continuously collected and later analyzed by Clocklab software (Actimetrics, Evanston, IL, USA). All routine animal husbandry practices during the dark phase were performed under dim red light. Anterograde tracing with cholera toxin White LED light embedded in contact lens recording electrodes were used as light sources. ERG recordings were done following the standards of the International Society for Clinical Electrophysiol- ogy of Vision (ISCEV; http://www.iscev.org/standards/erg1999. html). After an additional 5 min of dark adaptation, rod response was measured by averaging 4 dim light flashes of 80 cd.m2260.12 msec at .2 sec interval. Then mice were further dark adapted for 3 min and maximal combined (rod and cone) response and oscillatory potential were determined from the average of responses to four flashes of 6,000 cd.m2260.5 msec at .10 sec inter-stimulus interval. Finally, after 10min light adaptation (25 cd.m22), average single-flash cone response was determined from 4 flashes of 6,000 cd.m2260.5 msec at .15 sec interval. For tracking the gradual changes in PLR response following DT injection, pupil area constrictions over 8 days were normalized to maximum constriction taken as 100%. PLR PLR was assessed as described in [40] with some modification. Mice were dark adapted for at least an hour prior to PLR assessment. Unanaesthetized mice were hand restrained with the left eye apposed to one of the outlets of an integrating sphere. The pupil of right eye was video monitored under infrared light with a consumer grade digital video camera. An additional +2 diaptor lens and an infrared filter were fitted to the light path of the Author Contributions Conceived and designed the experiments: SP. Performed the experiments: CS SP TJ MH HL NT SK. Analyzed the data: SP MH VC. Contributed reagents/materials/analysis tools: CS TJ. Wrote the paper: SP MH. Conceived and designed the experiments: SP. Performed the experiments: CS SP TJ MH HL NT SK. Analyzed the data: SP MH VC. Contributed reagents/materials/analysis tools: CS TJ. Wrote the paper: SP MH. Acknowledgments We would like to thank Dr. Ari Waisman for providing R26iDTR/+ mouse strain, Van M. Lee, Victoria Piamonte, Kathryn Spencer and Dusko Trajkovic for technical assistance, and members of the Panda lab for critical discussion of the results. Visual cliff test The visual cliff test was assessed as described in [39]. Half of a 31 cm length645 cm width650 cm tall clear acrylic box had a black and white checkerboard pattern on the top (the ‘‘safe’’ side) and the other side had a clear top (the ‘‘unsafe’’ side/cliff side). The unsafe side had a checkerboard pattern on the bottom to give an illusion of added depth. Black paper was placed on the inside of the walls to reduce reflections. A block of 31 cm length63.5 cm width62.5 cm height was placed at the center of the box to separate these two sides. The mouse was placed on the block and allowed to make a spontaneous step down between the safe side and the unsafe side. Data was measured as positive when the mouse chose to step down onto the horizontal checkered surface (the safe side) and as negative onto the clear side (the unsafe side). Each mouse was tested for 10 consecutive trials. Fifty percent positive indicated that the mouse moved off the ridge onto the two sides with random chance. 12. Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, et al. (2003) Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424: 76–81. 8. Foster RG, Provencio I, Hudson D, Fiske S, De Grip W, et al. (1991) Circadian photoreception in the retinally degenerate mouse (rd/rd). J Comp Physiol [A] 169: 39–50. 1. Provencio I, Rodriguez IR, Jiang G, Hayes WP, Moreira EF, et al. (2000) A novel human opsin in the inner retina. J Neurosci 20: 600–605. p J 2. Gooley JJ, Lu J, Chou TC, Scammell TE, Saper CB (2001) Melanopsin in cells of origin of the retinohypothalamic tract. Nat Neurosci 4: 1165. p 6. 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Monitoring of Dual CRISPR/Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell
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Original Research published: 25 October 2017 doi: 10.3389/fendo.2017.00289 Monitoring of Dual crisPr/cas9- Mediated steroidogenic acute regulatory Protein gene Deletion and cholesterol accumulation Using high-resolution Fluorescence In Situ hybridization in a single cell Jinwoo Lee1,2* and Colin Jefcoate1,2,3,4* 1 Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI, United States, 2 Endocrinology and Reproductive Physiology Program, University of Wisconsin, Madison, WI, United States, 3 Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI, United States, 4 Molecular and Cellular Pharmacology, University of Wisconsin, Madison, WI, United States Keywords: steroidogenic acute regulatory protein, CRISPR, fluorescence in situ hybridization, cholesterol, lipid droplets Edited by: Edited by: Michael Schumacher, Institut national de la santé et de la recherche médicale, France Institut national de la santé et de la recherche médicale, France Reviewed by: Vassilios Papadopoulos, University of Southern California, United States Emilie Lesport, Institut Professeur Baulieu, France *Correspondence: Jinwoo Lee jwlee7@wisc.edu; Colin Jefcoate jefcoate@wisc.edu Reviewed by: Vassilios Papadopoulos, University of Southern California, United States Emilie Lesport, Institut Professeur Baulieu, France Reviewed by: Vassilios Papadopoulos, University of Southern California, United States Emilie Lesport, Institut Professeur Baulieu, France Specialty section: This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology Received: 25 June 2017 Accepted: 11 October 2017 Published: 25 October 2017 Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing technology, provide opportunities for understanding gene regulation at the single-cell level. The application of direct imaging shown here provides an in  situ side-by-side comparison of CRISPR/Cas9-edited cells and adjacent unedited cells. We apply this methodology to the steroidogenic acute regulatory protein (StAR) gene in Y-1 adrenal cells and MA-10 testis cells. StAR is a gatekeeper protein that controls the access of cholesterol from the cytoplasm to the inner mitochondria. The loss of this mitochon- drial cholesterol transfer mediator rapidly increases lipid droplets in cells, as seen in StAR−/− mice. Here, we describe a dual CRISPR/Cas9 strategy marked by GFP/mCherry expression that deletes StAR activity within 12 h. We used single-molecule fluorescence in situ hybridization (sm-FISH) imaging to directly monitor the time course of gene editing in single cells. We achieved StAR gene deletion at high efficiency dual gRNA targeting to the proximal promoter and exon 2. Seventy percent of transfected cells showed a slow DNA deletion as measured by PCR, and loss of Br-cAMP stimulated transcription. This DNA deletion was seen by sm-FISH in both loci of individual cells relative to non-target Cyp11a1 and StAR exon 7. sm-FISH also distinguishes two effects on stimulated StAR expression without this deletion. Br-cAMP stimulation of primary and spliced StAR RNA at the gene loci were removed within 4 h in this dual CRISPR/Cas9 strategy before any effect on cytoplasmic mRNA and protein occurred. StAR mRNA disappeared between 12 and 24 h in parallel with this deletion, while cholesterol ester droplets increased four- fold. These alternative changes match distinct StAR expression processes. This dual gRNA and sm-FISH approach to CRISPR/Cas9 editing facilitates rapid testing of editing strategies and immediate assessment of single-cell adaptation responses without the perturbation of clonal expansion procedures. INTRODUCTION course of deletion by PCR amplification of the targeted StAR gene segment and by probing the edited StAR DNA segment with sm- FISH after RNase removal of all RNA. We compared this targeted StAR deletion to a non-targeted region of the StAR locus (exon 7) or to another similarly expressed gene (Cyp11a1 loci). We used this Cas9 procedure to examine the immediate consequences of StAR deletion. The StAR transfer of cholesterol from LDs to the cleavage enzyme, Cyp11a1, located on the inner face of the inner membrane, is integrated with the cleavage of cholesterol esters (CEs) by hormone-sensitive lipase (HSL), under control of protein kinase A (PKA) (18). In StAR−/− mice and human deficiency, the loss of steroidogenesis in the adrenal glands, testes, and ovaries is matched by large accumulations of CE (3, 19). The capacity to resolve individual RNA species in single cells by single-molecule Fluorescence in Situ Hybridization (sm-FISH) (1, 2) now provides the means to examine the CRISPR/Cas9 gene editing in single cells. Here, we describe a dual CRISPR/ Cas9 cleavage of steroidogenic acute regulatory protein (StAR), the prime regulator of cholesterol metabolism, in Y-1 adrenal cells and MA-10 testis cells. We used direct sm-FISH to compare StAR expression in dual-transfected CRISPR (+) cells to non- transfected (NT) adjacent cells. The goal was to separate the tim- ing, respectively, of the transfection, editing, and gene expression processes. We also measured the subsequent adaptation resulting from the loss of StAR function. We demonstrated dramatic increases of lipid droplets (LDs) that mimic the human adrenal deficiency condition (3).h In this report, we show that editing was extensive within 12 h and that cholesterol trafficking was then rapidly redirected from intra-mitochondrial oxidation to the formation of cytoplasmic CE accumulations. The cholesterol captured as esters in these accumulations was much greater than the small amounts diverted from StAR-dependent steroid synthesis. StAR has been linked to several cell types to cholesterol export (20), to transcription controlled by hydroxy-metabolites through liver X receptors (LXR) (21, 22), and to other mitochondrial processes (23).h i This single-cell detection depends on sm-FISH, which uses multiple fluorescent 20-base oligomers (4) to detect primary transcripts (p-RNA) and spliced transcripts (sp-RNA) at gene loci and, then, to detect mRNA as single molecules in the cyto- plasm (1, 2). INTRODUCTION cAMP analogs extensively induce these StAR RNA species in the Y-1 adrenal and MA-10 testis cells that we used here (5, 6). The Y-1 cells are distinguished by basal StAR mRNA expression, which was sufficient for maximum stimulation by cAMP within 10 min of steroid synthesis (7). sm-FISH imaging of StAR expression showed that the loci responded asymmetrically to cAMP stimulation within asynchronous cell populations. Stimulation of StAR transcripts at the gene loci not only increased the levels of different types of RNA but also decreased inter-cell differences. These direct sm-FISH analyses of editing can be completed within 24 h of transfection. The speed this procedure provides the opportunity for a relatively quick optimization of the CRISPR/ Cas9 editing strategy, notably for gRNA selection and validation. We have designed gRNA sequences with an online CRISPR design tool and used the fast algorithm Cas-OFFinder, to searches for potential off-target sites of Cas9 (24). Various PCR-based meth- odologies are available for single-guide RNA (sgRNA) validation, including the following: mismatch cleavage assays (25), indel detection by amplicon analysis (IDAA) (26), and digital PCR (27). However, a complete workflow of gRNA validation in single-cell colony expansion is time intensive. This direct combined sm-FISH and CRISPR/Cas9 strategy provided an intermediate step in the isolation of appropriately targeted clonal lines. f Understanding the effects of CRISPR/Cas9 on StAR expres- sion requires an appreciation of the editing process. The CRISPR/ Cas9 technology was developed from bacterial adaptive immune systems (8–10). Cas9 is an RNA-guided DNA endonuclease that fuses with a guide RNA (gRNA). The gRNA includes a four-base endonuclease cleavage sequence and a Cas9 recognition site [protospacer adjacent motif (PAM)] at the 3′end (11–13). The association of Cas9 and gRNA directs specific localization to complementary DNA sequences selected for gene editing (14, 15). Here, we used a dual Cas9 vector strategy in which mCherry and GFP expression marked the respective deliveries of the 5′- and 3′- gRNA sequences. The guided Cas9 creates a double-stranded break (DSB) 3 bp upstream of the PAM sites, within the gRNA hybrid- ized sequence (13, 16, 17). The dual cleavages this design provided lead to an excision and re-ligation to produce an edited StAR gene lacking the early proximal promoter, exon 1, and intron 1. This deletion removed the possibility of functional mRNA expression. We directly assessed the deletion by measuring the deletion time Citation: Lee J and Jefcoate C (2017) Monitoring of Dual CRISPR/ Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell. Front. Endocrinol. 8:289. doi: 10.3389/fendo.2017.00289 October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 1 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Abbreviations: 3′EU, oligomer set targeting extended 3′UTR; Cas9, CRISPR- associated protein 9; CE, cholesterol ester; CRISPR, the clustered regularly inter- spaced short palindromic repeat; DSB, double-strand breaks; FISH, fluorescence in situ hybridization; gRNA, guide RNA; HSL, hormone-sensitive lipase; LD, lipid droplets; N-SIM, Nikon’s Structured Illumination Microscope; PAM, protospacer adjacent motif; PKA, protein kinase A; p-RNA, primary RNA; Single Molecule Fluorescence in Situ Hybridization (sm-FISH); sp-RNA, spliced primary RNA; StAR, steroidogenic acute regulatory protein. Cell Linesh The MA-10 mouse Leydig tumor cell line (a gift of Dr. Mario Ascoli) was derived from the Leydig tumor, M5480P (28). The Y-1 cell line (a gift of Dr. Bernard Schimmer) was derived from a mouse adrenocortical tumor (29). General procedures for cell culture were as described previously (1). A day before the experiment, approximately 3–6 × 105 cells were seeded onto the coverslip (Corning) coated with ploy-Lysine (Sigma) in wells of a six-well plate (Corning). Real-time RT-PCR and Genomic DNA-PCR for Analysis of StAR Primer design and cDNA synthesis were performed as previ- ously described (1, 30). qPCR was performed using BioRad CFX96™ Real-Time PCR Detection System. The qPCR protocolfor StAR expression time courses was done as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of Plasmid Preparation p Digested plasmids [plasmid (1  µg), FastDigest Bbsl (1  µl), FastAP (1 µl), 10× FastDigest Buffer (2 µl), ddH2O (total 20 µl), 30 min, 37°C] were purified using QIAquick Gel extraction kit. The oligo pairs encoding the guide sequences were annealed and ligated into the plasmids. We prepared the mixture [sgRNA left (100 µM, 1 μ), sgRNA right (100 µM, 1 µl), 10× T4 ligation buffer l (1 µl), T4 PNK (1 µl), ddH2O (total 10 µl)] for phosphorylating, and annealing the sgRNA oligos in a thermocycler (37°C for 30 min; 95°C for 5 min; ramp down to 25°C at 5°C min−1). We set up a ligation reaction (Bbsl), and the mixture were incubated for 10 min at RT. We also prepared a no-insert pSpCas9 (BB)- only negative control for ligation. DH5 alpha cells were used for transformation. The product (2 µl) was added to ice-cold DH5 alpha cells (50  µl), incubated the mixture on ice for 30  min, heat-shocked the cell mixture at 42°C for 45 s, and returned it immediately to ice for 2 min. Then, we added 500 µl of SOC medium and incubated for 1 h before plating the cells onto an LB plate containing 100 µg ml−1 ampicillin. After an overnight incubation at 37°C, we picked 10 colonies to check for the cor- rect insertion of the sgRNA. We ran the colony PCR reaction with the guide sequence (forward) and a primer on the plasmid backbone (reverse: CAG_enhancer region). The sm-FISH method has been shown to be effective at visualizing well-expressed mRNA transcripts in most cells. Glass coverslips with plated cells were washed with sterile PBS and placed in six-well plates. Cells were plated on glass cover- slips, fixed with 0.4% paraformaldehyde, stored at 4°C in 70% ethanol overnight, and hybridization was performed the next day. For single mRNA detection, sm-FISH probe sets consist of multiple singly labeled oligonucleotides (generally 30–48 bases long) designed to hybridize along targeted RNA transcripts. Hybridization was performed in a dark humidified condi- tion. Samples were re-suspended in 2× SSC and added GLOX buffer without enzymes for equilibration, incubated, and then re-suspended in GLOX buffer with enzymes (glucose oxidase and catalase). Again, antifade reagent was used prior to Z-stack imaging. Transfection DNA was transfected with 7.5 µl of Lipofectamine 3000 reagent, 5 µl P3000, and 2.5 µg of plasmid DNA cocktail following the manufacturer’s instructions. CRISPR (+) cells under conditions used here are present in 25–30% of the cells 24 h post-transfection. We analyzed all NT cells in randomly selected fields. The smaller proportion of CRISPR (+) cells necessitates analysis of all GFP (+) cells in wider fields. Design of sgRNA Sequence The term sm-FISH has been used to distinguish the use of a probe set consisting of 40 fluorescent 20mers. The RNA probe sets for StAR were generated by using the Stellaris probe design- er.4 Samples were prepared according to a previously described method (1). Freshly prepared StAR p-RNA (Quasar-570, Biosearch Technology), StAR sp-RNA (Quasar-670, Biosearch Technology), StAR 3′ EU (Quasar-570, Biosearch Technology), and Cyp11a1 p-RNA (Quasar-670, Biosearch Technology) probe sets and antibody for StAR were used. A clean coverslip was placed over the sample to prevent drying of the RNA hybridization solution [10% dextran sulfate (Sigma), 10% deionized formamide (Ambion), 2× SSC] during the incuba- tion. Samples were incubated in a dark humidified chamber at 37°C overnight. After a 30-min wash in wash buffer, samples were incubated in DAPI nuclear stain (wash buffer with 5 ng/ ml DAPI) to counterstain the nuclei for 30 min. In the case of the combined FISH, an extra washing step is needed with the secondary antibody. Samples were processed according to as previously described (1).hf g g q pSpCas9 (BB)-2A-GFP (Addgene plasmid # 48138) and pU6- (BbsI)_CBh-Cas9-T2A-mCherry (Addgene plasmid # 64324) were used for the sgRNA expression plasmids (31). pSpCas9 (BB)-2A-GFP express a chimeric gRNA plus EGFP and human codon-optimized Cas9. EGFP was replaced into Cherry in pU6- (BbsI)_CBh-Cas9-T2A-mCherry. The components of type II CRISPR/Cas9 system crRNA and tracrRNA were fused to gener- ate a sgRNA. For the S. pyogenes system, the target sequence leads a 5′-NGG PAM and the guide sequence pairs with the opposite strand at ~3 bp upstream of the PAM. The targeted sequences are as follows: StAR exon 2 GGTGGATGGGTCAAGTTCGACGT CGG and StAR promoter AGTCATCAGTCATTGTGCAG AGG. To clone the guide sequence into the sgRNA scaffold, we designed guide sequences with online CRISPR design tools.1,2 To search for potential off-target sites of Cas9, we used Cas-OFFinder.3 Real-time RT-PCR and Genomic DNA-PCR for Analysis of StAR Primer design and cDNA synthesis were performed as previ- ously described (1, 30). qPCR was performed using BioRad CFX96™ Real-Time PCR Detection System. The qPCR protocolfor StAR expression time courses was done as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 2 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate 10 s at 95°C and 30 s at 60°C. Primer sequences are as follows: Star E1/I1, 5′-AGACATATGCGGAATATGAAAGGTG-3′ and 5′-CCCAAGAGCTTTCCCACAAA-3′; E5/E6, 5′-GAGTGGTG TCATCAGAGCTGAAC-3′ and 5′-TGAGTTTAGTCTTGGAG GGACTTCC-3′. Genomic DNA extraction was done with TRIzol following the manufacturer’s protocol. The genomic DNA PCR protocol was done as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of 15 s at 95°C, 30 s at 58°C, and 150 s at 72°C. Primer sequences are as follows: 5′-CCTCTGCACAATG ACTGATGACT-3′ and 5′-GGATGGGTCAAGTTCGACGTC GG-3′. PCR products were separated on a 0.5% agarose gel. 4 http://www.biosearchtech.com/products/rna-fish 1 http://tools.genome-engineering.org. 2 http://chopchop.cbu.uib.no. 3 http://www.rgenome.net/cas-offinder/. Image Acquisition and Analysis To detect and visualize p-RNA, sp-RNA, and mRNA, we used the Olympus wide-field fluorescence microscope (Model IX81) and the Nikon’s Structured Illumination Microscope (N-SIM) for higher resolution images according to a previously described method (1, 33). We used two microscope settings representing high and low sensitivity to compare p-RNA and sp-RNA at loci under, respectively, basal conditions and after 1 and 3 h of stim- ulation by Br-cAMP (Figure S1B in Supplementary Material). The high sensitivity setting is also used to image mRNA, which appears as single molecules. The N-SIM uses different opti- cal processing methods but provided results that were fully consistent with the Olympus IX81. Five fluorescence imaging filter set (DAPI, GFP, Orange, mCherry, and Red) was used for IX81 in this study. GFP (488ext/507emi nm) and mCherry (587/610 nm) channels were used for the detection of Cas9- GFP and Cas9-mCherry. Orange (538/559 nm, pseudocolored green) and red (618/637 nm, pseudocolored red) channels were used for the detection of StAR p-RNA and sp-RNA/mRNA. Yellow color indicates colocalization of p-RNA and sp-RNA. We used the “StAR Search” software developed by the Arjun Raj lab at University of Pennsylvania.5 Statistical significance was determined by Student’s t-test or ANOVA; p < 0.05 was considered statistically significant where *p < 0.05, **p < 0.01, and ***p  <  0.001. Data were analyzed by using the PRISM software (San Diego, CA, USA). We have previously reported a set of sm-FISH oligomers (1, 2) that image specific segments of the StAR gene (intron 1 and the 3′end of exon 7) and equivalent sequences in RNA transcription products. We have resolved p-RNA and sp-RNA of StAR gene loci and single mRNA molecules in the cytoplasm (Figure 1C). Although mRNA expression in individual cells was variable, image analyses of sets of adjacent cells provided a mean expres- sion as copy numbers per cell that matched equivalent qPCR analyses based on several million cells in a culture well (1, 2). Here, we have applied these sm-FISH probes to in situ single-cell analysis of CRISPR/Cas9 editing.h y g The StAR sm-FISH images obtained from Y-1 adrenal and MA-10 testis cells differ by over 50-fold ranging. We have pre- viously reported single hybridizations for StAR gene DNA, for each resolved mRNA molecule and minimum p-RNA expres- sion in basal Y-1 loci (2, 33). Plasmid Preparation For DNA sm-FISH, cells were treated with RNase A at 37°C for 1 h, washed and equilibrated with PBS for 5 min before dehy- dration by consecutive 5 min incubations in 70, 85, and 100% ethanol. After air-drying, cells were heated at 80°C for 5 min in hybridization mixture (50% formamide, 10% dextran, 0.5  µM EDTA, and 4× SSC), and then washed using an ethanol series (ice old 70, 80, and 95%). After air-drying, cells were placed in RNA hybridyzation with probes for overnight. October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 3 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate targeted by CRISPR/Cas9 and the associated impact on StAR expression. We tested both basal, and Br-cAMP-stimulation of StAR expressed either as RNA at gene loci or as mRNA and protein in the cytoplasm. The final adaptation phase was indicated by fluorescence imaging of significant increases in CEs that form the major constituents in cytoplasmic LDs and also accumulate in late endosomes (34). These accumulations were readily detected in single cells by ORO, which delivered a low- intensity fluorescence (32). ORO (+) droplets were increased extensively in CRISPR (+) cells. We measured the time course of this response in individual cells in relation to the StAR RNA editing responses. Oil Red O (ORO) (Lipid Stain) Staining Oil Red O stock solution was prepared by dissolving 0.3 g of ORO (MP Biomedicals, OH, USA) in 100  ml of isopropanol. ORO working solution was prepared by mixing three parts (30 ml) of ORO stock solution with two parts (20 ml) of water and filtered through 0.2 µm filters. Cells were washed with water and incu- bated with 60% isopropanol for 5 min. After 5 min, the wells were completely dry. ORO working solution was added to the cells, and the cells were incubated for 10 min in the solution. Stained cells were washed three times with water. Again, the antifade reagent was used prior to Z-stack imaging. ORO is a fat-soluble dye and can be used for fluorescence microscopy wherein a lipid environ- ment has enough fluorescence (32). g p CRISPR/Cas9 editing produces sequence deletions that remove expression or activity when two gRNA sequences target double-strand DNA cleavage sites that span a functionally essential part of the gene locus. Plasmid Preparation The StAR gene sequence between these two gRNAs is lost, and ligation between the 5′- and 3′-ends restores the integrity of the gene. We have selected a 5′ target that overlaps C/EBPβ-binding site (−90 ~ −80 bp) (35) in the StAR promoter and a site on the reverse strand at the end of the exon 2 region. A deletion of key promoter elements then ensues, including the transcription StAR site, exon 1, intron 1, and most of intron 2 (Figure 1B). To test the efficiency of gRNA and to visualize the impact of gene deletion, we have combined this dual CRISPR/Cas9 strategy with sm-FISH analyses of the various steps in gene expression. 5 http://www.rajlab.seas.upenn.edu/StarSearch/launch.html. Image Acquisition and Analysis Peak unresolved fluorescence for, respectively, p-RNA and sp-RNA is seen in these loci after 1 h stimulation by Br-cAMP. StAR protein expression determined by immunohistochemistry has a similar range of expression, which corresponds to accumulation in mitochondria. The comparison of image intensities required the adjustment of microscope settings (Figures S1A–C in Supplementary Material). We also showed that the 3D distribution of loci and mRNA in these cells was appreciably different. Fortunately, the active StAR loci were found close to the nuclear midline thus facilitating detection (Z axis). Most of the mRNAs were found closer to the adherent sur- face in the widest part of the cell (Figure S1D in Supplementary Material). Optimum sensitivity for single DNA/RNA molecules was obtained with single probe sets where pixels can be directly counted with the aid of an imaging program (star search) (Figure S1E in Supplementary Material). Experimental Design for CRISPR/Cas9 Deletion from the StAR Genehi CRISPR (+) cells expressed both gRNAs and Cas9 conjugated with mCherry (PX330) or GFP (PX458), which indicates gRNA expression. (C) Characterization of editing: high- resolution fluorescence in situ hybridization probes were used for identification of altered StAR p-RNA and sp-RNA at loci during CRISPR/Cas9 editing. Targeted StAR DNA at gene locus was also visualized by the p-RNA probe for intron 1 after the RNase treatment. Single molecules of mRNA were detected by sp-RNA probes. StAR antibody was also added for immunofluorescence (IF) images. Before any stimulation, Y-1 cells showed a consistent basal expression of p-RNA and sp-RNA/mRNA, which were unre- solved when measured by q-PCR. Sm-FISH resolved this expres- sion to highly variable levels of p-RNA and sp-RNA at the loci in the individual cells (Figure 2B). About one-third of the cells showed mRNA in the cytoplasm, and about one-third show none. There is little relationship between expression of sp-RNA at the loci and the mRNA in the cytoplasm. However, the expression at the loci turns over much more quickly than the cytoplasmic mRNA. In MA-10 cells, this basal level p-RNA and sp-RNA was undetectable (2, 36). Experimental Design for CRISPR/Cas9 Deletion from the StAR Genehi This study has two general goals; first, to separate the timing of CRISPR/Cas9 editing of StAR through the temporal resolution in individual cells of transfection, editing, and gene expression; second, to measure the direct adaptation of the cells due to loss of StAR function (Figure 1A). The progress in editing was measured in whole cultures by PCR analysis of the targeted seg- ment of StAR DNA and then in single cells by direct sm-FISH imaging. We examined both the StAR DNA sequence that was October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 4 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 1 | Design for CRISPR/Cas9 editing of steroidogenic acute regulatory protein (StAR). (A) The sequence of steps used for dual CRISPR/Cas9 deletion of the StAR DNA from Y-1 and MA-10 cells. Transfection and DNA editing by Cas9 were assessed by impacts on StAR RNA at loci, on StAR mRNA and protein in the cytoplasm, plus evaluation of effects on StAR activity based on Oil Red O (ORO) staining of cholesterol in lipid droplets (LDs). Side-by-side comparison of NT and CRISPR (+) cells was performed. (B) Dual CRISPR/Cas9 design for StAR. Two gRNAs were used to generate a large deletion of the gene. CRISPR (+) cells expressed both gRNAs and Cas9 conjugated with mCherry (PX330) or GFP (PX458), which indicates gRNA expression. (C) Characterization of editing: high- resolution fluorescence in situ hybridization probes were used for identification of altered StAR p-RNA and sp-RNA at loci during CRISPR/Cas9 editing. Targeted StAR DNA at gene locus was also visualized by the p-RNA probe for intron 1 after the RNase treatment. Single molecules of mRNA were detected by sp-RNA probes. StAR antibody was also added for immunofluorescence (IF) images. Figure 1 | Design for CRISPR/Cas9 editing of steroidogenic acute regulatory protein (StAR). (A) The sequence of steps used for dual CRISPR/Cas9 deletion of the StAR DNA from Y-1 and MA-10 cells. Transfection and DNA editing by Cas9 were assessed by impacts on StAR RNA at loci, on StAR mRNA and protein in the cytoplasm, plus evaluation of effects on StAR activity based on Oil Red O (ORO) staining of cholesterol in lipid droplets (LDs). Side-by-side comparison of NT and CRISPR (+) cells was performed. (B) Dual CRISPR/Cas9 design for StAR. Two gRNAs were used to generate a large deletion of the gene. Frontiers in Endocrinology  |  www.frontiersin.org Characterization of StAR Expression in Untransfected Y-1 Cells To assess the impact of CRISPR/Cas9 editing in Y-1 cells, we first used q-PCR to characterize the normal expression of StAR p-RNA and sp-RNA after Br-cAMP stimulation. p-RNA increased 10-fold over 1  h to a steady state, while sp-RNA showed a delay of about 20 min [Figure 2A (a,b)]. We previously characterized the delay in sp-RNA as a pause in elongation at the end of the terminal exon that is coupled with splicing (1, 33). This increase is biphasic, corresponding first to the generation of sp-RNA from the splicing of p-RNA at the gene locus, and then to a slower process that corresponds to export of the processed mRNA from the loci to the cytoplasm. The primary p-RNA StAR transcripts were only found in the nucleus at the gene loci (1, 2). p-RNA and sp-RNA were expressed at the same loci in rep- resentative Y-1 cells at low sensitivity. Such robust expression was only visible in a few select basal cells. Two loci exhibited this robust expression in most Y-1 cells after of stimulation. There was October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 5 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate no further increase at 3 h [Figure 2C (a)]. The dot blots indicated the range of expression at Y-1 loci under basal conditions, and with stimulations for 1 and 3 h [Figure 2C (b)]. MA-10 cells where cytoplasmic mRNA is scarcely detectable at 1 h even as StAR protein exhibits peak translation. A major factor in this discrepancy is that StAR protein accumulates in mitochondria with slow turnover. By contrast, low levels of StAR mRNA, which can sustain peak StAR mitochondrial activity (7), may turnover more rapidly than the excess that induced by high concentrations of Br-cAMP. We compared sm-FISH images of cytoplasmic mRNA under basal conditions and after 1 and 3 h of stimulation by Br-cAMP [Figure 2D (a)]. There was no net increase in cytoplasmic mRNA in the initial 1 h, despite substantial increases of sp-RNA in the loci. The cytoplasmic mRNA rose extensively at 3  h. We have previously shown (1, 33) that cytoplasmic mRNA increased to this steady-state level after 2  h. The sm-FISH image showed a significant increase in StAR protein at 1 and 3 h, which evidently preceded the increase in mRNA. This anomaly is even clearer in CRISPR Cas9 Editing of Basal and Stimulated StAR We examined NT and CRISPR (+) cells under basal conditions or after 1 h stimulation by Br-cAMP. This stimulation provides optimal responses of p-RNA and sp-RNA at the loci. mCherry and GFP fluorescence mark the expression of 5′ gRNA and 3′ gRNA, respectively. Their dual fluorescence is therefore indicative of CRISPR (+) cells. We first examined p-RNA and sp-RNA in Y-1 cells under basal conditions. We examined NT and CRISPR (+) cells under basal conditions or after 1 h stimulation by Br-cAMP. This stimulation provides optimal responses of p-RNA and sp-RNA at the loci. mCherry and GFP fluorescence mark the expression of 5′ gRNA and 3′ gRNA, respectively. Their dual fluorescence is therefore indicative of CRISPR (+) cells. We first examined p-RNA and sp-RNA in Y-1 cells under basal conditions. to be processed to provide a statistically valid assessment of the CRISPR impact on basal Y-1 expression. To improve the detection of StAR transcripts, we took advan- tage of the rapid stimulation of StAR p-RNA and sp-RNA at loci in Y-1 cells. After 1 h of Br-cAMP stimulation, p-RNA and sp-RNA were visible even at low sensitivity settings. In five rep- resentative NT cells, all loci expressed both p-RNA and sp-RNA with 3/5 exhibiting two active loci and 2/5 with a single active locus. No CRISPR (+) cell showed an active locus [Figure 3B (a)]. However, a lower proportion of CRISPR (+) cells retained active loci as shown [Figure 3B (b)]. Examination of all CRISPR (+) cells from several microscope fields showed that 33/149 (22%) retained some p-RNA expression, compared to 315/394 (80%) in NT cells [Figure 3B (c)]. Similar proportions were shown for sp-RNA [CRISPR (+) 14/55 (25%); NT 175/222 (78%)]. Thus, the ratio of primary to spliced StAR transcripts retained in the active CRISPR (+) loci appears similar to the NT loci, consistent with no effect of dual CRISPR/Cas9 on StAR splicing. High-sensitivity microscope settings were needed to detect the expression at these minimally active loci (Figure S2A in Supplementary Material). The transfected cells, which were identified by the expression of both mCherry and GFP, did not show expression at the level reached by the subset of higher-expressing basal cells (3/7 Figure 3A; inserts a–c). Only one CRISPR (+) cell, out of 10 examined cells, retained active StAR loci [Figure 3A (b)]. CRISPR Cas9 Editing of Basal and Stimulated StAR We applied this sm-FISH analysis to basal and induced Y-1 cells 24  h after application of the dual CRISPR/Cas9 transfection. Figure 2 | Continued Figure 2 | Continued October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 6 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 2 | Characterization of steroidogenic acute regulatory protein (StAR) expression in Y-1 cells. (A) qPCR analysis of StAR transcription in Y-1 adrenal cells after stimulation by Br-cAMP. (a) Location of PCR primers to detect the primary transcripts [pRNA(E1/I1)] and spliced transcripts [spRNA(E5/E6)]. (b) Time course for the stimulation of StAR RNA as determined by q-PCR. (B) High sensitivity sm-FISH detection of basal expression of sp-RNA at loci and mRNA in cytoplasm. (C) (a) Low sensitivity paired sm-FISH images of the time-dependent appearance of p-RNA and sp-RNA at loci after stimulation by Br-cAMP (1 mM). (b) Relative integral density (IntDen) was measured based on the integrated density subtracted with background readings. (D) High sensitivity sm-FISH of StAR mRNAs (a) and IF of protein (b) after 1 and 3 h Br-cAMP (1 mM) stimulation. The “Star search” program was used for counting fluorescence of mRNA and protein immunohistochemistry in individual cells. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. Figure 2 | Characterization of steroidogenic acute regulatory protein (StAR) expression in Y-1 cells. (A) qPCR analysis of StAR transcription in Y-1 adrenal cells after stimulation by Br-cAMP. (a) Location of PCR primers to detect the primary transcripts [pRNA(E1/I1)] and spliced transcripts [spRNA(E5/E6)]. (b) Time course for the stimulation of StAR RNA as determined by q-PCR. (B) High sensitivity sm-FISH detection of basal expression of sp-RNA at loci and mRNA in cytoplasm. (C) (a) Low sensitivity paired sm-FISH images of the time-dependent appearance of p-RNA and sp-RNA at loci after stimulation by Br-cAMP (1 mM). (b) Relative integral density (IntDen) was measured based on the integrated density subtracted with background readings. (D) High sensitivity sm-FISH of StAR mRNAs (a) and IF of protein (b) after 1 and 3 h Br-cAMP (1 mM) stimulation. The “Star search” program was used for counting fluorescence of mRNA and protein immunohistochemistry in individual cells. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. Frontiers in Endocrinology  |  www.frontiersin.org CRISPR Cas9 Editing of Basal and Stimulated StAR Overall, in the basal NT cells, 27/40 expressed p-RNA, and 23/47 expressed sp-RNA with most expressing both in varying ratios [Figure 3A (c)]. To confirm the specificity of these low-level CRISPR responses as well as the sensitivity of p-RNA and sp-RNA detection in the presence of a background, we examined expression in Y-1 cells with CRISPR/Cas9 transfection carried out in the absence of gRNAs (Figure S2B in Supplementary Material). Without gRNAs, the basal expression of p-RNA and sp-RNA was not hindered in the mCherry-positive cells. Nevertheless, the variability and low detection mean that still larger numbers of CRISPR (+) need f To establish dual sgRNA-directed gene deletion, we designed PCR primer sets that amplifies the StAR genomic DNA which we expect to be deleted by the dual CRISPR editing [Figure 3C (a)]. The full StAR gene product (WT, 1.8 kb) and deleted gene product [CRISPR (+), 0.65 kb] were separated on a 0.5% agarose gel. In the experiment, the deletion shown by a 0.65 kb band was about 25% at 12 h and at least 50% at 24 h [Figure 3C (b)]. October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 7 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 3 | CRISPR Cas9 editing of basal and stimulated steroidogenic acute regulatory protein (StAR). (A) (a) CRISPR/Cas9 StAR deletion under the basal condition at the locus. The boxes indicate the active StAR loci (p-RNA, sp-RNA), and the dashed line (circle) shows the CRISPR/Cas9-positive cell. GFP and mCherry were used as a marker for gRNA expression. (b) a CRISPR (+) cell that retained an active StAR loci. (c) Percent of NT basal cells expressing p-RNA and sp-RNA at loci (the segment is shown in blue). Only 1/10 CRISPR (+) cells had detectable RNA at the locus. (B) (a) CRISPR/Cas9 StAR deletion was measured at the loci after 1 h stimulation with Br-cAMP (1 mM). (b) An example of a CRISPR (+) cell that retained active StAR loci. (c) Percent of non-transfected cells (NT, left) and CRISPR (+) cells (right) that expressed active StAR loci (p-RNA and sp-RNA) (segment shown in blue). (C) (a) PCR strategy to validate StAR gene deletion. (b) PCR amplification in wild type (WT) and transfected (24 h) cells with various CRISPR/Cas9 plasmids. (D) (a) Detection of p-RNA (StAR, cyp11a1) after 1 h stimulation with Br-cAMP in Y-1 cells. Application of gRNA to Manipulation of StAR Editing A similar probing with the StAR exon 7 DNA probe (3′EU) showed that StAR DNA outside the targeted segment was retained in the CRISPR (+) cells to the same extent as in the adjacent NT cells (Figure 5D; Figure S5B in Supplementary Material). Again the 3′EU sequence was detected at one locus in each of the CRISPR (+) cells. Thus, both StAR 3′EU DNA and Cyp11a1 shared similar detection efficiencies. The locus DNA hybridization that requires denaturation at a much higher temperature than the locus-associated RNA is likely to be easier for regions of the chromatin that are actively transcribed. A distal StAR promoter sequence with similar base composition requires still a harsher denaturation (Figure S5C in Supplementary Material). The 3D spatial distribution of StAR loci, mRNA, and protein which is central to the sm-FISH assessment of gene expression was best seen in MA-10 cells. Thus, StAR protein and mRNA after 3 h of stimulation showed very characteristic 3D organiza- tions (38) (Figure 4B). The StAR protein in these mitochondria is located in the matrix and is inactive in promoting cholesterol transfer to Cyp11a1. StAR is only active immediately after the translation of mRNA at the mitochondrial surface (7). We next assessed whether this dual CRISPR/Cas9 system is needed for this removal of StAR sp-RNA/mRNA. MA-10 cells were transfected with the dual gRNAs, with each gRNA singly or without gRNAs and then assessed for mRNA expression. Without the gRNA, the three Cas9/GFP (+) cells retained the same StAR mRNA distribution as surrounding NT  cells [Figure  4C (a)], with no statistical difference between NT and GFP (+) cells in the extent of expression [Figure 4C (b)]. Consistent with the Y-1 experiments, StAR mRNA was completely removed in the dual gRNA CRISPR (+) MA-10 cells [Figure 4D (a,b)]. Single 5′-gRNA/Cas9 or 3′-gRNA/Cas9 transfections of MA-10 cells for 24 h were marked by, respectively, mCherry or GFP. 5′gRNA CRISPR transfections produced similar effects after 24 h to the dual protocol. PCR analyses showed that neither single CRISPR/Cas9 produced any of the DNA deletion seen for the dual editing [Figure 3C (c)]. Out of eight mCherry (+) cells, six cells showed complete loss of transcription while two retained active StAR loci [Figure S4A (a,b) in Supplementary Material]. One of these cells with active loci also showed loss of mRNA, while one retained the mRNA. Application of gRNA to Manipulation of StAR Editing To verify whether excision of the targeted sequence had occurred in individual loci of StAR, DNA sm-FISH was performed with the p-RNA probe, which hybridizes to intron 1 which is targeted by the dual gRNACRISPR/Cas9. We compared this hybridiza- tion to non-target DNA either within the StAR locus (distal promoter-3′EU/exon 7) or in Cyp11a1, another expressed gene (intron 3/Cyp11a1) (Figure 5A). The key steps in distinguishing DNA sm-FISH from RNA sm-FISH are the RNase removal of all RNA and the stronger denaturation conditions needed to open the chromatin structure for hybridization (Figure 5B). The gene hybridization targets a single DNA sequence at each locus whereas the RNA expression also detected by p-RNA may vary from 1 to >50 transcripts. Single mRNA particles are harder to image than the accumulated RNA at loci, particularly in Y-1 cells, which slowly round up when stimulated by Br-cAMP. This cell rounding which is larger in Y-1 cells than in MA-10 cells arises from activation of PTPases that affect adhesion complexes (37). Overall, the stimulation of StAR p-RNA and sp-RNA in MA-10 cells by Br-cAMP was similar to that seen in Y-1 cells (Figure 2A) except that MA-10 cells were devoid of basal expression measured either by qPCR (Figure 4A) or sm-FISH (Figure S3 in Supplementary Material). Again, q-PCR showed that Br-cAMP stimulation was characterized by a steady state for p-RNA from 1 to 3 h. The formation of sp-RNA, which included increases first at the locus and then as mRNA in the cytoplasm, showed a 25-min delay.h We compared StAR and Cyp11a1 DNA in two CRISPR (+) cells with four adjacent NT-cells. CRISPR (+) cells (#1 and #2) each showed a pair of Cyp11a1 loci but no StAR intron 1 (Figure 5C). Overall, these CRISPR (+) cells showed that about 20% retained at least 1 StAR locus that retained intron 1. Of the four adjacent NT cells, Cyp11a1 loci are seen in three with an average of one locus detected per cell. The NT cells each show at least the intron 1 DNA segment from StAR loci (Figure S5A in Supplementary Material). A similar heterogeneity in sm-FISH hybridization was seen in the Cyp11a1 transcription (Figure 3D). CRISPR Cas9 Editing of Basal and Stimulated StAR (b) Quantitation of active loci per cell for StAR and Cyp11a1 in NT cells and CRISPR (+) cells after 1 h Br-cAMP stimulation. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. Figure 3 | CRISPR Cas9 editing of basal and stimulated steroidogenic acute regulatory protein (StAR). (A) (a) CRISPR/Cas9 StAR deletion under the basal condition at the locus. The boxes indicate the active StAR loci (p-RNA, sp-RNA), and the dashed line (circle) shows the CRISPR/Cas9-positive cell. GFP and mCherry were used as a marker for gRNA expression. (b) a CRISPR (+) cell that retained an active StAR loci. (c) Percent of NT basal cells expressing p-RNA and sp-RNA at loci (the segment is shown in blue). Only 1/10 CRISPR (+) cells had detectable RNA at the locus. (B) (a) CRISPR/Cas9 StAR deletion was measured at the loci after 1 h stimulation with Br-cAMP (1 mM). (b) An example of a CRISPR (+) cell that retained active StAR loci. (c) Percent of non-transfected cells (NT, left) and CRISPR (+) cells (right) that expressed active StAR loci (p-RNA and sp-RNA) (segment shown in blue). (C) (a) PCR strategy to validate StAR gene deletion. (b) PCR amplification in wild type (WT) and transfected (24 h) cells with various CRISPR/Cas9 plasmids. (D) (a) Detection of p-RNA (StAR, cyp11a1) after 1 h stimulation with Br-cAMP in Y-1 cells. (b) Quantitation of active loci per cell for StAR and Cyp11a1 in NT cells and CRISPR (+) cells after 1 h Br-cAMP stimulation. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. To test the specificity of genome editing introduced by CRISPR/ Cas9, we examined whether CRISPR (+) and NT cells maintained normal expression of Cyp11a1 p-RNA. StAR p-RNA was not sup- pressed in CRISPR (+) cells [Figure 3D (a)]. The four NT cells expressed Cyp11a1 in 3/4 cells, with two showing both loci and one only a single locus. Two CRISPR (+) cells showed one cell with two active Cyp11a1 loci and one without Cyp11a1 expres- sion. Neither CRISPR (+) cell showed StAR expression. Overall, October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 8 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate in exon 2. Application of gRNA to Manipulation of StAR Editing By contrast, the pattern of responses for 3′gRNA indicated much lower locus disruption [Figure S4B (a,b) in Supplementary Material]. Ten GFP (+) single transfections showed six that retained locus transcription, five of which expressed mRNA. Thus, half of the 3′gRNA-Cas9 transfections remain similar to the surrounding NT cells. Clearly, DSB introduced around the 5′ PAM site, located in the proximal promoter, more readily introduce mutations that remove StAR expression than DSB introduced around the 3′PAM site located An analysis of the StAR gene in 277 cells showed recognition of at least 1 StAR intron 1 sequence in 157/226 UT cells (70%) but in only 11/51 CRISPR (+) cells (20%) (Figure 5E). The CRISPR (+) cells that retain StAR intron 1 closely parallel the 25% that retain p-RNA and sp-RNA transcription without mRNA and protein. CRISPR Cas9 Editing of Basal and Stimulated StAR The only difference between the 5′gRNA CRISPR (+) cells and the dual edit is that 1/8 of these cells retain both locus transcription and the full processing to mRNA. in 39 NT cells, StAR was expressed in an average of 1.4 loci per cell, with Cyp11a1 in 1.3 loci per cell. In CRISPR (+) cells, the Cyp11a1 expression decreased to 0.85 loci per cell, whereas StAR loci were undetected in these 13 CRISPR (+) cells [Figure 3D (b)]. Frontiers in Endocrinology  |  www.frontiersin.org Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells We have addressed the time dependence of several key steps: the entry of editing CRISPR vectors into the cells, their expression as marked by GFP and mCherry, the deletion of the targeted StAR DNA, and the several changes in StAR expression. We examined each at 4, 12, and 24 h after transfection. The editing responses were compared after a 2 h Br-cAMP stimulation [Figure 6A (a)]. Sm-FISH provides an assessment of the impact of increasing October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 9 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 4 | Role of gRNA in steroidogenic acute regulatory protein (StAR) editing; Br-cAMP stimulation of MA-10 Cells and suppression of mRNA. (A) qPCR analysis of StAR transcription in MA-10 testis cells. (B) XY and XZ projection of StAR mRNA and protein in MA-10 cells after 3 h of stimulation by Br-cAMP (1 mM). (C) CRISPR/Cas9 system without gRNA. (a) The high sensitivity of FISH image to StAR mRNA with sp-RNA probes. The dotted circles indicate the dual-transfected cells (left). GFP and mCherry’s dual fluorescence (right). (b) Quantitation of mRNA copy numbers in NT and GFP/mCherry (+) cells after 3 h of stimulation by Br-cAMP (1 mM). Expression differences were not significant in the t-test (ns). (D). Dual CRISPR/Cas9 system with gRNAs. (a) High sensitivity to sp-RNA detection of mRNA (left) with GFP and mCherry detection of the transfected cells (right). Expression difference was significant by Student’s t-test ***p < 0.001. Scale bar represents 10 µm. Figure 4 | Role of gRNA in steroidogenic acute regulatory protein (StAR) editing; Br-cAMP stimulation of MA-10 Cells and suppression of mRNA. (A) qPCR analysis of StAR transcription in MA-10 testis cells. (B) XY and XZ projection of StAR mRNA and protein in MA-10 cells after 3 h of stimulation by Br-cAMP (1 mM). (C) CRISPR/Cas9 system without gRNA. (a) The high sensitivity of FISH image to StAR mRNA with sp-RNA probes. The dotted circles indicate the dual-transfected cells (left). GFP and mCherry’s dual fluorescence (right). (b) Quantitation of mRNA copy numbers in NT and GFP/mCherry (+) cells after 3 h of stimulation by Br-cAMP (1 mM). Expression differences were not significant in the t-test (ns). (D). Dual CRISPR/Cas9 system with gRNAs. (a) High sensitivity to sp-RNA detection of mRNA (left) with GFP and mCherry detection of the transfected cells (right). Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells The dashed lines (circle) indicate the nucleus. Blue loci/dots are Cyp11a1, and red loci/dots are StAR intron 1. Orange channel (pseudocolored red) was used for the detection of StAR int1 and StAR oligomer set targeting extended 3′UTR (3′EU). Red channel (pseudocolored blue) was used for Cyp11a1 int3. (D) Probes hybridizing CRISPR non-target sequences in StAR (3′EU) and cyp11a1 were applied in CRISPR (+) and NT cells. Red loci/dots are StAR exon 7. (E) Quantitation of StAR intron 1. The blue segment in the bar denotes the percentage of cells with the active locus (StAR p-RNA). Scale bar represents 10 µm. Figure 5 | Detection of DNA deletion from the steroidogenic acute regulatory protein (STAR) loci of Y-1 cells by Cas9. (A) Probe design of DNA FISH. The probe hybridizes the StAR intron 1 (538/559 nm, pseudocolored red) in the CRISPR targeted sequence compared to non-target exon 7 [UTR/oligomer set targeting extended 3′UTR (3′EU), 538/559 nm, pseudocolored red] and Cyp11a1 int3 (618/637 nm, pseudocolored blue). (B) Procedure for DNA FISH. After RNase treatment, the slide was heated to denature DNA. Probes for CRISPR target StAR (int1) and non-target Cyp11a1 show the difference between NT and CRISPR (+) cells (top). Non-target StAR (3′EU) and Cyp11a1 were used as a control (bottom). (C) StAR intron1 was deleted in CRISPR (+) cells. StAR intron 1 compared to Cyp11a1 locus DNA retained in NT (a–d) cells. GFP and mCherry were used as markers for gRNA expression. The dashed lines (circle) indicate the nucleus. Blue loci/dots are Cyp11a1, and red loci/dots are StAR intron 1. Orange channel (pseudocolored red) was used for the detection of StAR int1 and StAR oligomer set targeting extended 3′UTR (3′EU). Red channel (pseudocolored blue) was used for Cyp11a1 int3. (D) Probes hybridizing CRISPR non-target sequences in StAR (3′EU) and cyp11a1 were applied in CRISPR (+) and NT cells. Red loci/dots are StAR exon 7. (E) Quantitation of StAR intron 1. The blue segment in the bar denotes the percentage of cells with the active locus (StAR p-RNA). Scale bar represents 10 µm. expression time profile. In Figure 5, we show that DNA hybridiza- tion in intron 1 remains heterogeneous in ways that may relate to the transcription access. The intensity of GFP fluorescence in each cell has been assessed for all cells in the fields of two CRISPR- transfected slide cultures at 4, 12, and 24 h [Figure 6A (b)]. Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells Expression difference was significant by Student’s t-test ***p < 0.001. Scale bar represents 10 µm. Figure 4 | Role of gRNA in steroidogenic acute regulatory protein (StAR) editing; Br-cAMP stimulation of MA-10 Cells and suppression of mRNA. (A) qPCR analysis of StAR transcription in MA-10 testis cells. (B) XY and XZ projection of StAR mRNA and protein in MA-10 cells after 3 h of stimulation by Br-cAMP (1 mM). (C) CRISPR/Cas9 system without gRNA. (a) The high sensitivity of FISH image to StAR mRNA with sp-RNA probes. The dotted circles indicate the dual-transfected cells (left). GFP and mCherry’s dual fluorescence (right). (b) Quantitation of mRNA copy numbers in NT and GFP/mCherry (+) cells after 3 h of stimulation by Br-cAMP (1 mM). Expression differences were not significant in the t-test (ns). (D). Dual CRISPR/Cas9 system with gRNAs. (a) High sensitivity to sp-RNA detection of mRNA (left) with GFP and mCherry detection of the transfected cells (right). Expression difference was significant by Student’s t-test ***p < 0.001. Scale bar represents 10 µm. DSB within a few minutes of their expression, which is marked by synthesis of GFP and mCherry proteins. In this event, the Cas9/ StAR editing is likely to occur on the leading edge of the GFP/RFP periods of CRISPR/Cas9 activity on StAR expression (locus sp- RNA versus cytoplasmic mRNA and protein). The gRNA/Cas9’s complex interacts with the StAR gene and generates the initial October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 10 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 5 | Detection of DNA deletion from the steroidogenic acute regulatory protein (STAR) loci of Y-1 cells by Cas9. (A) Probe design of DNA FISH. The probe hybridizes the StAR intron 1 (538/559 nm, pseudocolored red) in the CRISPR targeted sequence compared to non-target exon 7 [UTR/oligomer set targeting extended 3′UTR (3′EU), 538/559 nm, pseudocolored red] and Cyp11a1 int3 (618/637 nm, pseudocolored blue). (B) Procedure for DNA FISH. After RNase treatment, the slide was heated to denature DNA. Probes for CRISPR target StAR (int1) and non-target Cyp11a1 show the difference between NT and CRISPR (+) cells (top). Non-target StAR (3′EU) and Cyp11a1 were used as a control (bottom). (C) StAR intron1 was deleted in CRISPR (+) cells. StAR intron 1 compared to Cyp11a1 locus DNA retained in NT (a–d) cells. GFP and mCherry were used as markers for gRNA expression. Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells We have used GFP fluorescence as the marker since mCherry expres- sion overlapped in all cases. The GFP expression was robust in some cells after 4 h and approximately doubled in both numbers and intensities after 12 h. Between 12 and 24 h the number of cells expressing GFP remained at about 30%, but the mean expression has doubled. We presented three GFP expression ranges: low/ background in which transfection is minimal, medium, which is sufficient to generate DSB and a higher level for which the editing impact is unknown [Figure 6A (c)]. We have used the PCR amplification of StAR DNA to ana- lyze the effects of the single and dual CRISPR/Cas9 processes on the StAR locus (Figure 3C). We examined the excision as a time course in relation to the CRISPR/Cas9 GFP transfection [Figure 6A (d)]. After 12 h, there was about a 15–20% excision compared to the 50% excision after 24 h. However, after 4 h, the October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 11 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 6 | (A) Time course of steroidogenic acute regulatory protein (StAR) editing in relation to Cas9 transfection. (a) Design for varying post-transfection time for CRISPR editing: after the transfection period, Br-cAMP (1 mM) was treated for 2 h. p-RNA, sp-RNA/mRNA, and protein in CRISPR (+) and NT cells were analyzed by high-resolution fluorescence in situ hybridization. (b) Duplicate transfections (#1 and #2) were used to show GFP expression in cells as a function of post- transfection time (4, 12, and 24 h). (c) Relative GFP intensity in individual cells was shown. Two dotted lines separate three levels of expression: lower—background; middle—effective CRISPR (+); upper—effective but in excess of the amount needed for CRISPR. (d) PCR strategy to validate StAR gene deletion. Time course experiment was performed (2–24 h). (B) Impact of CRISPR on StAR editing at various transfection times: (a) StAR locus (sp-RNA), 4 versus 12 h; (b,c) loss of mRNA and protein, 4, 12, and 24 h. (d) The impact of CRISPR/Cas9 on StAR mRNA and protein. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey Scale bar represents 10 µm Figure 6 | (A) Time course of steroidogenic acute regulatory protein (StAR) editing in relation to Cas9 transfection. Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells (a) Design for varying post-transfection time for CRISPR editing: after the transfection period, Br-cAMP (1 mM) was treated for 2 h. p-RNA, sp-RNA/mRNA, and protein in CRISPR (+) and NT cells were analyzed by high-resolution fluorescence in situ hybridization. (b) Duplicate transfections (#1 and #2) were used to show GFP expression in cells as a function of post- transfection time (4, 12, and 24 h). (c) Relative GFP intensity in individual cells was shown. Two dotted lines separate three levels of expression: lower—background; middle—effective CRISPR (+); upper—effective but in excess of the amount needed for CRISPR. (d) PCR strategy to validate StAR gene deletion. Time course experiment was performed (2–24 h). (B) Impact of CRISPR on StAR editing at various transfection times: (a) StAR locus (sp-RNA), 4 versus 12 h; (b,c) loss of mRNA and protein, 4, 12, and 24 h. (d) The impact of CRISPR/Cas9 on StAR mRNA and protein. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. October 2017  |  Volume 8  |  Article 289 12 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate transfection (Figure 7C). To assess this adaptation to the Cas9 editing of StAR, we examined ORO staining in Y-1 cells at 12, 24, and 48 h after the CRISPR transfection. Each time, Br-cAMP was added for the final 1 h to match the conditions used for the assessment of StAR editing. A comparison of ORO staining in CRISPR (+) and adjacent NT cells shows a large increase in CE/ ORO in the CRISPR (+) cells that lack StAR activity (Figure 7D). excision fragment was not detectable despite CRISPR/Cas9 trans- fection at about half the 12 h level. The 0.65 kb product should be readily detectable at 10% of the 24 h level representing 5% excision. Overall, the excision process matched the intensity of CRISPR/Cas9 transfection. We examined the effects of editing in CRISPR (+) cells at the three times after CRISPR transfection followed by a 2 h Br-cAMP stimulation. Threefold increases in sp-RNA/mRNA in Y-1 cells produced by Br-cAMP after 2 h (Figure 2C) matched the increase in cytoplasmic mRNA (Figure 2D) and previous assessments of this response (1, 33). Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells After 12  h, the suppression of locus sp-RNA continued, but there was now a modest decline in StAR mRNA and protein [Figure 6B (b–d)]. However, 24 h after transfec- tion, there was the complete removal of StAR mRNA and protein in CRISPR (+) cells. The decline in StAR protein follows the loss of mRNA. The NT cells again showed modestly progressive increases. The more rapid effect on the expression at the loci compared to the mRNA and protein indicated an editing mechanism that discriminates between locus expression and mRNA generation. The complete losses of locus RNA, mRNA, and protein at 24 h match the measured DNA deletion shown in Figure 6A (d). The diminished effects on mRNA and protein at 4 and 12 h correspond well with the diminished DNA deletion at these earlier times. The extensive suppression of sp-RNA at 4 and 12 h far exceed the DNA deletion at these times and therefore arise from another effect of the CRISPR/Cas9. As noted earlier, the single 5′gRNA CRISPR/Cas9 can impact StAR without any deletion. h The more rapid effect on the expression at the loci compared to the mRNA and protein indicated an editing mechanism that discriminates between locus expression and mRNA generation. The complete losses of locus RNA, mRNA, and protein at 24 h match the measured DNA deletion shown in Figure 6A (d). The diminished effects on mRNA and protein at 4 and 12 h correspond well with the diminished DNA deletion at these earlier times. The extensive suppression of sp-RNA at 4 and 12 h far exceed the DNA deletion at these times and therefore arise from another effect of the CRISPR/Cas9. As noted earlier, the single 5′gRNA CRISPR/Cas9 can impact StAR without any deletion. f g p The relationship between ORO and StAR editing remains simi- lar to the plot for GFP accumulation. The accumulation of GFP in vacuoles raised a possibility of interference with CE trafficking. To check this possibility, we followed the conditions shown in Figure 4C and omitted both the gRNAs that are necessary for the Cas9 cleavage of StAR without affecting GFP expression. We meas- ured the ORO changes in NT and GFP (+) cells. Without gRNA to initiate StAR editing, the GFP (+) cells showed no stimulation of ORO staining to match the high cytoplasmic GFP expression [Figure S7A in Supplementary Material]. Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells This control was con- firmed for multiple gRNA (−) cells [Figure S7B in Supplementary Material]. The significant increase in CE accumulations, therefore, directly derives within a few hours from the redirection of cyto- plasmic cholesterol dynamics resulting from the removal of StAR. Time Course of CRISPR Initiation and StAR Editing in Y-1 Cells Thus, at least 75% of the RNA at the end of each 2 h Br-cAMP treatment was produced within this period and after the CRISPR–Cas9 transfection.t A high-resolution N-SIM image of ORO in adjacent NT and CRISPR (+) cells showed CE/ORO lining the inside of vacuoles [Figure 7E (a)]. We further examined the NT/CRISPR (+) pair [Figure 7E (a)] with a 3D compilation [Figure 7E (b)]. For the most part, the ORO accumulations are separate from the GFP accumulations and are positioned above them. There are also many vacuoles lined with GFP. The second set of three CRISPR (+) cells [Figure 7E (c)] showed appreciable local heterogeneity in the stimulation of ORO (+) CE accumulations. There was little difference in the accumulation of CE regardless of the presence or absence of Br-cAMP, which activates both StAR and HSL. The regions of high ORO staining in NT  cells again avoided regions of StAR expression in these cells [Figure  7F (a)]. We also tested the effect of a 1 h addition of the Cyp11a1 inhibitor aminoglutethimide (AMG), which fully inhibits mitochondrial cholesterol metabolism (39). AMG also increased ORO staining in NT cells in regions devoid of StAR. After the AMG treatment, 60% of the basal cells showed increased ORO with enlarged LDs or CE-laden endosomes, much like what was seen in StAR deficiency in CRISPR (+) cells. The AMG treatment of CRISPR (+) cells produced additive increases in ORO [Figure 7F (b)]. The effect of AMG is remarkably rapid, raising the question of whether AMG is affecting CE distribution at an additional point.h When the stimulation was initiated after 4 h, we saw the complete loss of locus sp-RNA expression from CRISPR (+) cells [Figure 6B (a)]. Surprisingly, mRNA expression was retained (Figure S6 in Supplementary Material). After 12  h, the suppression of locus sp-RNA continued, but there was now a modest decline in StAR mRNA and protein [Figure 6B (b–d)]. However, 24 h after transfec- tion, there was the complete removal of StAR mRNA and protein in CRISPR (+) cells. The decline in StAR protein follows the loss of mRNA. The NT cells again showed modestly progressive increases.hf When the stimulation was initiated after 4 h, we saw the complete loss of locus sp-RNA expression from CRISPR (+) cells [Figure 6B (a)]. Surprisingly, mRNA expression was retained (Figure S6 in Supplementary Material). LDs Accumulate in CRISPR (+) Y-1 Cells but Only after StAR mRNA Deletionf but Only after StAR mRNA Deletion We cannot directly image the effect of StAR on steroid synthesis, but LDs, which can be readily imaged by ORO, may provide an alternative target (19). In steroidogenic cells, these droplets mostly comprise CE. The CE content of LDs reflects the complex choles- terol dynamics of the cytoplasm, which involves transport through endosomes, biosynthesis from acetyl-CoA, and acylation by cho- lesterol acyltransferase (34). LDs include some endosomes that are filled with CE derived from low-density lipoprotein (LDL) but in rodent cells mostly comprise CE droplets that derive from high- density lipoprotein (HDL) taken up by Srb1. These droplets are surrounded by organizing proteins that also control interactions with processing enzymes such as HSL and StAR (18). A change of CE in LDs in CRISPR (+) cells visualized by ORO may, therefore, represent an adaptation to StAR deletion CRISPR (+) (Figure 7A). There appears to be an inverse relationship between the levels and location of ORO (+) droplets and the mitochondria that express StAR protein. Y-1 cells with high StAR have low ORO stain- ing, and this inverse relationship extends to the cells (Figure 7B). We have shown that CRISPR (+) cells that are marked by GFP show substantial increases in ORO, which start 12 h after CRISPR We cannot directly image the effect of StAR on steroid synthesis, but LDs, which can be readily imaged by ORO, may provide an alternative target (19). In steroidogenic cells, these droplets mostly comprise CE. The CE content of LDs reflects the complex choles- terol dynamics of the cytoplasm, which involves transport through endosomes, biosynthesis from acetyl-CoA, and acylation by cho- lesterol acyltransferase (34). LDs include some endosomes that are filled with CE derived from low-density lipoprotein (LDL) but in rodent cells mostly comprise CE droplets that derive from high- density lipoprotein (HDL) taken up by Srb1. These droplets are surrounded by organizing proteins that also control interactions with processing enzymes such as HSL and StAR (18). A change of CE in LDs in CRISPR (+) cells visualized by ORO may, therefore, represent an adaptation to StAR deletion CRISPR (+) (Figure 7A).h Frontiers in Endocrinology  |  www.frontiersin.org DISCUSSION This report describes the direct imaging with sm-FISH of the dual-gRNA-directed CRISPR/Cas9 inactivation of the key steroidogenic gatekeeper gene StAR. PCR analyses showed the excision of a central segment of proximal promoter/exon 1/intron 1 from the StAR genomic DNA. In addition, sm-FISH provided comprehensive single-cell imaging of CRISPR editing at the level of primary and spliced transcripts at gene loci and single molecules of cytoplasmic mRNA in relation to expressed protein and gene function. The sm-FISH method has also been extended to provide novel direct single-cell imaging of the DNA deletion in There appears to be an inverse relationship between the levels and location of ORO (+) droplets and the mitochondria that express StAR protein. Y-1 cells with high StAR have low ORO stain- ing, and this inverse relationship extends to the cells (Figure 7B). We have shown that CRISPR (+) cells that are marked by GFP show substantial increases in ORO, which start 12 h after CRISPR October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 13 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 7 | Cholesterol esters (CEs) in lipid droplets (LDs) appear selectively and rapidly in cells with CRISPR steroidogenic acute regulatory protein (StAR) deletion. (A) Model for StAR cytoplasmic activity. Protein kinase A activates CE conversion to cholesterol (CH) mediated by the hormone-sensitive lipase; transfer into the mitochondria-mediated by StAR leading to CYP-mediated metabolism (Cyp11a1 > Cyp27a1). StAR may also impact cholesterol uptake into the cell mediated by the high-density lipoprotein (HDL) receptor Srb1; internal transfer in and out of LDs and exit from the cell due to an ATP-dependent pump activity (Abca1, Abcg1). StAR deletion increases CE-containing LDs visualized and quantified through Oil Red O (ORO) staining. (B) CE in LDs measured by ORO in Y-1 cells after 24 h of CRISPR transfection separated from StAR protein expression in NT cells. CRISPR (+) cells are circled. (C) ORO staining in Y-1 cells at 12 and 24 h after the CRISPR transfection. (D) Quantitation of ORO intensity in individual cells at 12–48 h after transfection in Y-1 cells. (E) N-SIM images of GFP expression and ORO droplets in Y-1 cells. (a) XY section of CRISPR (+) cells expressing GFP and showing high ORO and an adjacent NT cell with typical low ORO. DISCUSSION We also observed this accumulation in humans with StAR deficiency and in StAR−/− mice (40). i However, in addition to excision, 30% of the CRISPR (+) cells retained this StAR sequence and normal p-RNA and sp-RNA tran- scripts at the loci, but remarkably, lost StAR mRNA and protein in the cytoplasm (Figure 3). We detected similar losses when using a single 5′gRNA (but not a 3′gRNA) (Figure S5 in Supplementary Material), thus establishing that alternative mutation-based mechanisms can operate in parallel with DNA excision.fi g y The 4-h CRISPR effect was consistent with disruption of the uncoupled process, which provided locus RNA but contributed little to mRNA. The slower 12–24 h alternative editing without excision targeted the rapid coupled process, which did not con- tribute to locus p-RNA or sp-RNA but generated most of the StAR mRNA and protein. This alternative to CRISPR-induced excision implies a distinct slower Cas9 intervention in the StAR promoter that may arise as an error in the excision ligation step (Figure S8B in Supplementary Material). The equivalent effects of the single 5′gRNA CRISPR/Cas9 strategy established that a single promoter intervention proximal to the 5′PAM site can produce such changes, presumably from mutations derived from errors in the initial NHEJ-type repair process (42). Interestingly, the endonuclease cleavage site overlapped the C/EBPβ-binding sequence, which likely mediated of the delayed coupled StAR transcription (41).h p p Y-1 cells exhibit modest basal StAR expression, which is suffi- cient to sustain maximum cholesterol metabolism when activated by Br-cAMP (7). This expression is diversely distributed among the individual cells and their loci [Figure 2 (b)]. The impact on basal expression in CRISPR (+) cells is consistent with deletion but currently, lacks a sufficient number of images from more active basal cells to achieve a statistical assessment. We overcame this dif- ficulty by applying CRISPR/Cas9 before the appreciable induction by Br-cAMP, when most cells exhibit strong locus and cytoplasmic responses (Figures 2 and 3). The precision of the sm-FISH in resolving transcription and splicing at the locus from processing to mRNA established distinct early (4 h) and delayed (12–24 h) alternative disruptions of the StAR locus without deletion that nevertheless retained transcription. Sm-FISH analyses of MA-10 cells differ in that they lack measurable basal locus and cytoplasm expression. This distinction facilitated our analysis of the complex StAR response to Br-cAMP stimulation (Figure 4; Figure S4 in Supplementary Material). DISCUSSION We applied this dual gRNA Cas9 targeting method to the StAR gene in Y-1 adrenal cells and MA-10 interstitial testis cells, each of which showed an acute PKA stimulation, thus providing extra insight into CRISPR’s impact. This strategy, which is applicable to any responsive gene, provides novel direct assessment of the immediate impact of CRISPR edit- ing on both the gene expression mechanism and the function.l These two alternative CRISPR editing effects were consistent with selective interventions in two StAR multistep expression processes that functioned in parallel after 2  h of Br-cAMP stimulation of both Y-1 or MA-10 cells (1, 33) (Figure S8 in Supplementary Material). StAR loci are initially activated with substantial restraints over splicing and possibly polyadenylation such that p-RNA and sp-RNA build up at the loci with the only slow transfer of mRNA to the cytoplasm. Protein translation, however, was robust despite this low cytoplasmic mRNA. This early stimulus corresponded to maximum cholesterol metabo- lism. This activity depends on the translation of a labile 3.5 kb mRNA that locates to mitochondria. This location depends on the extended 3′UTR of StAR, which delivers cleavage and polyadenylation at specific sites (30). After about 1 h, a much faster transcription process began in which formation of p-RNA at loci is bypassed through a coupling of splicing to transcription. We suspect that this surge derived from Br-cAMP induction of additional StAR transcription factors, notably c/EBPβ and NR4a1 (6, 41). The rapid sp-RNA processing meant that sp-RNA remained low in the loci while mRNA accumulated extensively in the cytoplasm, particularly at the mitochondria. At the end of a 2-h Br-cAMP stimulus, both transcription mechanisms were functioning equally. We marked CRISPR (+) cells using mCherry and GFP fluo- rescence (Figures 2, 4 and 6), which additionally provides a time course for effective transfection (Figure 6). PCR shows that half of the StAR DNA exhibited the predicted 1.1 kb deletion 24 h after transfection (Figure 6), while sm-FISH shows a complete loss of StAR expression of mRNA and protein from CRISPR (+) cells (Figure 3). This loss was matched by excisions of the dual CRISPR target sequence as shown by sm-FISH assessment of intron 1 (Figure 5). This loss of StAR expression was matched by a large increase in CE accumulation in LDs and endosomes, which we imaged using ORO. DISCUSSION (b) The 3D assemblage of the same cell (c) different CRISPR cell with more extensive ORO staining but heterogeneous distribution. (F) Distribution of ORO and StAR in NT cells under basal conditions and after 1 h stimulation by Br-cAMP or aminoglutethimide. Left and right panels show same cells marked by, respectively, GFP and ORO (left) and StAR protein. In each case, ORO several large LDs locate together and displace StAR/mitochondria. Figure 7 | Cholesterol esters (CEs) in lipid droplets (LDs) appear selectively and rapidly in cells with CRISPR steroidogenic acute regulatory protein (StAR) deletion. (A) Model for StAR cytoplasmic activity. Protein kinase A activates CE conversion to cholesterol (CH) mediated by the hormone-sensitive lipase; transfer into the mitochondria-mediated by StAR leading to CYP-mediated metabolism (Cyp11a1 > Cyp27a1). StAR may also impact cholesterol uptake into the cell mediated by the high-density lipoprotein (HDL) receptor Srb1; internal transfer in and out of LDs and exit from the cell due to an ATP-dependent pump activity (Abca1, Abcg1). StAR deletion increases CE-containing LDs visualized and quantified through Oil Red O (ORO) staining. (B) CE in LDs measured by ORO in Y-1 cells after 24 h of CRISPR transfection separated from StAR protein expression in NT cells. CRISPR (+) cells are circled. (C) ORO staining in Y-1 cells at 12 and 24 h after the CRISPR transfection. (D) Quantitation of ORO intensity in individual cells at 12–48 h after transfection in Y-1 cells. (E) N-SIM images of GFP expression and ORO droplets in Y-1 cells. (a) XY section of CRISPR (+) cells expressing GFP and showing high ORO and an adjacent NT cell with typical low ORO. (b) The 3D assemblage of the same cell (c) different CRISPR cell with more extensive ORO staining but heterogeneous distribution. (F) Distribution of ORO and StAR in NT cells under basal conditions and after 1 h stimulation by Br-cAMP or aminoglutethimide. Left and right panels show same cells marked by, respectively, GFP and ORO (left) and StAR protein. In each case, ORO several large LDs locate together and displace StAR/mitochondria. October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 14 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate CRISPR (+) cells and adjacent NT cells. The extent of this StAR deletion is compared to retentions of, respectively, a non-targeted segment of the StAR gene and the Cyp11a1 gene, the immediate partner in the steroidogenic pathway. Frontiers in Endocrinology  |  www.frontiersin.org DISCUSSION The time course for CRISPR/Cas9 effects showed that editing was effective within 4 h in removing StAR expression at the locus, even though mRNA and protein were retained. Examination of single 5′end targeting was selec- tively effective within 24 h in suppressing StAR expression without DNA deletion suggesting that this 4 h disruption may arise from an error in the early DSB repair of the 5′gRNA Cas9 process in the proximal promoter. Alternatively, direct inhibition of StAR tran- scription factors such as CREB or C/EBPβ may be produced by this high affinity Cas9 complex. Removal of this complex through the slow mutation will remove this direct disruption. The CRISPR editing machinery appears to function more rap- idly than expected based on the in vitro modeling (43). However, a novel recent approach to the measurement of the dissociation of gRNA/Cas9 complexes in cells has indicated much faster rates (44). These cell dissociation rates vary appreciably, probably because of different competing binding effects from transcription factors. Here, the 5′gRNA interaction in the StAR proximal promoter region overlapped several functional transcription factor sites.h The role of StAR as a regulator of cholesterol metabolism in mitochondria has been extensively documented (7, 45). Here, the CRISPR/Cas9 removal of the StAR activity between 12 and 24 h has been linked to a large increase in CE in LDs (Figure 7), which StAR affects through interactions with a HSL and activation of alternative transcription processes mediated by LXRa (22, 46). This accumulation in single cells imaged by ORO staining provides a measure of the cell adaptation to StAR loss (Figure 7A). Increases in CE in LD and late endosomes in these cells were enriched in regions where StAR protein and associated mitochondria were low (Figure 7). The Cyp11a1/cholesterol metabolism inhibitor AMG October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 15 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 8 | Schematic diagrams of selective CRISPR/Cas9 impact on synthesis of steroidogenic acute regulatory protein (StAR) mRNA and cholesterol transfer activity in the cytoplasm. CRISPR/Cas9 editing of StAR increases accumulation of cholesterol esters (CEs) detected by Oil Red O (ORO). Newly translated p37 StAR becomes phosphorylated by protein kinase A and then imported into mitochondria concomitant with cholesterol (CH) transfer and metabolism by Cyp11a1 (52). FUNDING This study was financially supported by NIH (RO1 DK074819, RO1 DK090249). This research was also supported by funding from the University of Wisconsin School of Medicine and Public Health Department of Cell and Regenerative Biology. AUTHOR CONTRIBUTIONS JL and CJ designed and analyzed the experiments; wrote the manuscript. JL carried out all experiments. DISCUSSION StAR deletion appears to affect enhance the complex cytoplasmic cholesterol exchange dynamics involving cholesterol movement between multiple orga- nelles. Interestingly, NPC/STARD3 mediates removal from late endosomes/lysosomes (LE/Ly), which are inhibited by lipophilic amines (48) that may potentially include AMG. NPC1/Stard3 mediates contacts between LE/Ly and endoplasmic reticulum (ER), which mediate cholesterol transfer between these organelles (49, 50). ER contacts with mitochondria facilitate their fusion, which is now recognized as a prime site of StAR activity (51). This rapid response to dual CRISPR Cas9 editing of StAR closely matched the cytoplasmic large CE accumulations in the adrenals, testes, and ovaries of StAR−/− mice, as well as in humans, who carry the loss of function StAR mutations (40).f the means to identify alternative variations, for example, changing the gRNA sites while also providing insight into the optimum conditions for more detailed single-cell cloning or sequencing. ACKNOWLEDGMENTS We thank Dr. Mario Ascoli for the MA-10 mouse Leydig tumor cell, and Dr. Bernard Schimmer for the Y-1 cell line. DISCUSSION Without phosphorylation, StAR may promote cytoplasmic steps that promote exit of excess CH from the cells; for example by stimulation of liver X receptor (LXR) induction of ATP-dependent cholesterol export pumps: AbcA1 to low-density lipoprotein (LDL) and AbcG1 to high-density lipoprotein (HDL) (21). Entry from LDL deposits cholesterol in the late endosomes/lysosomes. Re-distribution, including endoplasmic reticulum or mitochondria, is directed by NPC1 and 2 together with StARD3/MLN64 (48). Entry from HDL deposits CE in LDs. CE is converted to CH by hormone-sensitive lipase (HSL) and in LE by acid-sensitive esterase (18, 53). Acylation by cholesterol acyltransferase (ACAT) produces the reverse formation of CE. StAR inactivation by Cas9 increases CE. This can arise through decreased removal from LE and LD and decreased export from the cell via AbcA1 and AbcG1. Aminoglutethimide (AMG) and StAR/Cas9 share similar effects on ORO/CE. Decreased CH metabolism cannot produce AMG effect (1 h). Other points of overlap include effects on mitochondrial cholesterol that reduce ATP, or decreased distribution of CE from LE. Lipophilic amines like AMG, notably imipramine, disrupt the release of CH from LE (54–56). StAR may also mediate the activation of LXR, notably to maintain AbcA1-mediated CH transport. Figure 8 | Schematic diagrams of selective CRISPR/Cas9 impact on synthesis of steroidogenic acute regulatory protein (StAR) mRNA and cholesterol transfer activity in the cytoplasm. CRISPR/Cas9 editing of StAR increases accumulation of cholesterol esters (CEs) detected by Oil Red O (ORO). Newly translated p37 StAR becomes phosphorylated by protein kinase A and then imported into mitochondria concomitant with cholesterol (CH) transfer and metabolism by Cyp11a1 (52). Without phosphorylation, StAR may promote cytoplasmic steps that promote exit of excess CH from the cells; for example by stimulation of liver X receptor (LXR) induction of ATP-dependent cholesterol export pumps: AbcA1 to low-density lipoprotein (LDL) and AbcG1 to high-density lipoprotein (HDL) (21). Entry from LDL deposits cholesterol in the late endosomes/lysosomes. Re-distribution, including endoplasmic reticulum or mitochondria, is directed by NPC1 and 2 together with StARD3/MLN64 (48). Entry from HDL deposits CE in LDs. CE is converted to CH by hormone-sensitive lipase (HSL) and in LE by acid-sensitive esterase (18, 53). Acylation by cholesterol acyltransferase (ACAT) produces the reverse formation of CE. StAR inactivation by Cas9 increases CE. This can arise through decreased removal from LE and LD and decreased export from the cell via AbcA1 and AbcG1. Aminoglutethimide (AMG) and StAR/Cas9 share similar effects on ORO/CE. DISCUSSION Decreased CH metabolism cannot produce AMG effect (1 h). Other points of overlap include effects on mitochondrial cholesterol that reduce ATP, or decreased distribution of CE from LE. Lipophilic amines like AMG, notably imipramine, disrupt the release of CH from LE (54–56). StAR may also mediate the activation of LXR, notably to maintain AbcA1-mediated CH transport. (39) rapidly increased these effects in NT  cells independent of StAR, making the involvement of cholesterol metabolism unlikely. Each ORO image following StAR deletion in CRISPR (+) cells and AMG stimulation in NT cells showed similar accumulations in cytoplasmic endosome-like vacuoles (Figure 7E). CE in LDs is supplied by HDL via SRB1 at the plasma membrane but in late endosomes by LDL through LDL receptors. Each import is balanced by ATP-dependent export pumps (Abcg1 and Abca1). Potential mechanisms include stimulation of ATP-dependent cho- lesterol export (47), mitochondrial ATP and apoptotic processes (46), and the effects of hydroxyl-cholesterol metabolites on LXR receptors (21). These cytoplasmic disruptions are represented in relation to CRISPR editing (Figure 8). StAR deletion appears to affect enhance the complex cytoplasmic cholesterol exchange dynamics involving cholesterol movement between multiple orga- nelles. Interestingly, NPC/STARD3 mediates removal from late endosomes/lysosomes (LE/Ly), which are inhibited by lipophilic amines (48) that may potentially include AMG. NPC1/Stard3 mediates contacts between LE/Ly and endoplasmic reticulum (ER), which mediate cholesterol transfer between these organelles (49, 50). ER contacts with mitochondria facilitate their fusion, which is now recognized as a prime site of StAR activity (51). This rapid response to dual CRISPR Cas9 editing of StAR closely matched the cytoplasmic large CE accumulations in the adrenals, testes, and ovaries of StAR−/− mice, as well as in humans, who carry the loss of function StAR mutations (40).f (39) rapidly increased these effects in NT  cells independent of StAR, making the involvement of cholesterol metabolism unlikely. Each ORO image following StAR deletion in CRISPR (+) cells and AMG stimulation in NT cells showed similar accumulations in cytoplasmic endosome-like vacuoles (Figure 7E). CE in LDs is supplied by HDL via SRB1 at the plasma membrane but in late endosomes by LDL through LDL receptors. Each import is balanced by ATP-dependent export pumps (Abcg1 and Abca1). 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This approach provides Frontiers in Endocrinology  |  www.frontiersin.org October 2017  |  Volume 8  |  Article 289 16 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate REFERENCES Characterization of the acute stimulation of steroidogenesis in primary bovine adrenal cortical cell cultures. J Biol Chem (1984) 259:10159–67. 19. Caron KM, Soo SC, Wetsel WC, Stocco DM, Clark BJ, Parker KL. Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia. Proc Natl Acad Sci U S A (1997) 94:11540–5. doi:10.1073/pnas.94.21.11540 f 40. Hasegawa T, Zhao L, Caron KM, Majdic G, Suzuki T, Shizawa S, et  al. Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol Endocrinol (2000) 14:1462–71. doi:10.1210/mend.14.9.0515 20. Borthwick F, Taylor JM, Bartholomew C, Graham A. 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Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 48. Borthwick F, Allen AM, Taylor JM, Graham A. Overexpression of STARD3 in human monocyte/macrophages induces an anti-atherogenic lipid phenotype. Clin Sci (Lond) (2010) 119:265–72. doi:10.1042/CS20100266 49. Raiborg C, Wenzel EM, Stenmark H. ER-endosome contact sites: molecular compositions and functions. EMBO J (2015) 34:1848–58. doi:10.15252/ embj.201591481 Copyright © 2017 Lee and Jefcoate. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribu- tion 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. 50. Wilhelm LP, Wendling C, Vedie B, Kobayashi T, Chenard MP, Tomasetto C, et  al. STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites. EMBO J (2017) 36:1412–33. Frontiers in Endocrinology  |  www.frontiersin.org REFERENCES doi:10.15252/ embj.201695917 October 2017  |  Volume 8  |  Article 289 Frontiers in Endocrinology  |  www.frontiersin.org 18
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Mining tissue specificity, gene connectivity and disease association to reveal a set of genes that modify the action of disease causing genes
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BioMed Central BioMed Central g Antonio Reverter*, Aaron Ingham and Brian P Dalrymple Address: Computational and Systems Biology, CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, B i b Q l d A li Address: Computational and Systems Biology, CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, Brisbane, Queensland 4067, Australia Email: Antonio Reverter* - Tony.Reverter-Gomez@csiro.au; Aaron Ingham - Aaron.Ingham@csiro.au; Brian P Dalrymple - Brian.Dalrymple@csiro.au * Corresponding author Email: Antonio Reverter* - Tony.Reverter-Gomez@csiro.au; Aaron Ingham - Aaron.Ingham@csiro.au; Brian P Dalrymple - Brian.Dalrymple@csiro.au Received: 7 January 2008 Accepted: 19 September 2008 Received: 7 January 2008 Accepted: 19 September 2008 BioData Mining 2008, 1:8 doi:10.1186/1756 0381 1 8 This article is available from: http://www.biodatamining.org/content/1/1/8 This article is available from: http://www.biodatamining.org/content/1/1/8 © 2008 Reverter et al; licensee BioMed Central Ltd. © 2008 Reverter 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. ticle distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2. d use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Ac Research Mining tissue specificity, gene connectivity and disease association to reveal a set of genes that modify the action of disease causing genes Antonio Reverter*, Aaron Ingham and Brian P Dalrymple Open Access Data resources, edits and nomenclature We merged three large datasets as follows: Firstly, we accessed expression data drawn from massively parallel signature sequencing (MPSS) covering 182,719 tag signa- tures across 32 tissues [2]. Tissues represented on the MPSS data included nine different central nervous system (CNS) areas (amygdale, caudate nucleus, cerebellum, cor- pus callosum, fetal brain, hypothalamus, thalamus, spinal cord, and pituitary gland) and 23 non-CNS organs (adre- nal gland, bladder, bone marrow, heart, kidney, lung, mammary gland, pancreas, placenta, prostate, retina, sali- vary gland, small intestine, spleen, stomach, testis, thy- mus, thyroid, trachea, uterus, colon, monocytes and peripheral blood lymphocytes). A total of 18,677 unique genes were represented on the MPSS data and the number of expressed genes per tissue averaged 8,943 and ranged from 5,845 in pancreas to 12,267 in testis. Among the high-throughput technologies, gene expres- sion profiling has led to the identification of genes that perform in a coordinated manner allowing researchers to reasonably predict the role of genes for which no biologi- cal function was attributed, based on the known perform- ance of other group members. These predictions rely on the guilt-by-association heuristic, widely invoked in genomics and with proven applicability [1]. At the same time, a comprehensive atlas of transcribed genes in humans has revealed that genes may be split into two broad categories based on the number of tissues they are expressed in [2]. Genes that are expressed in many tis- sues are designated as housekeeping (HK) while those that are expressed in few tissues are termed tissue-specific (TS). Secondly, we downloaded a set of 55,606 true positive interactions among 7,197 genes that were defined from functional studies [15]. This interactions dataset was built including 2,788 confirmed, direct, physical protein-pro- tein interactions derived from the Biomolecular Interac- tion Network Database (BIND; http://binddb.org) [16], 18,176 confirmed human protein interactions from the Human Protein Reference Database (HPRD; http:// www.hprd.org/) [17], 22,012 direct functional interac- tions from the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg) [18], and 16,295 interactions derived from Reactome http:// www.reactome.org[19]. Tissue specificity has subsequently been linked to a number of significant outcomes including level of expres- sion [3], ability to detect cis-acting and trans-acting expression- quantitative trait loci [4], and differential rates of polymorphism [5], evolution [6] and disease-associa- tion [7]. Abstract Background: The tissue specificity of gene expression has been linked to a number of significant outcomes including level of expression, and differential rates of polymorphism, evolution and disease association. Recent studies have also shown the importance of exploring differential gene connectivity and sequence conservation in the identification of disease-associated genes. However, no study relates gene interactions with tissue specificity and disease association. Methods: We adopted an a priori approach making as few assumptions as possible to analyse the interplay among gene-gene interactions with tissue specificity and its subsequent likelihood of association with disease. We mined three large datasets comprising expression data drawn from massively parallel signature sequencing across 32 tissues, describing a set of 55,606 true positive interactions for 7,197 genes, and microarray expression results generated during the profiling of systemic inflammation, from which 126,543 interactions among 7,090 genes were reported. Results: Amongst the myriad of complex relationships identified between expression, disease, connectivity and tissue specificity, some interesting patterns emerged. These include elevated rates of expression and network connectivity in housekeeping and disease-associated tissue-specific genes. We found that disease-associated genes are more likely to show tissue specific expression and most frequently interact with other disease genes. Using the thresholds defined in these observations, we develop a guilt-by-association algorithm and discover a group of 112 non-disease annotated genes that predominantly interact with disease-associated genes, impacting on disease outcomes. Conclusion: We conclude that parameters such as tissue specificity and network connectivity can be used in combination to identify a group of genes, not previously confirmed as disease causing, that are involved in interactions with disease causing genes. Our guilt-by-association algorithm should be useful for the discovery of additional modifiers of genetic diseases, and more generally, for the ability to associate genes of unknown function to clusters of genes with defined functions allowing for novel biological inference that can be subsequently validated. Page 1 of 11 (page number not for citation purposes) http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Background disease, may impact the development of diseases, includ- ing cancers, and hypothesize that many other members of this list will ultimately be confirmed as modifiers of vari- ous genetic diseases. g The understanding of the biology underlying phenotype is still a limiting factor in delivering the promise of high throughput genomics. However, as new datasets are avail- able, new data mining methods are developed and the goal appears ever more achievable. The impact of tissue-specificity Among the myriad of complex relationships, some inter- esting patterns emerged. Consistent with previous find- ings [3], we observed a strong relationship between the number of tissues in which a gene was expressed and its level of expression (Table 1). Importantly, this relation- ship was unaffected by disease or interaction status. Results and discussion Initial gene groupings and unknown biological processes Initial gene groupings and unknown biological processes Figure 1 illustrates the way in which the 15,050 genes were simultaneously annotated as either disease-associ- ated or included in the true positive interactions and the inflammation datasets. These genes were further classified as either TS, NS or HK, and the number of disease-associ- ated and/or interacting genes contained within each of the resulting 12 categories was determined. The proportion of genes with unknown biological process was also regis- tered. These criteria resulted in 15,050 genes [see Additional file 1] of which 5,198 and 4,950 were included in the true positive interactions and the inflammation datasets, respectively, and with 2,499 genes in common. In addi- tion, a total of 6,151 (41%) of the genes were associated with disease according to OMIM database [22] as of Sep- tember 19, 2007; and with 1,445 of them defined as dis- ease-causing (i.e., associated with either known disease phenotype or polymorphic sequence known). As expected, the discovery of interactions as well as dis- ease-association for a given gene provides additional bio- logical knowledge, allowing inferences as to its genomic functionality. Nevertheless, the biological process of about 10% of these presumably well-characterized genes remains to be elucidated. On the other extreme, and high- lighting the extent to which further research is needed, as many as 85% of NDIS, NINT genes and across the three expression categories (TS, NS and HK) belong to an unknown biological process. Hereafter, we refer to DIS to indicate the 6,151 genes from our resulting dataset that are disease-associated according to OMIM, and to NDIS to indicate the remaining 8,899 non-disease-associated genes also according to OMIM. Similarly, we refer to INT (and NINT) to indicate genes in our dataset for which interactions have (and have not) been reported. Data mining approaches In order to further characterize the relationship existing between tissue specificity, gene connectivity and disease association, the 15,050 genes were classified as either TS or HK. To ensure that these two categories together repre- sented the majority of the genes, we searched for category limits from either extreme of the distribution of the number of genes expressed in one, two, and up to 32 tis- sues, until equivalent categories were defined, cumula- tively representing > 50% of the total number of genes. In doing so, there were 4,232 (28%) TS genes expressed in 1 to 4 tissues, and 4,006 (27%) HK genes expressed in more than 25 tissues. The remaining 6,812 (45%) genes were classified as non-specific (NS). Overall, the distribution of the expression of genes among tissues was grossly bimodal. However, this bimodality vanished when the distribution was examined separately for INT and NINT genes (Figure 2). INT genes are over- represented among HK genes, while NINT genes are pre- dominantly TS. We conclude that the more tissues a gene is expressed in, the higher its chances of interacting with at least one other gene, irrespective of the tissue-specificity of this second gene. Figure 3 presents the relationship between tissue specifi- city and proportion of disease-associated genes. The over- all Pearson correlation coefficient (PCC) was moderate (0.53) yet significant (P = 0.0019) indicating an increase in the number of DIS genes among broadly expressed genes. Computing the PCC conditional on interaction sta- tus results in a non-significant PCC of -0.26 (P = 0.1459) for NINT genes, and a strong negative PCC of -0.73 (P < 0.0001) for INT genes. This counterintuitive pattern of correlation is representative of the Simpson's Paradox [23] with the paradox being that, although INT genes tend to be expressed in many tissues, those that are expressed in a tissue specific manner are more likely to be DIS. This is likely due to the increased number of relationships an Finally, and in order to identify novel candidate genes impacting disease, we developed a guilt-by-association algorithm. Selection thresholds based on the average number of known interactions combined with the average proportion of DIS genes among their interactors were determined from DIS genes. These thresholds were then applied to genes in the NDIS category. Genes exceeding both thresholds were identified as likely disease-associ- ated candidates. Data resources, edits and nomenclature In addition, we [8] and others [9,10] have dem- onstrated the importance of exploring differential gene connectivity in the identification of disease-associated genes using microarray gene expression data. More recently, the combination of text mining with gene inter- action network analysis has been proposed to infer unknown gene-disease associations [11]. Furthermore, genes with a high degree of connectivity (network hubs) have been shown to be conserved across species [12] and their knockout phenotype more likely to be lethal [13]. Finally, based on sequence conservation across species, a computational algorithm has been devel- oped to identify genes associated with disease [14]. How- ever, no study relates gene interactions with tissue specificity and its subsequent likelihood of association with disease. Finally, we used the microarray expression results gener- ated during the profiling of systemic inflammation across 44,924 probe sets [20] and from which 126,543 interac- tions among 7,090 genes were reported [8]. The microar- ray experiment used 92 Affymetrix GeneChips (Affymetrix, Santa Clara, CA) to examine gene expression profiles in whole blood leukocytes immediately before and at 2, 4, 6, 9 and 24 h after intravenous administration of bacterial lipopolysaccharide (LPS) endotoxin to four healthy human subjects. For the control (placebo) data, four additional subjects were studied under identical con- ditions but without LPS administration. To address this situation, we mined three large independ- ent datasets and classified transcribed human genes based on transcript abundance, tissue specificity, gene connec- tivity and disease association. We discuss how these fac- tors relate to each other and, based on this new knowledge, implement a simple yet powerful guilt-by- association algorithm that allows us to identify several candidate genes that, while not previously associated with For the present study, and to enable the merging of the three datasets, a number of edits were performed as fol- lows: For the MPSS data, tags not expressed at more than 5 transcripts per million (tpm), in at least one tissue, were Page 2 of 11 (page number not for citation purposes) BioData Mining 2008, 1:8 http://www.biodatamining.org/content/1/1/8 Page 3 of 11 (page number not for citation purposes) Results and discussion I i i l i d disregarded. The threshold of 5 tpm corresponds to the sensitivity of MPSS technology as claimed by the manu- facturers and independently assessed in our laboratory [21]. Also, when the same gene was represented by more than one MPSS tag, the reading from the most abundant tag, summed across all tissues, was assigned to that gene. Finally, for the true positive interactions and the inflam- mation datasets, interactions involving genes not sur- veyed in the MPSS data were also discarded. Identification of candidate disease genes via guilt-by- association Given our measurement confirming that like associates with like, we developed a guilt-by-association algorithm with the aim of identifying candidate genes among the previously classified non-disease subset. Our guilt-by- association algorithm starts by examining the connectivity properties of the DIS genes. In this context, DIS genes were found to be involved, on average, in 12 interactions (ranging from 0 to 176). Also on average, their interactors were themselves DIS genes in 75% of instances. Impor- tantly, while only 1,132 (or 18.4%) of DIS genes had > 12 interactions (revealing the skewedness in the number of interactions), 651 (or 57.5%) of them interacted with DIS genes > 75% of the time. When these same thresholds (i.e., > 12 interactions and > 75% of DIS genes among interactors) were applied to NDIS genes, we revealed the presence of 112 genes [see Additional file 2], including 26 TS, 50 NS and 36 HK, that while not being associated with disease, have higher than average connectivity degree (> Data mining approaches Page 3 of 11 (page number not for citation purposes) Page 3 of 11 (page number not for citation purposes) http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Gene groupings Figure 1 Gene groupings. Genes were classified as tissue-specific (TS), non-specific (NS) or housekeeping (HK). Among each class, the number of interacting and disease-associated genes is noted, and for each of the resulting 12 categories, the percentage of genes with unknown biological process ontology is given. HK Y Y HK N Y HK Y N HK N N NS Y Y NS N Y NS Y N NS N N TS Y Y TS N Y TS Y N TS N N 0 10 20 30 40 50 60 70 80 90 100 Interaction Disease # Genes % Unknown Biological Process 1,581 219 1,288 918 2,034 775 1,325 2,678 954 588 467 2,223 Gene grou Figure 1 Gene groupings Figure 1 Gene groupings. Genes were classified as tissue-specific (TS), non-specific (NS) or housekeeping (HK). Among each class, the number of interacting and disease-associated genes is noted, and for each of the resulting 12 categories, the percentage of genes with unknown biological process ontology is given. genes (Figure 4). Consistent with our results, genes associ- ated with similar disorders have been shown to have higher likelihood of physical interactions between their products and a higher expression profiling similarity for their transcripts [24]. interacting HK gene would have compared to a TS equiv- alent, thereby increasing the likelihood of a mutation leading to a detrimental and potentially lethal outcome, as previously determined [6]. We conclude that it is not so much that TS genes are more likely to be associated with disease, but rather that HK genes associated with disease are rarely observed. Page 4 of 11 (page number not for citation purposes) Gene interactions in the context of tissue-specificity and disease association Our analyses revealed that interacting HK genes are more likely to interact with genes that are also HK (PCC = 0.89; P < 0.0001) and vice-versa (i.e., TS genes are more likely to interact among themselves). Importantly, this correla- tion remained strong when conditioning on disease status (Table 1). Also, interactions between two HK genes were 12.8 times more frequent (P < 0.0001) and 3.3 times more cohesive (P < 0.0001) as measured by the clustering coefficient, than interactions between two TS genes. The clustering coefficient is a measure of network cohesive- ness and captures how many neighbours of a given gene are connected to each other. Similarly, interactions between two DIS genes were 3.1 times more frequent (P < 0.0001) and 1.6 times more cohesive (P < 0.001) than interactions between two NDIS Page 4 of 11 (page number not for citation purposes) Page 4 of 11 (page number not for citation purposes) http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Table 1: Relationship between the number of tissues in which a gene is expressed and a series of variables. Variable Correlation Regression Expression: Overall genes 0.706 2.034 Non-Interacting (NI) genes only 0.709 1.802 Interacting genes only 0.707 2.107 Non-Disease (ND) 0.707 1.769 Disease (D) 0.709 2.382 Non-Interacting and Non-Disease 0.691 1.759 Non-Interacting and Disease 0.764 2.039 Interacting and Non-Disease 0.719 1.803 Interacting and Disease 0.702 2.438 Proportion of interacting genes: Overall genes 0.949 0.012 Non-Disease genes only 0.942 0.013 Disease genes only 0.917 0.008 Proportion of disease genes: Overall 0.527 0.002 Non-Interacting genes only -0.263 -0.001 Interacting genes only -0.733 -0.004 Tissue specificity of interactors: Overall genes 0.887 0.112 Non-Disease genes only 0.736 0.062 Disease genes only 0.872 0.151 Proportion of disease genes among interactors: Overall genes 0.229 0.000 Non-Disease genes only -0.048 0.000 Disease genes only 0.575 0.001 Table 1: Relationship between the number of tissues in which a gene is expressed and a series of variables. Table 1: Relationship between the number of tissues in which a gene is expressed and a series of variable In order to further ascertain the optimality of various loca- tion parameters to be used as thresholds in the guilt-by- association algorithm, we explored the proportion of truly disease associated genes from the total number of cap- tured genes and the results are presented in Table 3. While the median performs slightly better (i.e. Gene interactions in the context of tissue-specificity and disease association by up to 1.03 times better, or 78.9 over 76.3) than the mean when used as a threshold for the proportion of disease genes among interactors, this improvement is at the expense of generat- ing substantially larger lists of candidate genes. When exploring the number of connections, the mean is very close to the 75th percentile, indicating the skewness in the connectivity distribution with most genes having few con- nections and few genes having many connections. Also, as a threshold for the number of connections, the mean per- forms favourably against either inter-quartile. 12 connections) and higher than average proportion (> 75%) of genes in OMIM among their connectors. Table 2 presents the number of genes in the contingency table underlying our guilt-by-association algorithm. To assess the optimality of our approach, we repeated the analyses using only the 1,445 DIS genes (out of the initial 6,151) with known disease phenotype and either sequence mutation or molecular basis known as those declared as truly disease-associated. The new thresholds for connectivity and proportion of DIS genes among inter- actors were 12 and 35%, respectively. The new list of can- didate genes included 127 genes of which 107 were assessed as DIS in the initial list of 6,151. Assuming the remaining 20 genes are indeed false positives, this implies a precision of at least 84%. However, the infeasibility of directly computing perform- ance measures associated with a given algorithm in the absence of negative examples should be acknowledged. That is, although one can be relatively sure that certain It should be noted that precision alone is not enough to assess the goodness of a classifier, as it is only concerned with the ratio of identified genes that are positive, but not with the total number of discovered genes. Page 5 of 11 (page number not for citation purposes) Page 5 of 11 (page number not for citation purposes) BioData Mining 2008, 1:8 http://www.biodatamining.org/content/1/1/8 Frequency histogram of gene expression Figure 2 Frequency histogram of gene expression. For each gene, the tissues showing expression at more than 5 transcript per million were counted and the histogram explored separately for non-interacting (green) and interacting genes (red). The two distributions are statistically different (Kolmogorov-Smirnov test P-value < 0.001). Gene interactions in the context of tissue-specificity and disease association Non-Interacting genes Interacting genes http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Non-Interacting genes Interacting genes Non-Interacting genes Interacting genes Frequency Figure 2 q y g g p g Frequency histogram of gene expression. For each gene, the tissues showing expression at more than 5 transcript per million were counted and the histogram explored separately for non-interacting (green) and interacting genes (red). The two distributions are statistically different (Kolmogorov-Smirnov test P-value < 0.001). (hypergeometric P = 7.5 × 10-16) than the 14% predicted by OMIM across the genome, with 2,549 genes defined as the basis of heritable disease out of the 18,091 total. genes are associated with a disease, it is not possible to ensure that a set of genes is not involved in any disease. In other words: Absence of evidence is not evidence of absence. On the other extreme, some of the genes anno- tated as disease associated by OMIM could also be false positives. In these situations, partially supervised learning algorithms have been proposed to address this issue and in the context of identifying disease genes [14]. Clusters of disease among candidate genes f g g In order to determine what diseases these genes might impact, we explored the gene networks spanned by the members of our guilt-by-association list, alone and in combination with their interactors. Based on the disease associations shown [see Additional file 2], each cluster was examined for a common disease. In this fashion, we identified two clusters of genes that impact on either breast or gastric cancer. Figure 5 depicts the Cytoscape [25] representation of the breast cancer cluster where seven of our guilt-by-association genes (APBA2BP, CCNA2, COBRA1, PCAF, RAD51, SMARCA4 and STAT5A) were linked to the well characterized human Nevertheless, a literature survey revealed that 44 of the 112 candidate genes [see Additional file 2] have been pre- viously associated with polymorphisms or differential gene expression leading to a modified risk of disease. A further 10 genes exist within chromosomal regions associ- ated with disease. The remaining 58 genes have no obvi- ous association to disease in any system. The 39% rate of disease association determined here is much higher Page 6 of 11 (page number not for citation purposes) Page 6 of 11 (page number not for citation purposes) BioData Mining 2008, 1:8 http://www.biodatamining.org/content/1/1/8 Disease association and tissue specificity Figure 3 Disease association and tissue specificity. Relationship between tissue specificity (x-axis) and proportion of disease-asso- ciated genes (y-axis) computed using all genes (blue pattern), and separate for non-interacting (green pattern) and interacting genes (red pattern). Number of tissues Proportion of disease genes 1 7 13 19 25 31 0.1 0.3 0.5 0.7 http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Disease as Figure 3 Disease association and tissue specificity Figure 3 Disease association and tissue specificity. Relationship between tissue specificity (x-axis) and proportion of disease-asso- ciated genes (y-axis) computed using all genes (blue pattern), and separate for non-interacting (green pattern) and interacting genes (red pattern). Conclusion 3.1 1.0 2.6 2.6 7.4 9.7 7.4 2.6 2.6 6.7 0.2 0.5 0.7 2.7 3.8 2.7 0.7 0.5 1.9 0.4 1.0 1.2 3.9 5.7 4.2 3.1 1.0 1.2 1.2 1.0 0.4 1.0 1.2 4.2 3.9 5.7 Disease…… Non-Disease Non-Disease Non-Disease Disease…… Disease…… TS NS HK TS NS HK 3.1 1.0 2.6 2.6 7.4 9.7 7.4 2.6 2.6 6.7 0.2 0.5 0.7 2.7 3.8 2.7 0.7 0.5 1.9 0.4 1.0 1.2 3.9 5.7 4.2 3.1 1.0 1.2 1.2 1.0 0.4 1.0 1.2 4.2 3.9 5.7 Disease…… Non-Disease Non-Disease Non-Disease Disease…… Disease…… TS NS HK TS NS HK Relating ge Figure 4 Relating gene connectivity with disease association and tissue specificity Figure 4 Relating gene connectivity with disease association and tissue specificity. Percentage of gene-gene interactions that exists between two groups of genes depending on their tissue specificity (TS: tissue-specific, NS: non-specific, and HK: house- keeping) and disease association. Colours indicate interactions between two disease-associated genes (red), between a disease- associated and a non-disease-associated gene (yellow), and between two non-disease-associated genes (green). The size of the rectangles indicates the relative number of interacting genes in each group. parameter in our guilt-by-association algorithm obviates the need to also include tissue specificity. members of this list will ultimately be confirmed as mod- ifiers of various genetic diseases. However, it should also be acknowledged that probability values associated with testing the null hypothesis of a given PCC not being statistically different from zero were computed assuming asymptotic normality and as such are prone to inaccuracies. With this in mind, we focussed on combining discrete parameters such as number of connec- tions and the association to disease-associated genes to identify a group of genes, not previously confirmed as dis- ease causing, that are involved in interactions with disease causing genes. The nature of these newly identified inter- actions could range from epistatic interactions (i.e., the action of one gene is suppressed by another such as the case of RAD51 and BRCA1) to physical gene-gene interac- tions to correlated co-expression. Based on bibliographi- cal validation and network re-construction we have identified several candidate genes that may impact the development of cancer and hypothesize that many other Finally, it should be noted that while new algorithms are being proposed in the literature on a rather frantic pace, the task of comprehensively comparing algorithms could be unattainable if not futile. Conclusion breast cancer susceptibility genes, BRCA1 and BRCA2. Although none of these genes are annotated as disease causing in OMIM, five have been previously associated with the development of breast cancer, for example, alle- les of RAD51 are epistatic with alleles of BRCA2. However, CCNA2 is only mentioned in a very small number of reports on breast cancer and APBA2BP is not a well stud- ied gene. Data mining approaches have allowed us to gain an insight into the complex relationships existing between gene expression, disease association, network connectivity and tissue specificity. We have identified elevated rates of expression and network connectivity among broadly expressed genes, and among disease-associated tissue-spe- cific genes. In particular, when exploring the relationship between tis- sue specificity and disease association, we found this rela- tionship most interesting. While there is a moderate positive relationship between the number of tissues in which a gene is expressed and the proportion of disease genes, we show that this relationship is reversed when only considering genes for which interactions have been reported. We present this phenomenon as an example of the well-reported Simpson's Paradox. To a great extent, the inclusion of number of interactions as a threshold For the case of gastric cancer, another cluster of seven genes (AKT3, KRAS, MAP2K4, PIK3CB, PLCB1, PIK3R5 and PPP3R2) was identified. Four of these genes have been previously associated with gastrointestinal disease while AKT3, PIK3CB and PIK3R5 have not, although the differential expression of AKT3 in gastric cancer is well defined [see Additional file 2]. We suggest these previ- ously non-associated genes are strong candidates for fur- ther study into the basis of these diseases and are potential prognostic markers. Page 7 of 11 (page number not for citation purposes) Page 7 of 11 (page number not for citation purposes) http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Relating gene connectivity with disease association and tissue specificity Figure 4 Relating gene connectivity with disease association and tissue specificity. Percentage of gene-gene interactions that exists between two groups of genes depending on their tissue specificity (TS: tissue-specific, NS: non-specific, and HK: house- keeping) and disease association. Colours indicate interactions between two disease-associated genes (red), between a disease- associated and a non-disease-associated gene (yellow), and between two non-disease-associated genes (green). The size of the rectangles indicates the relative number of interacting genes in each group. Conclusion Instead, we claim that our conservative thresholds for predicting disease association is justified because using thresholds of known disease genes increases our likelihood of success given any estima- tion process is going to have a degree of false positives. We acknowledge the list does not exhaust all possible disease genes but merely gives researchers the best short list for further study. Page 8 of 11 (page number not for citation purposes) Competing interests The authors declare that they have no competing interests. Abbreviations HK: housekeeping; MPSS: massively parallel signature sequencing; NS: non-specific; PCC: Pearson correlation coefficient; TS: tissue-specific; DIS: genes in our dataset that are disease-associated according to OMIM as of Sep- tember 19, 2007; NDIS: genes in our dataset that are non- Page 8 of 11 (page number not for citation purposes) Page 8 of 11 (page number not for citation purposes) BioData Mining 2008, 1:8 Table 2: Contingency table underlying the guilt-by-association algorithm Disease Associated? Number of Connections % Disease-associated genes among interactors ≤ 75 > 75 Yes ≤ 12 3,112 1,907 > 12 481 651 No ≤ 12 7,853 705 > 12 229 112 Number of disease- and non-disease-associated genes by thresholds on number of connections and percentage of disease-associated genes among interactors. The thresholds are obtained from exploring disease-associated genes and correspond to the average number of connections (12) among disease-associated genes and the average proportion of disease-associated genes (75%) among their interactors. The 112 non-disease- associated genes (bottom right cell) form the basis of the newly reported disease-associated genes [see Additional file 2]. Table 2: Contingency table underlying the guilt-by-association algorithm Number of disease- and non-disease-associated genes by thresholds on number of connections and percentage of disease-associated genes among interactors. The thresholds are obtained from exploring disease-associated genes and correspond to the average number of connections (12) among disease-associated genes and the average proportion of disease-associated genes (75%) among their interactors. The 112 non-disease- associated genes (bottom right cell) form the basis of the newly reported disease-associated genes [see Additional file 2]. disease-associated genes also according to OMIM; INT: genes in our dataset for which interactions have been reported; NINT: genes in our dataset for which interac- tions have not been reported. Authors' contributions AR conceived the study, carried out the data mining approaches and drafted the manuscript. AI directed the design and coordination of the biological/immunological relevance of the results and drafted the manuscript. BD participated in the coordination of the whole study and drafted the manuscript. All authors read and approved the final manuscript. Table 3: Precision analysis of the guilt-by-association algorithm Threshold for number of connections (TC) Threshold for % disease genes among interactors (TD) Q1 TC = 1 Q2 TC = 4 Q3 TC = 13 Mean TC = 12 Q1 TD = 12.8 N Captured 1,943 1,391 638 683 % Known 73.3 75.0 76.5 76.4 Q2 TD = 28.6 N Captured 1,024 563 195 219 % Known 74.8 78.9 85.1 84.9 Q3 TD = 50.0 N Captured 251 118 16 19 % Known 70.5 67.8 75.0 78.9 Mean TD = 35.0 N Captured 748 409 109 127 % Known 73.4 76.3 84.4 84.2 The optimality of various location parameters to be used as thresholds in the guilt-by-association algorithm was explored by computing the proportion of known (% Known) disease associated genes from the total number of captured genes (N Captured). The analysis was performed using only the 1,445 genes (out of the initial 6,151) with known disease phenotype as the set of truly disease causing, and with the remaining 4,706 declared as disease associated. The three inter-quartiles (Q1: 25th percentile; Q2: 50th percentile or median; and Q3: 75th percentile) plus the mean were used as thresholds. Table 3: Precision analysis of the guilt-by-association algorithm Threshold for number of connections (TC) The optimality of various location parameters to be used as thresholds in the guilt-by-association algorithm was explored by computing the proportion of known (% Known) disease associated genes from the total number of captured genes (N Captured). The analysis was performed using only the 1,445 genes (out of the initial 6,151) with known disease phenotype as the set of truly disease causing, and with the remaining 4,706 declared as disease associated. The three inter-quartiles (Q1: 25th percentile; Q2: 50th percentile or median; and Q3: 75th percentile) plus the mean were used as thresholds. 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Additional file 2 ™ 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 22. McKusick VA: Online Mendelian Inheritance in Man, OMIM™. [http://www.ncbi.nlm.nih.gov/Omim]. 23. Simpson EH: The interpretation of interaction in contingency tables. J Royal Stat Soc Ser B 1951, 13:238-241. J y 24. Goh K, Cusick ME, Valle D, Childs B, Vidal M, Barabási AL: The human disease network. Proc Natl Acad Sci USA 2007, 104:8685-8690. 25. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T: Cytoscape: a software environment for integrated models of biomolecular interaction networks. 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https://refubium.fu-berlin.de/bitstream/fub188/28908/1/ZSE_article_55733_en_1.pdf
English
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New insights gained from museum collections: Deep-sea barnacles (Crustacea, Cirripedia, Thoracica) in the Muséum National d’Histoire Naturelle, Paris, collected during the Karubar expedition in 1991
Zoosystematics and evolution/Mitteilungen aus dem Museum für Naturkunde in Berlin. Zoologische Reihe
2,020
cc-by
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Abstract An examination of the deep-sea barnacles (Cirripedia, Thoracica) collected by the Karubar expedition to Indonesia (1991) and deposited in the Muséum National d’Histoire Naturelle, Paris, identified 40 species contained in three families of stalked and five families of acorn barnacles. Information on these species is presented, including descriptions, updated distributions and images to aid species identification. Thirty of the species, treated herein, are new records for the Indonesian Kei Islands and Tanimbar Island, which increases the total number of species recorded from Kei Islands, Aru Island and Tanimbar Island to 40. This study demonstrates the value of museum collections as a resource in biodiversity science. New insights gained from museum collections: Deep-sea barnacles (Crustacea, Cirripedia, Thoracica) in the Muséum National d’Histoire Naturelle, Paris, collected during the Karubar expedition in 1991 Pipit Pitriana1,2,5, Diana S. Jones3, Laure Corbari4, Kristina von Rintelen1 1 Museum für Naturkunde – Leibniz Institute for Evolution and Biodiversity Science, Invalidenstrasse 43, 10115, Berlin, Germany 2 Research Centre for Deep-sea, Indonesian Institute of Science (LIPI), Jl. Y. Syaranamual, Poka, Tlk. Ambon, Kota Ambon, Maluku, Indonesia 3 The Western Australian Museum, 49 Kew Street, Welshpool WA 6106, Locked Bag 49, Welshpool DC WA 6986, Australia 4 Muséum national d’Histoire naturelle, Institut de Systématique, Évolution, Biodiversité ISYEB – UMR 7205 – CNRS, MNHN, UPMC, EPHE, 57 rue Cuvier, CP 26, 75005, Paris, France 5 Institute of Geological Sciences, Freie Universität Berlin, Malteserstrasse 74–100 Building C and D, 12249, Berlin, Germany 5 Institute of Geological Sciences, Freie Universität Berlin, Malteserstrasse 74–100 Building C and D, 12249, Berlin, Germany http://zoobank.org/DF25E94F-EDEC-4FD4-BA1D-E4AC288282AD Corresponding author: Pipit Pitriana (pipit.pitriana@mfn.berlin) Academic editor: Martin Husemann  ♦  Received 23 June 2020  ♦  Accepted 13 August 2020  ♦  Published 28 September 2020 Academic editor: Martin Husemann  ♦  Received 23 June 2020  ♦  Accepted 13 August 2020  ♦  Published 28 Sep Key Words acorn barnacles, Indonesian biodiversity, new record, stalked barnacles, taxonomy Copyright Pipit Pitriana 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. Zoosyst. Evol. 96 (2) 2020, 649–698  |  DOI 10.3897/zse.96.55733 Zoosyst. Evol. 96 (2) 2020, 649–698  |  DOI 10.3897/zse.96.55733 New insights gained from museum collections: Deep-sea barnacles (Crustacea, Cirripedia, Thoracica) in the Muséum National d’Histoire Naturelle, Paris, collected during the Karubar expedition in 1991 Introduction present). This programme was a collaboration between the Muséum National d’Histoire Naturelle (MNHN), Par­ is and the Institut de Recherche pour le Développement (IRD) (formerly ORSTOM), to explore the deep-sea fau­ na of the tropical Indo-Pacific. As the programme was inspired and guided by carcinologists, it is not surprising that ~ 33% of the papers resulting from these cruises con­ cern crustaceans, especially crabs, lobsters and shrimps (Richer de Forges et al. 2013). In 1991, scientists from France and Indonesia conducted collaborative research through the Karubar expedition in Indonesia. The acronym for this expedition, which collect­ ed the material reported on herein, is a contraction of the names of the Kei, Aru and Tanimbar Islands. These Islands attracted attention after Professor Th. Mortensen’s Danish expedition to the Kei Islands (1914–16). Mortensen sug­ gested that the Islands were an ideal place for a marine laboratory to study deep-sea fauna, as he had found stalked crinoids, elasipods and other abyssal creatures at depths of 200–400 m around the Kei Islands (Crosnier et al. 1997). Publications on the thoracic barnacles collected by the Karubar expedition are those of Buckeridge (1994, 1997) who focused on barnacles from the suborder Verru­ comorpha. He reported six species of verrucomorph bar­ nacles: Newmaniverruca albatrossiana (Pilsbry, 1912), Altiverruca navicula (Hoek, 1913), Brochiverruca dens The Karubar expedition was part of the MUSOR­ STOM-Tropical Deep-Sea Benthos programme (1976– Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 650 (Broch, 1932), Metaverruca recta (Aurivillius, 1898), Rostratoverruca intexta (Pilsbry, 1912) and R. kruegeri (Broch, 1922). Here, we present a study of the remaining barnacle material collected by the Karubar expedition, to cover the entire taxonomic range of deep-sea barnacles collected by this expedition. two genera and species; Pachylasmatidae: one genus and species; Archaeobalanidae: three genera, six species; Pyr­ gomatidae: one genus and species; Balanidae: one genus and species). A list of the species of Cirripedia collected during the Karubar expedition and deposited in the Muséum National d’Histoire Naturelle (MNHN) Paris is present­ ed, including additional information on substrate, as well as the depth where the sample was found (Suppl. material 1: Table S1). Material and methods In total, 459 specimens were studied at the Muséum Na­ tional d’Histoire Naturelle (MNHN), of which 89 speci­ mens were studied in detail at the Museum für Naturkunde (MfN), Berlin, Germany. Species were determined on ex­ ternal shell morphology and mouthpart and arthropodal characters, as described in Darwin (1852, 1854), Hoek (1883, 1907, 1913), Pilsbry (1890, 1907, 1916), Annan­ dale (1905, 1906, 1908, 1909, 1910, 1913, 1916), Broch (1916, 1922, 1931), Foster (1974, 1978, 1980, 1981), Newman and Ross (1971, 1976), Rosell (1981, 1989, 1991), Calman (1918, 1919), Ren (1989), Chan et al. (2009, 2010) and Shalaeva and Boxshall (2014). Genus Heteralepas Pilsbry, 1907 Alepas Darwin, 1852: 156, pl. III, figs 5, 6. Heteralepas (Heteralepas) Pilsbry, 1907a: 100. Heteralepas s. str. – Newman, 1960: 109. Alepas Darwin, 1852: 156, pl. III, figs 5, 6. Heteralepas (Heteralepas) Pilsbry, 1907a: 100. Heteralepas s. str. – Newman, 1960: 109. Type genus. Heteralepas Pilsbry, 1907a. Type genus. Heteralepas Pilsbry, 1907a. Remarks. The family consists of two genera: Heterale­ pas Pilsbry, 1907a and Paralepas Pilsbry, 1907a. Suborder HETERALEPADOMORPHA New­ man, 1987 Diagnosis. Capitulum and peduncle without calcareous armament. According to Buhl-Mortensen and Mifsud (2017), Heteralepadomorpha consists of seven families of poorlyknown Pedunculata. In the “Taxonomic account” section, the diagnosis is followed by description, type species and localities and known distributions for each species are given. Each species is listed under its current valid binomen and all known synonyms are also provided. Photographs of each species are provided to facilitate their recognition. Family HETERALEPADIDAE Nilsson-Cantell, 1921 Heteralepadidae Nilsson-Cantell, 1921: 245 Lepadidae Darwin, 1852: 8 (part.) In the “Material examined” section, there are four station codes dependent on the type of dredge used and are listed as follows: DW for dredge Warén, ED (drague épibenthique) for epibenthic dredge, CP (chalut à perche) for beam trawl and CC (chalut à crevettes) for otter trawl (shrimp). Diagnosis. Capitulum naked, thick-walled, globular, sup­ ported by stout peduncle; filamentary appendage at base of cirrus I; caudal appendage multi-segmented; cirri cten­ opod or acanthopod, rarely lasiopod. Measurements were made using digital callipers (ac­ curate to 0.1 mm) as follows: for acorn barnacles, bas­ al length of shell (LB), basal width (WB), orifice length (LO), orifice width (WO) and carinal height (H); for stalked barnacles, total height (TH), capitular height (CH), diameter of base of capitulum (DBC), distance be­ tween carina and scutum (CS), scutal length (LS), scutal width (WS), tergal length (LT) and tergal width (WT). Taxonomic account Class HEXANAUPLIA Oakley, Wolfe, Lind­ gren & Zaharoff, 2013 Subclass THECOSTRACA Gruvel, 1905 Infraclass CIRRIPEDIA Burmeister, 1834 (= Cirrhipèdes Lamarck, 1806) Superorder THORACICA Darwin, 1854 zse.pensoft.net Order LEPADIFORMES Buckeridge & New­ man, 2006 Hard body parts, such as the shell (parietes) and the opercular plates, were studied using a Leica M125 stereo­ microscope. Soft body parts, such as mouthparts and cir­ ri, were studied using an Axioskop 20 light microscope. Mouthparts and cirri were mounted on glass slides and examined under a light microscope. Images were taken using a stereomicroscope with a digital camera (Leica Microsystems M205C and Leica Z16 APo-A). Suborder HETERALEPADOMORPHA New­ man, 1987 Heteralepas japonica (Aurivillius, 1892) Figure 1 Alepas japonica Aurivillius, 1892: 125. – 1894: 28, pl. II figs 14, 15, pl. VIII, figs 3, 7, pl. IX, fig. 3. Alepas indica Gruvel, 1901: 259. – 1905a: 162, fig. 179. Heteralepas (Heteralepas) japonica. – Pilsbry, 1907a: 101. Heteralepas (Heteralepas) japonica var. alba Krüger, 1911: 34, pl. 1, fig. 2b. Heteralepas (Heteralepas) dubia Broch, 1922: 288, fig. 38. Heteralepas japonica. – Pilsbry 1911: 71, fig. 4. – Zevina et al. 1992: 31, fig. 19. – Chan et al. 2009a: 61. – Chan et al. 2009b: 88–91, figs 2A–D, 3A–D, 4, 5. – Pitriana et al. 2020: 12, fig. 3. Type locality. Hirado Strait, Japan, 146 m depth (Auriv­ illius 1892). Genus Paralepas Pilsbry, 1907 Material examined. Tanimbar Island: 1 specimen, MNHN-IU-2019-4877, stn. CP 46, 08°01'S, 132°51'E, 271–273 m depth, 29 October 1991. Alepas Darwin, 1852: 156 (part.). Heteralepas (Paralepas) Pilsbry, 1907a: 100, fig. 34A. Paralepas s. str. – Newman, 1960: 108. Diagnosis. Capitulum rounded, plates absent; wall of ca­ pitulum thick; orifice crenulated with no more than two crests on carinal region; cirrus VI with caudal appendage; maxillule strongly notched. Diagnosis. Maxillule slightly notched; filamentary ap­ pendages well-developed; posterior and anterior rami of cirri V and VI similar in length; segments of cirri V and VI square-shaped, each armed with semi-circular brush of many setae on anterior face. Description. Capitulum and peduncle yellowish. Orifice slightly protuberant, crenulated, occupying one half to one third capitular length, parallel to or at oblique angle to capitulum; integument thick, chitinous. Cirrus I with anterior rami (20-segmented) shorter than posterior rami (25-segmented); cirrus VI with long caudal appendage, 24-segmented, 2/3 length of anterior ramus. Mandibles with four large teeth excluding inferior angle; maxillule strongly notched, two large setae at upper angle, blade- shaped setae on cutting margin; labrum concave with nu­ merous teeth. Measurements of specimen: height of ca­ Type species. Alepas percarinata Pilsbry, 1907c: 185, fig. 2, pl. IV, fig. 8; type locality: Molokai, Hawaiian waters. Type species. Alepas percarinata Pilsbry, 1907c: 185, fig. 2, pl. IV, fig. 8; type locality: Molokai, Hawaiian waters. Results Morphological analyses of all the samples revealed 40 species from three families of stalked barnacles (Heter­ alepadidae: two genera, three species; Poecilasmatidae: four genera, five species; Scalpellidae: twelve genera, 21 species) and five families of acorn barnacles (Verrucidae: Diagnosis. Filamentary appendage present at base of cir­ rus I, short; posterior rami of cirri V and VI shorter and more slender than anterior rami. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 651 pitulum 18.50 mm, width 12.82 mm, thickness 5.97 mm; length of peduncle 15.26 mm, width 5.55 mm. pitulum 18.50 mm, width 12.82 mm, thickness 5.97 mm; length of peduncle 15.26 mm, width 5.55 mm. Type species. Alepas rex Pilsbry, 1907c: 186, fig. 3, pl. IV fig. 7; type locality: Kauai Island, Hawaiian waters. Distribution. Singapore; Indo-west Pacific: Indian Ocean; Australia; Malay Archipelago; Vietnam; Condor Island; South China Sea; East China Sea; Taiwan, Philip­ pines; South Japan; NE New Zealand; fouling hard rock substrata, crabs, gorgonians, antipatharians, deep-sea ca­ bles; 48–500 m depth (Jones and Hosie 2016). Recently, the species has also been recorded from the Lifamatola Sea and the Halmahera Sea, Indonesia (Pitriana et al. 2020). In this study, Heteralepas japonica was found at Tanimbar Island, Indonesia. Type locality. Mediterranean Sea (Chan et al. 2009b). Paralepas minuta. – Stubbings 1967: 240. – Newman 1960: 109. – Chan et al. 2009b: 66, figs 55–57. Paralepas minuta. – Stubbings 1967: 240. – Newman 1960: 109. – Chan et al. 2009b: 66, figs 55–57. Remarks. Paralepas minuta (Philippi, 1836) can be dis­ tinguished from Paralepas ovalis (Hoek, 1907) by the presence of triangular scutal areas and from Paralepas nodulosa Broch, 1922 by the absence of “well defined, scanty, almost thorn-like warts” (Broch 1922). Material examined. – Kei Islands: 6 specimens, MNHN- IU-2019-4882, Stn. CP 25, 05°30'S, 132°52'E, 336–346 m depth, 26 October 1991, attached to dead stem of gorgonian. – Tanimbar Island: 4 specimens, MNHN-IU-2019-4883, Stn. CP 85, 09°22'S, 131°14'E, 240–245 m depth, 4 No­ vember 1991, attached to spine of sea urchin. – Tanimbar Island: 4 specimens, MNHN-IU-2019-4883, Stn. CP 85, 09°22'S, 131°14'E, 240–245 m depth, 4 No­ vember 1991, attached to spine of sea urchin. Paralepas minuta (Philippi, 1836) Figure 2 Figure 2 Alepas minuta Philippi, 1836: 254, pl. 12, fig. 23. Alepas (Paralepas) minuta. – Weltner, 1897: 239. Figure 1. Heteralepas japonica (Aurivillius, 1892) (MNHN-IU-2019-4877). a. left lateral view showing the capitulum and pedun­ cle; b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, capitulum and peduncle. Scale bar: 6 mm (a–d). Figure 1. Heteralepas japonica (Aurivillius, 1892) (MNHN-IU-2019-4877). a. left lateral view showing the capitulum and pedun­ cle; b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, capitulum and peduncle. Scale bar: 6 mm (a–d). zse.pensoft.net 652 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 2. Paralepas minuta (Philippi, 1836) (MNHN-IU-2019-4882). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. anterior view, orifice. Scale bar: 5 mm (a–c). Figure 2. Paralepas minuta (Philippi, 1836) (MNHN-IU-2019-4882). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. anterior view, orifice. Scale bar: 5 mm (a–c). Heteralepas (Paralepas) minuta. – Broch, 1927b: 18, fig. 4, pl. 1, figs 1, 2. Paralepas minuta nipponica Utinomi, 1970: 342. Paralepas minuta. – Stubbings 1967: 240. – Newman 1960: 109. – Chan et al. 2009b: 66, figs 55–57. Heteralepas (Paralepas) minuta. – Broch, 1927b: 18, fig. 4, pl. 1, figs 1, 2. Paralepas minuta nipponica Utinomi, 1970: 342. Type locality. Mediterranean Sea (Chan et al. 2009b). Genus Dianajonesia Koçak & Kemal, 2008 Temnaspis Fischer, 1884: 357. – Broch 1931: 30. Poecilasma (part). – Darwin, 1852: 99. – Pilsbry 1907a: 82. – Nils­ son-Cantell 1921: 253. Dianajonesia Koçak & Kemal, 2008: 2. Temnaspis Fischer, 1884: 357. – Broch 1931: 30. Paralepas morula (Hoek, 1907) Figure 3 Alepas morula Hoek, 1907: 35, pl. IV, figs 9–12. Heteralepas (Paralepas) morula. – Broch, 1922: 28, fig. 34. Paralepas morula. – Newman, 1960: 109. Diagnosis. Capitulum yellowish with globular shape; ante­ rior and posterior rami of cirrus V and VI similar in length. Description. Capitulum yellowish, globose, small-sized, externally smooth; orifice crenulated; peduncle rather short. Segments of rami of cirrus I wide, anterior ramus (9-segmented) shorter than posterior ramus (13-seg­ mented); cirrus VI with short caudal appendage, 8-seg­ mented. Penis annulated, setae sparsely distributed over most of surface, a few longer setae towards tip. Maxillule notched, two large and one small setae at upper angle; mandible with four major teeth; labrum concave, with numerous teeth. Measurements of specimen: height of capitulum 8.34 mm, width 6.67 mm, thickness 4.53 mm; length of peduncle 3.84 mm, width 2.60 mm. Material examined. – Tanimbar Island: 45 specimens, MNHN-IU-2019-4878, Stn. CP 54, 08°21'S, 131°43'E, 836–869 m depth, 30 October 1991, attached to spine of sea urchin. Material examined. – Tanimbar Island: 45 specimens, MNHN-IU-2019-4878, Stn. CP 54, 08°21'S, 131°43'E, 836–869 m depth, 30 October 1991, attached to spine of sea urchin. Diagnosis. Capitulum globular with numerous tubercles on surface, scuta absent, orifice small, not protuberant, peduncular length more than half capitular length. Description. Capitulum and peduncle yellowish. Capit­ ulum globular with thick, chitinous, large warts all over surface. Peduncle rather long, narrower than capitulum; surface bearing tubercles, smaller than those of capitulum. Cirrus I placed rather far from cirrus II; cirri II–VI similar, length and number of segments slightly increasing from second to sixth; cirrus VI with 7-segmented caudal append­ age. Penis indistinctly segmented, few long hairs towards tip. Maxillule notched, two large setae on upper notch; mandibles with three major teeth, inferior angle considered Distribution. Java Sea (Indonesia); Mediterranean Sea; West Africa; Indo-west Pacific: Indian Ocean; Australia; Malay Archipelago; Taiwan; Japan; Philippines; North­ ern New Zealand; 485–736 m depth; attached to spines of cidarids (Jones and Hosie 2016). In this study, Para­ lepas minuta was found at Kei Islands and Tanimbar Is­ land, Indonesia. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 653 Figure 3. Paralepas morula (Hoek, 1907) (MNHN-IU-2019-4878). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle. Scale bar: 3 mm (a, b). Figure 3. Paralepas morula (Hoek, 1907) (MNHN-IU-2019-4878). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle. Suborder LEPADOMORPHA Pilsbry, 1916 Poecilasma (part). – Darwin, 1852: 99. – Pilsbry 1907a: 82. – Nils­ son-Cantell 1921: 253. Poecilasma (part). – Darwin, 1852: 99. – Pilsbry 1907a: 82. – Nils­ son-Cantell 1921: 253. Lepadomorpha Pilsbry, 1916: 14 (Lepadidae sensu Darwin, 1852). Lepadoidea Darwin, 1852: 8 (nom. trans. Zevina 1978a, b). Dianajonesia Koçak & Kemal, 2008: 2. Diagnosis. Capitulum with five plates, approximate or variously reduced; scutum divided into two parts; apex of carina extending to, or slightly overlapping basal end of terga, basally terminating in disc. Paralepas morula (Hoek, 1907) Figure 3 Scale bar: 3 mm (a, b). Trilasmatidae Nilsson-Cantell, 1934: 40. – Hiro 1937a: 79. – Krüger 1940: 29. as fourth tooth; labrum with continuous row of numer­ ous short, strong, blunt teeth. Measurements of specimen: height of capitulum 5.14 mm, width 3.69 mm, thickness 1.81 mm; length of peduncle 5.08 mm, width 1.31 mm. Diagnosis. Capitulum with up to five plates, including tergum and scutum; scutum in some species split into two (resulting in seven plates); some or all plates may be degenerate or absent; umbos of terga apical, those of carina and scuta fundamentally basal; peduncle without calcareous scales; maxillule not stepped; cirrus I widely separated and much shorter than posterior cirri; caudal appendages uniarticulate, spinose. Diagnosis. Capitulum with up to five plates, including tergum and scutum; scutum in some species split into two (resulting in seven plates); some or all plates may be degenerate or absent; umbos of terga apical, those of carina and scuta fundamentally basal; peduncle without calcareous scales; maxillule not stepped; cirrus I widely separated and much shorter than posterior cirri; caudal appendages uniarticulate, spinose. Distribution. Flores Sea (Indonesia); Philippines; Bass Strait, N of Tasmania; attached to spines of echinoids, for example, Histocidaris elegans (Agassiz, 1879); 182– 538 m depth (Jones and Hosie 2016). In this study, Para­ lepas morula was found at Tanimbar Island, Indonesia. Type locality. Bali Sea, North of Lombok; 538 m depth (Hoek 1907). Type genus. Poecilasma Darwin, 1852: 99. Type genus. Poecilasma Darwin, 1852: 99. Type genus. Poecilasma Darwin, 1852: 99. Remarks. Most of the smaller specimens do not show the warts that are characteristic of this species. Type species. Poecilasma fissa Darwin, 1852: 109, pl. II, fig. 4, pl. X, fig. 29. Dianajonesia excavatum (Hoek, 1907) Figure 5 Poecilasma excavatum Hoek, 1907: 10, pl. I, figs 5–10. Poecilasma excavatum Hoek, 1907: 10, pl. I, figs 5–10. Poecilasma excavatum Hoek, 1907: 10, pl. I, figs 5–10. Poecilasma tridens. – Weltner 1922: 80, pl. 4, fig. 19 (non Aurivillius Poecilasma (Temnaspis) excavatum. – Nilsson-Cantell 1925: 16, fig. pl. 1, fig. 1. Material examined. – Tanimbar Island: 2 specimens, MNHN-IU-2019-4861, Stn. CP 79, 09°16'S, 131°22'E, 239–250 m depth, 3 November 1991, attached to crab leg. Diagnosis. Capitulum broadly oval, with five smooth plates; carina not extending to area between terga; pedun­ cle with circles of small protuberances; cirri short. Material examined. – Tanimbar Island: 2 specimens, MNHN-IU-2019-4861, Stn. CP 79, 09°16'S, 131°22'E, 239–250 m depth, 3 November 1991, attached to crab leg. Poecilasma tridens. – Weltner 1922: 80, pl. 4, fig. 19 (non Aurivillius).i Poecilasma (Temnaspis) excavatum. – Nilsson-Cantell 1925: 16, fig. 5, pl. 1, fig. 1. Trilasmis (Temnaspis) excavatum. – Hiro 1937b: 412. Diagnosis. Capitulum broadly oval, with five smooth plates; carina not extending to area between terga; pedun­ cle with circles of small protuberances; cirri short. Dichelaspis (Dichelaspis) tridens. – Stubbings 1936: 7, fig. 2. (non Au­ rivillius). Trilasmis excavatum. – Nilsson-Cantell 1938: 9. Temnaspis excavatum. – Broch 1931: 31, fig. 10. – Dong et al. 1982: 77. Description. Capitulum yellowish, with five tranparent, smooth plates. Scutum bilobed, occludent segment nar­ row, bow-shaped; tergum sub-triangular, nearly half as broad as long. Carina very narrow, slightly curved down­ wards, end blunt. Peduncle yellow, plainly ringed, spines absent. Cirrus I with anterior ramus wider than posterior ramus. Maxillule notched, with two strong teeth in upper part; mandible with four teeth, fourth pectinated, placed very close to inferior angle; labrum with row of minute teeth. Measurements of specimen: basal diameter of ca­ pitulum 0.94 mm; capitular height 7.03 mm; total height 10.40 mm; scutal width 4.11 mm; scutal length 5.85 mm; tergal width 1.00 mm; tergal length 3.13 mm. Temnaspis excavatum. – Broch 1931: 31, fig. 10. – Dong et al. 1982: 77. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Material examined. – Tanimbar Island: 3 specimens, MNHN-IU-2019-4859, Stn. CP 78, 09°06'S, 131°24'E, 284–295 m depth, 3 November 1991. Diagnosis. Family POECILASMATIDAE Annandale, 1910 Lepadidae Darwin, 1852: 8 (part.). – Pilsbry 1907a: 72 (part.). Lepadinae. – Gruvel, 1905: 104 (part.). Poecilasmatinae Annandale, 1910a: 84. Poecilasmatidae. – Nilsson-Cantell, 1921: 253. Lepadidae Darwin, 1852: 8 (part.). – Pilsbry 1907a: 72 (part.). Lepadinae. – Gruvel, 1905: 104 (part.). Poecilasmatinae Annandale, 1910a: 84. Poecilasmatidae. – Nilsson-Cantell, 1921: 253. Type species. Poecilasma fissa Darwin, 1852: 109, pl. II, fig. 4, pl. X, fig. 29. Type species. Poecilasma fissa Darwin, 1852: 109, pl. II, fig. 4, pl. X, fig. 29. zse.pensoft.net zse.pensoft.net 654 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Dianajonesia amygdalum (Aurivillius, 1894) Figure 4 Philippines; South Japan; tropical West and central Pacific Ocean to Fiji and Hawaii; attached to decapod crustaceans; shallow water (Jones and Hosie 2016). In this study, Diana­ jonesia amygdalum was found at Tanimbar Island, Indonesia. Philippines; South Japan; tropical West and central Pacific Ocean to Fiji and Hawaii; attached to decapod crustaceans; shallow water (Jones and Hosie 2016). In this study, Diana­ jonesia amygdalum was found at Tanimbar Island, Indonesia. Poecilasma amygdalum Aurivillius, 1894: 10, pl. 1, figs 4–6, pl. 8, fig. 4. – Nilsson-Cantell 1921: 262, fig. 46d–g, pl. 3, fig. 6. Poecilasma fissum (non Darwin). – Hoek, 1907: 8 (part.). Trilasmis fissum hawaiense. – Pilsbry, 1928: 306, pl. 24, figs 1–8. Trilasmis (Temnaspis) amygdalum. – Hiro, 1937a: 85, fig. 69. Temnaspis amygdalum amygdalum. – Zevina, 1982: 70, fig. 62. Temnaspis amygdalum. – Utinomi, 1966: 5. – Dong et al. 1982: 77. Dianajonesia amygdalum. – Koçak & Kemal, 2008: 2. Type locality. Nordwachter Island, Thousand Islands, Java Sea (Aurivillius 1894). Dianajonesia excavatum (Hoek, 1907) Figure 5 Capitulum with five plates; scutum with two distinct segments, united at rostral angle; carina generally extending up between terga, terminating downwards as imbedded disc (fork- or cup-shaped). Type species. Octolasmis warwicki Gray, 1825: 100. – 1830: pl. VI, fig. 16. Type species. Octolasmis warwicki Gray, 1825: 100. – 1830: pl. VI, fig. 16. Dichelaspis orthogonia Darwin, 1852 Figure 6 – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell 1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2, figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings 1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26, fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Type locality. Siboga station 253; 5°48.2'S, 132°15'E; depth: 304 m; bottom: grey clay, hard and crumbly (Hoek 1907). Material examined. – Kei Islands: 4 specimens, MNHN-IU-2019-4864, Stn. CP 27, 05°33'S, 132°51'E, 314–304 m depth, 27 October 1991. – Tanimbar Island: 6 specimens, MNHN- IU-2019-4862, Stn. DW 24, 05°32'S, 132°51'E, 230– 243 m depth, 26 October 1991. Material examined. – Kei Islands: 4 specimens, MNHN-IU-2019-4864, Stn. CP 27, 05°33'S, 132°51'E, 314–304 m depth, 27 October 1991. Dianajonesia excavatum (Hoek, 1907) Figure 5 Capitulum with scutum divided into two seg­ ments parallel to occludent margin; tergum with excava­ tion in scutal margin near occludent margin, receiving tip of occludent segment of scutum. Description. Capitulum oval, apex pointed, slightly thick, swollen. Scutum with larger segment strongly bowed, basal margin short, apex pointed; smaller segment bowed, termi­ nating in point at base, tergal margin rounded, fitting exactly Distribution. Indo-west Pacific: Indian Ocean; Madagascar through Malaysia, Hong Kong, South China Sea; Taiwan; Figure 4. Dianajonesia amygdalum (Aurivillius, 1894) (MNHN-IU-2019-4861). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 4. Dianajonesia amygdalum (Aurivillius, 1894) (MNHN-IU-2019-4861). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 655 Figure 5. Dianajonesia excavatum (Hoek, 1907) (MNHN-IU-2019-4859). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 5. Dianajonesia excavatum (Hoek, 1907) (MNHN-IU-2019-4859). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Octolasmis Gray, 1825: 100. – Pilsbry 1907a: 93. Octolasmis Gray, 1825: 100. – Pilsbry 1907a: 93. into excavation of tergum. Tergum triangular, characteristic excavation at scutal margin near occludent margin. Carina narrow, terminating in spatula-shaped disc. Cirrus I with anterior and posterior rami subequal (each five-segment­ ed); cirri II–VI longer, more slender; cirrus VI with caudal appendages. Penis thick, ringed, especially mid-length, ter­ minating in narrower, curved part. Maxillule notched, two large teeth on upper side; mandible with four teeth, largedis­ tance between first and second teeth. Labrum convex, with numerous blunt teeth. Measurements of specimen: basal diameter of capitulum 1.06 mm; capitular height 7.12 mm; total height 12.69 mm; scutal width 3.08 mm; scutal length 6.09 mm; tergal width 1.30 mm; tergal length 3.47 mm. Diagnosis. Capitulum with five plates; scutum with two distinct segments, united at rostral angle; carina generally extending up between terga, terminating downwards as imbedded disc (fork- or cup-shaped). Diagnosis. Dichelaspis orthogonia Darwin, 1852 Figure 6 Dichelaspis orthogonia Darwin, 1852: 130, pl. II, fig. 10a, b. Dichelaspis versluysi Hoek, 1907: 28, pl. III, figs 8–13. Octolasmis (Dichelaspis) orthogonia. – Pilsbry, 1907a: 94. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11, figs 6, 7. – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell 1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2, figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings 1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26, fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Dichelaspis orthogonia Darwin, 1852: 130, pl. II, fig. 10a, b. Dichelaspis orthogonia Darwin, 1852: 130, pl. II, fig. 10a, b. Distribution. Indo-west Pacific: East coast of Africa (Zanzibar); Gulf of Aden; Indonesia; Malay Archipela­ go; South China Sea; East China Sea; Philippines; South Japan (Goto Island); attached to echinoid spines, crusta­ ceans; palinurids and cirripedes; 189–600 m depth (Jones and Hosie 2016). In this study, Dianajonesia excavatum was found at Tanimbar Island, Indonesia. Dichelaspis versluysi Hoek, 1907: 28, pl. III, figs 8–13. Dichelaspis versluysi Hoek, 1907: 28, pl. III, figs 8–13. Octolasmis (Dichelaspis) orthogonia. – Pilsbry, 1907a: 94. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11, figs 6, 7. – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell 1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2, figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings 1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26, fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11, figs 6, 7. – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell 1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2, figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings 1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26, fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11, figs 6, 7. Type locality. Unknown. Description. Capitulum and peduncle yellowish. Scutum with basal segment narrow, L-shaped; tergum triangular, three prominent ridges at scutal margin; base of carina crescent-formed cup. Cirrus I with anterior ramus (6-seg­ mented) shorter than posterior ramus (8-segmented); cir­ rus VI with small caudal appendage. Maxillule notched, three large setae on upper notch; mandible with four teeth, inferior angle produced into single strong spine; labrum concave, with numerous teeth. Measurements of specimen: basal diameter of capitulum 1.89 mm; capitu­ lar height 8.69 mm; total height 10.47 mm; scutal width 3.5 mm; scutal length 7.16 mm; tergal width 0.80 mm; tergal length 6.53 mm. Remarks. The type locality of this species is unknown. The species was re-discovered in the Malay Archipelago by the Siboga Expedition in 40–112 m depth (Hoek 1907) and the Albatross Expedition, off Kagoshima Gulf, Japan, in 87 m depth (Pilsbry 1907d). Genus Dichelaspis Darwin, 1852 – Tanimbar Island: 6 specimens, MNHN- IU-2019-4862, Stn. DW 24, 05°32'S, 132°51'E, 230– 243 m depth, 26 October 1991. Dichelasmis Darwin, 1852: 115. Heptalasmis. Agassiz, 1842: 178. zse.pensoft.net 656 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 6. Dichelaspis orthogonia Darwin, 1852 (MNHN-IU-2019-4864). а. left lateral view capitulum and peduncle; b. right lateral view, capitulum and peduncle. Scale bar: 2 mm (a–b). Figure 6. Dichelaspis orthogonia Darwin, 1852 (MNHN-IU-2019-4864). а. left lateral view capitulum and peduncle; b. right lateral view, capitulum and peduncle. Scale bar: 2 mm (a–b). Diagnosis. Capitulum flattened; five plates, separated by thin membrane; scutum consisting of two distinct seg­ ments, with point of junction perfectly calcified; base of carina oval. sometimes fouling; 14–818 m depth (Jones and Hosie 2016). In this study, Dichelaspis orthogonia was found at Kei Islands and Tanimbar Island, Indonesia. Type species. Megalasma (Glyptelasma) subcarinatum Pilsbry, 1907a: 91, pl. VII, figs 1–5; type locality: Atlan­ tic Ocean, East of New Jersey, USA. Genus Glyptelasma Pilsbry, 1907 Glyptelasma Pilsbry, 1907a: 87. Glyptelasma gracile (Hoek, 1883) Figure 7 long hairs towards tip. Maxillule notched, with strong teeth on upper side; mandible with four teeth; labrum with row of small teeth on crest. Measurements of spec­ imen: basal diameter of capitulum 1.45 mm; capitular height 10.44 mm; total height 13.99 mm; scutal width 4.79 mm; scutal length 9.01 mm; tergal width 1.87 mm; tergal length 5.07 mm. Poecilasma gracile Hoek, 1883: 46, pl. II, figs 2–4. Megalasma gracile gracilius. – Pilsbry, 1907a: 88, pl. V, fig. 16, pl. VII, figs 6–9. Megalasma gracile. – Nilsson-Cantell 1938: 10. Megalasma (Glyptelasma) gracile. – Zevina 1982: 84, fig. 74. Glyptelasma gracile. – Jones et al. 2001: 239. Poecilasma gracile Hoek, 1883: 46, pl. II, figs 2–4. Poecilasma gracile Hoek, 1883: 46, pl. II, figs 2–4. Megalasma gracile gracilius. – Pilsbry, 1907a: 88, pl. V, fig. 16, pl. VII, figs 6–9. Megalasma gracile. – Nilsson-Cantell 1938: 10. Megalasma (Glyptelasma) gracile. – Zevina 1982: 84, fig. 74. Glyptelasma gracile. – Jones et al. 2001: 239. Megalasma gracile. – Nilsson-Cantell 1938: 10. Megalasma (Glyptelasma) gracile. – Zevina 1982: 84, fig. 74. Glyptelasma gracile. – Jones et al. 2001: 239. Distribution. West-southwest Pacific: from Australia (Sydney) to Indonesia (Sumbawa) and the Philippines (northwest Panay Island); East Indian Ocean; attached to glassy spicule of hexactinellid sponge, fragments of sea urchin tests, spines of cidarids; 395–935 m depth (Jones and Hosie 2016). In this study, Glyptelasma gracile was found at Tanimbar Island, Indonesia. Material examined. – Tanimbar Island: 14 specimens, MNHN-IU-2019-4873, Stn. CP 54, 08°21'S, 131°43'E, 836–869 m depth, 30 October 1991, attached to spine of sea urchin; 1 specimen, MNHN-IU-2019-4875, Stn. CC 57, 08°19'S, 31°53'E, 603–620 m depth, 31 Octo­ ber 1991; 3 specimens (1 adult, 2 juveniles), MNHN- IU-2019-4876, Stn. CP 73, 08°29'S, 131°33'E, 840– 855 m depth, 2 November 1991, attached to spines of sea urchins. Type locality. Challenger expedition Station 164a (off Sydney, Australia); 34°13'S, 151°38'E; depth 125 m; bot­ tom: grey ooze (Hoek 1883). Diagnosis. Capitulum with five plates; carina enlarged, keel-shaped; tergum with basal point truncated; max­ illule notched; caudal appendages with relatively-long spines at tip. Glyptelasma Pilsbry, 1907a: 87. Diagnosis. Capitulum with five approximate, fully cal­ cified plates; scutum quadrangular, umbone subcentral; carina becoming larger towards base; peduncle short. Distribution. Indo-west Pacific: East and South African Coast, Indian Ocean, Australia, Indonesia, Malay Ar­ chipelago, Vietnam, South China Sea, East China Sea, Philippines, Taiwan, South Japan; attached to hydroid, gorgonians, antipatharians, sea urchin spines, nylon cord, zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 657 Type species. Megalasma striatum Hoek, 1883: 51, pl. II, figs 5–9, pl. VII, figs 8, 9. Genus Megalasma Hoek, 1883 Megalasma Hoek, 1883: 50. – Pilsbry 1907a: 87. Megalasma Hoek, 1883: 50. – Pilsbry 1907a: 87. Figure 8 Figure 8 Megalasma striatum Hoek, 1883: 51, pl. II, figs 5–9, pl. VII, figs 8, 9. – Broch 1931: 270, figs 29, 30. Megalasma (Megalasma) elegans. – Zullo & Newman, 1964: 355, fig. 2a–i. Megalasma (Megalasma) striatum. – Zevina 1982: 80, fig. 71. – Chan 2009: 66, figs 2A, 16 A–H. Distribution. West-southwest Pacific, Indo-west Pacific, East coast of Africa, Indian Ocean, north Australia, Indo­ nesia, Malay Archipelago, East China Sea, South China Sea, Taiwan, Philippines, south Japan to New Zealand; attached to echinoid spines, antipatharians, gorgonians, glassy spicule of hexactinellid sponges, corallines; 125– 984 m depth (Jones and Hosie 2016). In this study, Meg­ alasma striatum was found at Kei Islands and Tanimbar Island, Indonesia. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4868, Stn. CP 09, 05°23'S, 132°29'E, 368– 389 m depth, 23 October 1991; 1 specimen, MNHN- IU-2019-4869, Stn. CP 16, 05°17'S, 132°50'E, 315–349 m depth, 24 October 1991, attached to sea urchin spine; 2 specimens, MNHN-IU-2019-4866, Stn. CP 17, 05°15'S, 133°01'E, 439–459 m depth, 24 October 1991. Type locality. Philippine Archipelago (Hoek 1883). – Tanimbar Island: 1 specimen, MNHN-IU-2019-4867, Stn. CP 46, 08°01'S, 132°51'E, 271–273 m depth, 29 Oc­ tober 1991; 4 specimens (3 attached to sea urchin spine), MNHN-IU-209-4870, Stn. DW 49, 08°00'S, 132°59'E, 206–210 m depth, 29 October 1991; 6 specimens, MNHN- IU-2019-4871, Stn. CP 85, 09°22'S, 131°14'E, 240–245 m depth, 4 November 1991, attached to spines of sea urchin. Order SCALPELLIFORMES Buckeridge & Newman, 2006 Scalpelloidea Pilsbry, 1916: 14 (nom. trans. Zevina, 1978). Scalpelliformes Buckeridge & Newman, 2006: 22. Scalpelloidea Pilsbry, 1916: 14 (nom. trans. Zevina, 1978). Scalpelliformes Buckeridge & Newman, 2006: 22. Diagnosis. Capitulum ovate, occludent margin almost same length as carinal margin; capitular plates heavily calcified, markedly sculptured with radial striations be­ tween grooves of growth lines; peduncle short, covered by capitulum. Megalasma Hoek, 1883: 50. – Pilsbry 1907a: 87. Diagnosis. Capitulum with five strong, calcified plates; scutal umbones subcentral; apex of carina sometimes slightly overlapping basal end of terga, progressively be­ coming broader towards base; peduncle very short. Description. Capitulum whitish, peduncle brownish. Capitulum compressed, striated, about twice as long as broad. Scutum with apex pointed; tergum basally trun­ cated, flat, oblong. Carina flat, very narrow, enlarged, keel-shaped in basal region. Cirrus I with anterior and posterior rami equal length; cirrus II–VI long, slender; cirrus VI with small caudal appendages. Penis with few Type species. Megalasma striatum Hoek, 1883: 51, pl. II, figs 5–9, pl. VII, figs 8, 9. Type species. Megalasma striatum Hoek, 1883: 51, pl. II, figs 5–9, pl. VII, figs 8, 9. Figure 7. Glyptelasma gracile (Hoek, 1883) (MNHN-IU-2019-4875). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 7. Glyptelasma gracile (Hoek, 1883) (MNHN-IU-2019-4875). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). zse.pensoft.net 658 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Megalasma striatum Hoek, 1883 Figure 8 Maxillule with three strong setae at upper angle separat­ ed by wide notch; mandible with four teeth, lower angle sharp. Measurements of specimen: basal diameter of ca­ pitulum 2.14 mm; capitular height 9.36 mm; total height 9.36 mm; scutal width 3.81 mm; scutal length 7.28 mm; tergal width 1.58 mm; tergal length 4.29 mm. Family SCALPELLIDAE Pilsbry, 1907 Pollicipedidae Gray, 1825: 100 (part.). – Annandale 1909: 63 (part.). Lepadidae Darwin, 1852: 8 (part.). Pollicipedidae Gray, 1825: 100 (part.). – Annandale 1909: 63 (part.). Pollicipedidae Gray, 1825: 100 (part.). – Annandale 1909: 63 (part.). Lepadidae Darwin, 1852: 8 (part.). Polyaspidae Gruvel, 1905: 8, 16 (part.; rejected by Pilsbry, 1907a, be­ cause family group name not derived from generic name). Polyaspidae Gruvel, 1905: 8, 16 (part.; rejected by Pilsbry, 1907a, be­ cause family group name not derived from generic name). Description. Capitulum white, with five calcified plates, surfaces strongly striated. Scutum with basal margin ro­ tated; tergum triangular in lateral view; carina with dorsal roof widening apically on either side of midline groove. Cirrus I with anterior ramus wider than posterior ramus. Scalpellinae Pilsbry, 1907a: 3 (part.). Scalpellidae. – Krüger, 1911: 7. – Pilsbry 1916: 4. – Nilsson-Cantell 1921: 162. – Foster 1978: 38. – Zevina 1978a: 999. – Buckeridge 1983: 27. – Gale 2016: 296. Figure 8. Megalasma striatum Hoek, 1883 (MNHN-IU-2019-4868). а. left lateral view, capitulum; b. right lateral, capitulum; c. anterior view, capitulum and peduncle; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 8. Megalasma striatum Hoek, 1883 (MNHN-IU-2019-4868). а. left lateral view, capitulum; b. right lateral, capitulum; c. anterior view, capitulum and peduncle; d. posterior view, carina. Scale bar: 2 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 659 Diagnosis. Capitulum with fully or partially calcified plates; peducle with calcareous or phosphorus scales. According to Gale (2016), the Scalpellidae have a max­ imum of 14 plates i.e. carina, rostrum, paired scuta, ter­ ga, upper latera, carinolatera, rostrolatera and inferior median latera; and rarely 13 plates, due to secondary loss of the rostrum. Diagnosis. Capitulum with 14 plates, smooth, white and reddish coloured; carina simply, strongly bowed, roof slightly convex with umbo at apex; rostrum triangular, distinct; upper latus quadrangular, large; infra-median la­ tus triangular. Description. Capitulum flat, rather broad, not covered by distinct membrane. Scutum with occludent margin arched, forming with tergal margin a triangular portion projecting over tergum. Tergum surpassing scutal area with occludent margin almost straight. Upper latus quad­ rangular, angle at apex between scutal and tergal margins distinctly projecting over scutum. Rostrum small, trian­ gular; rostrolatus very low, quadrangular; infra-median latus small, triangular, umbo at apex; carinal latus larger than other latera with carinal margin arched. Diotascalpellum rubrum (Hoek, 1883) Figure 9 Scalpellum rubrum Hoek, 1883: 91, pl. IV fig. 18. – Pilsbry 1911: 62, text-fig. 1, pl. VIII, figs 1–4. – Calman 1918b: 122–123. – Broch 1922: 237. – Nilsson-Cantell 1927: 745–747, text-fig. 2. – 1931: 2. Trianguloscalpellum rubrum. – Zevina 1981: 316–317, fig. 240. – Rosell 1986: 89, text-fig. 1a. – 1991: 22, fig. 1f, g. – Huang 1994: 517. – 2001: 318. – Jones et al. 2001: 254. – Young 2001: 464, fig. 7. – Jones 2007: 292. – Shalaeva and Boxshall 2014: 49, fig. 34. Diotascalpellum rubrum. – Gale 2016: 297, figs 2A, 6U–Y, 11Q–T, 13K–M, 18A–G. Scalpellum rubrum Hoek, 1883: 91, pl. IV fig. 18. – Pilsbry 1911: 62, text-fig. 1, pl. VIII, figs 1–4. – Calman 1918b: 122–123. – Broch 1922: 237. – Nilsson-Cantell 1927: 745–747, text-fig. 2. – 1931: 2. Trianguloscalpellum rubrum. – Zevina 1981: 316–317, fig. 240. – Rosell 1986: 89, text-fig. 1a. – 1991: 22, fig. 1f, g. – Huang 1994: 517. – 2001: 318. – Jones et al. 2001: 254. – Young 2001: 464, fig. 7. – Jones 2007: 292. – Shalaeva and Boxshall 2014: 49, fig. 34. Diotascalpellum rubrum. – Gale 2016: 297, figs 2A, 6U–Y, 11Q–T, 13K–M 18A–G Type species. Scalpellum regium Thomson, 1873; type locality: Stn 61: Atlantic, Western Central (North Western Atlantic Basin); 5,210 m depth. Type species. Scalpellum regium Thomson, 1873; type locality: Stn 61: Atlantic, Western Central (North Western Atlantic Basin); 5,210 m depth. Genus Diotascalpellum Gale, 2016 Diotascalpellum Gale, 2016: 297. Type locality. Philippines, 12°43'N, 122°10'E; depth, 180 m and 207 m; bottom, mud (near Luzon) (Hoek 1883). Diagnosis. Scalpellines without specialised contact be­ tween carina and carinolatus, merely a cresentic concavi­ ty on interior of plate that forms a rim slightly overlapping the upper latus; umbo of carinolatus upright, only slightly incurved, dorsal margin gently convex; inframedian sur­ face of carinolatus well demarcated, slightly inset. Family SCALPELLIDAE Pilsbry, 1907 Cirrus I with anterior and posterior rami almost same length; cirrus VI with long caudal appendages. Maxillule not notched, with large spine on upper side, cutting edge almost straight; mandible with three large teeth excluding inferior an­ gle. Measurements of two specimens: height of capitu­ lum 12.51–20.57 mm, width 7.39–10.55 mm, thickness 4.09–6.51 mm; length of peduncle 5.10–7.76 mm, width 4.36–6.62 mm. Subfamily SCALPELLINAE Pilsbry, 1907 Scalpellinae Pilsbry, 1907a: 4. – Zevina 1978a: 1002. – Zevina 1981: 93. – Gale 2016: 296. Scalpellinae Pilsbry, 1907a: 4. – Zevina 1978a: 1002. – Zevina 1981: 93. – Gale 2016: 296. Scalpellinae Pilsbry, 1907a: 4. – Zevina 1978a: 1002. – Zevina 1 93. – Gale 2016: 296. Pollicipedidae. – Annandale, 1909: 63 (part.). Pollicipedidae. – Annandale, 1909: 63 (part.). Pollicipedidae. – Annandale, 1909: 63 (part.). Diagnosis. Formerly, the subfamily was characterised by a subapical carinal umbo, inflexed carina and subapi­ cal umbones of the upper and inframedian latus (Zevina 1978a). Gale (2016) characterised the subfamily by the broad, low, straplike and gently incurved rostrolatus. The rostrum is broader than high, rectangular, trapezoidal or triangular and its large, triangular, lateral surfaces contact the interior of the rostrolatus. The articulation surface be­ tween the rostrum and rostrolatus extends over the entire height of both plates. Distribution. Java Sea, Indonesia. Pacific: western cen­ tral and northwest; Philippines; Kagoshima Sea and Sag­ ami Bay, Japan; 133–551 m depth (Shalaeva and Box­ shall 2014). In this study, Diotascalpellum rubrum was found at Kei Islands, Indonesia. Type genus. Scalpellum Leach, 1817: 68. Type genus. Scalpellum Leach, 1817: 68. Type genus. Scalpellum Leach, 1817: 68. Regioscalpellum Gale, 2016: 298. Regioscalpellum Gale, 2016: 298. Diagnosis. Concave scalpellines; internal surface upper latus covered by epidermis, short carinal margin present on carinolatus; carinolatus with incurved umbones, later­ al margin strongly convex. Trianguloscalpellum moluccanum. – Jones, 2012: 371, table 2. – Sha­ laeva and Boxshall 2014: 45, fig. 31. Regioscalpellum moluccanum (Hoek, 1883) Figure 10 Diotascalpellum rubrum. – Gale 2016: 297, figs 2A, 6U–Y, 11Q–T, 13K–M, 18A–G. Scalpellum moluccanum Hoek, 1883: 104, pl. V figs 3, 4; 1907: 56, 85, pl. VII, fig. 13. – Gruvel 1905: 76, fig. 85. – Nilsson-Cantell 1927: 747, fig. 3. Material examined. – Kei Islands: 2 specimens, MNHN-IU-2019-4925, Stn. CP 09, 05°23'S, 132°29'E, 368–389 m depth, 23 October 1991; 1 specimen, MNHN- IU-2019-4926, Stn. CP 16, 05°17'S, 132°50'E, 330– 350 m depth, 24 October 1991, attached to gorgonian. Arcoscalpellum moluccanum. – Newman & Ross, 1971: 60. – Jones et al. 1990: 6, 27. – Jones 1992: 172–175, figs 18, 19. Trianguloscalpellum moluccanum. – Jones, 2012: 371, table 2. – Sha­ laeva and Boxshall 2014: 45, fig. 31. Trianguloscalpellum moluccanum. – Jones, 2012: 371, table 2. – Sha­ laeva and Boxshall 2014: 45, fig. 31. zse.pensoft.net 660 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 9. Diotascalpellum rubrum (Hoek, 1883) (MNHN-IU-2019-4925). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 5 mm (a–d). Figure 9. Diotascalpellum rubrum (Hoek, 1883) (MNHN-IU-2019-4925). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 5 mm (a–d). Regioscalpellum moluccanum. – Gale, 2016: 298. mandible with three large teeth excluding inferior an­ gle. Measurements of five specimens: height of capitu­ lum 7.88–14.89 mm, width 4.81–10.65 mm, thickness 2.15–5.24 mm; length of peduncle 3.38–9.13 mm, width 2.09–6.04 mm. Material examined. – Tanimbar Island: 3 specimens, MNHN-IU-2019-4909, Stn. CP 38, 07°40'S, 132°27'E, 620–666 m depth, 28 October 1991; 1 specimen, MNHN- IU-2019-4910, Stn. DW 49, 08°00'S, 132°59'E, 206–210 m depth, 29 October 1991; 1 specimen, MNHN-IU-2019–4911, Stn. CC 57, 08°19'S, 131°53'E, 603–620 m depth, 31 Octo­ ber 1991; 1 specimen, MNHN-IU-2019-4912, Stn. CP 52, 08°03'S, 131°48'E, 1244–1266 m depth, 30 October 1991. Distribution. Pacific, Central and Southwest; Tasman Sea, north-eastern Australia; 788–2,745 m depth (Jones 2012; Shalaeva and Boxshall 2014). In this study, Re­ gioscalpellum moluccanum was found at Tanimbar Is­ land, Indonesia. Distribution. Pacific, Central and Southwest; Tasman Sea, north-eastern Australia; 788–2,745 m depth (Jones 2012; Shalaeva and Boxshall 2014). In this study, Re­ gioscalpellum moluccanum was found at Tanimbar Is­ land, Indonesia. Diagnosis. Regioscalpellum moluccanum (Hoek, 1883) Figure 10 Capitulum with 14 plates, surface covered by chitinous, almost smooth membrane; carina simply, not strongly bowed, apex projecting freely, roof not flat; umbo of the carina apical; upper latus almost triangular. Type locality. Banda Sea (4°21'S, 129°7'E); Challenger stn 195; 2,606 m depth: bottom temp. 3 °C; substrate grey ooze (Hoek 1883). Description. Capitulum inflated, subtriangular, apices of plates projecting freely through membrane, growth lines fairly distinct. Scutum trapezioid, umbo apical. Tergum large, oval, truncated, very pointed, extending between upper latus and carina. Carina simply, slightly bowed, roof slightly convex, apex projecting freely. Upper latus quadrangular; rostrum very small, carinate; rostral latus trapeziform; infra-median latus triangular, moderately small; carinal margins of carinal latera convex, touching each other at base and below middle of carina. Peduncle stout, slightly thinner below apex. Cirrus I unequal, an­ terior ramus oval, posterior ramus more slender; cirrus VI with long caudal appendages. Maxillule not notched, large spine on upper side, cutting edge almost straight; zse.pensoft.net Genus Scalpellum Leach, 1817 Scalpellum Leach, 1817: 68. – Darwin 1852: 21. – Hoek 1883: 59. – Gruvel 1905: 23. – Pilsbry 1907d: 181. – Tarasov and Zevina 1957: 126. – Zevina 1978a: 1002. – 1981: 94. – Gale 2016: 297. Strictoscalpellum Broch, 1924: 14. Diagnosis. Scalpellines with carinal and scutal umbones subapical; carinolatus with horn-like projection, often recurved, extending beyond carinal margin; inframedian latus rectangular to slightly trapezoidal, with low umbo; upper latus rhomboidal with subapical umbo; rostrum rectangular, pyramidal, with large sub-umbonal surface. Zoosyst. Evol. 96 (2) 2020, 649–698 661 Figure 10. Regioscalpellum moluccanum (Hoek, 1883) (MNHN-IU-2019-4909). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral view showing the capitulum and peduncle. Scale bar: 4 mm (a–d). Figure 10. Regioscalpellum moluccanum (Hoek, 1883) (MNHN-IU-2019-4909). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral view showing the capitulum and peduncle. Scale bar: 4 mm (a–d). Type species. Lepas scalpellum Linnaeus, 1767: 1109. Type species. Lepas scalpellum Linnaeus, 1767: 1109. pitulum 15.90 mm, width 10.29 mm, thickness 4.58 mm; length of peduncle 6.97 mm, width 5.67 mm. pitulum 15.90 mm, width 10.29 mm, thickness 4.58 mm; length of peduncle 6.97 mm, width 5.67 mm. Type locality. Unknown. Distribution. In this study, Scalpellum sp. was found in Tanimbar Island, Indonesia. Scalpellum stearnsi Pilsbry, 1890 Figure 12 left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 7 mm (a–d). Figure 11. Scalpellum sp. (MNHN-UI-2019-4929). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 7 mm (a–d). MNHN-IU-2019-4891, Stn. CP 69, 08°42'S, 131°53'E, 356–368 m depth, 2 November 1991; 5 specimens, MNHN-IU-2019-4892, Stn. CC 41, 07°45'S, 132°42'E, 393–401 m depth, 28 October 1991; 5 specimens, MNHN-IU-2019-4893, Stn. CP 59, 08°20'S, 132°11'E, 399–405 m depth, 31 October 1991, 2 specimens attached to glass rope sponge. MNHN-IU-2019-4891, Stn. CP 69, 08°42'S, 131°53'E, 356–368 m depth, 2 November 1991; 5 specimens, MNHN-IU-2019-4892, Stn. CC 41, 07°45'S, 132°42'E, 393–401 m depth, 28 October 1991; 5 specimens, MNHN-IU-2019-4893, Stn. CP 59, 08°20'S, 132°11'E, 399–405 m depth, 31 October 1991, 2 specimens attached to glass rope sponge. north east Australia; Malay Archipelago; Celebes; Sulu Arch.; Sulu Sea; South China Sea; East China Sea; Phil­ ippines; Taiwan; east coast of south Japan, Sagami Bay, off Hondo; attached to mollusc shells, anchor filaments of hexactinellid sponges, telegraph cables, carapaces of crabs, stones; 146–2117 m depth (Jones and Hosie 2016). In this study, Scalpellum stearnsi was found at Tanimbar Island and Kei Islands, Indonesia. – Kei Islands: 8 specimens, MNHN-IU-2019-4894, Stn. CP 06, 05°49'S, 132°21'E, 287–298 m depth, 22 Oc­ tober 1991. Type locality. East coast of Japan, between the Bay of Tokyo and the Inland Sea (Jones 1992). Diagnosis. Capitulum rhomboidal, compressed; carina strongly angled at umbo; scutum large, umbo at tergo-oc­ cludent angle; tergum triangular; inframedian latus pen­ tagonal; carinolateral horn-shaped. Remarks. For the first time, Scalpellum stearnsi was found in Japan and described by Pilsbry (1890). During the Siboga expedition (1899), S. stearnsi was collected from different locations in the Malay Archipelago with the depths varying from 204 m to 450 m. Hoek (1907) found intraspecific variations of the shell plate mor­ phology. He then divided S. stearnsi into two groups, i.e. variety robusta and var. gemina, which differed in the shape of the tergum. The species S. stearnsi in this study belongs to the group of var. gemina because of the V-shaped tergum. Description. Capitulum with 14 fully calcified plates. Scutum large, longer than broad, apex slightly projecting over tergum; tergum large, triangular, apex erect, slightly recurved. Scalpellum stearnsi Pilsbry, 1890 Figure 12 Material examined. – Tanimbar Island: 1 specimen, MNHN-UI-2019-4929, Stn. DW 49, 08°00'S, 132°59'E, 206–210 m depth, 29 October 1991. Figure 12 Figure 12 Scalpellum magnum Darwin, 1852: 18, pl. I, fig. 1. Scalpellum magnum Darwin, 1852: 18, pl. I, fig. 1. Scalpellum calcariferum Fischer, 1891: 116. Scalpellum calcariferum Fischer, 1891: 116. Scalpellum stearnsii var. gemina and var. robusta Hoek, 1907: 69, pl. VI, figs 2, 3, 8–12. Diagnosis. Capitulum with 14 fully calcified plates; ter­ gum triangular; inframedian latus broad, quadrilateral with umbo near to basal; upper latus rhomboidal, umbo sub-apical; carinal lateral horn-shaped, with angle ex­ tending beyond carina. Scalpellum stearnsii var. inerme Annandale, 1916a: 293. Scalpellum stearnsii var. inerme Annandale, 1916a: 293. Scalpellum stearnsii forma typica Broch, 1931: 16. Scalpellum stearnsii forma typica Broch, 1931: 16. Scalpellum stearnsii Pilsbry, 1890a: 96. – 1890b: 441, pl. IV, figs 1–5. – 1907a: 14, pl. 4, figs 1–5. – Nilsson-Cantell 1921: 175. – 1934: 33. – Broch 1922: 235, fig. 6. – Hiro 1933: 22, fig. 4, pl. 1, figs 5, 5a. – 1939a: 237. – Zevina 1981: 98, fig. 68. – Rosell 1991: 15. – Jones 1992: 146, figs 1, 2. – Liu and Ren 2007: 226, fig. 95. – Chan et al. 2009b: 89, figs 73–76. – Chan et al. 2010: 24, figs 2C, 18A–G. Scalpellum stearnsi: Gruvel, 1905: 44, fig. 46. – Gale 2016: fig. 5, table 1. Description. Capitulum subtriangular, covered by mem­ brane. Tergum triangular, occludent and basal margins slightly convex; scutum quadrangular, basal and upper latus margins concave; upper latus pentagonal; rostrolatus quadrangular; inframedian latus broad, rectangular, umbo close to basal margin; carinolatus horn-shaped with slight­ ly deep transverse furrows, umbo at basi-carinal angle; carina convex, umbo apical. Cirrus I with rami unequal, anterior ramus broader than posterior ramus; both rami densely covered with long setae. Maxilla subtriangular, with dense long setae; maxillule notched, with numerous setae; mandible with three teeth, inferior angle with large cuspidate setae. Measurements of specimen: height of ca­ Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4888, Stn. CP 71, 08°38'S, 131°44'E, 477–480 m depth, 2 November 1991; 1 specimen, MNHN-IU-2019-4889, Stn. CC 58, 08°19'S, 132°02'E, 457–461 m depth, 31 October 1991; 6 specimens, MNHN-IU-2019–4890, Stn. CC 42, 07°53'S, 132°42'E, 350–354 m depth, 28 October 1991; 3 specimens, zse.pensoft.net 662 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 11. Scalpellum sp. (MNHN-UI-2019-4929). а. Scalpellum stearnsi Pilsbry, 1890 Figure 12 Carina distinctly bent near middle, umbo dis­ tant from apex. Cirrus I with anterior ramus oval (12-seg­ ments), posterior ramus slender, long (15-segments); cir­ ri II–VI slender, long, rami almost equal length; cirrus VI with short caudal appendages (six segments). Max­ illule not notched, with numerous setae; mandible with six teeth excluding inferior angle. Measurements of five specimens: height of capitulum 31.69–52.00 mm, width 28.20–40.11 mm, thickness 11.19–18.56 mm; peduncle length 28.29–86.59 mm, width 11.39–17.94 mm. Scalpellum stearnsi has a low period of larval develop­ ment (Ozaki et al. 2008) and a slow growth rate (Yusa et al. 2018). This can result in the broad geographical distribution of this species. Recently, Lin et al. (2020) examined the diversity and genetic differentiation of populations of S. stearnsi from the East China Sea, West Philippine Basin, Sulu Sea and Caroline Trenches, which resulted in four distinct clades of S. stearnsi. Distribution. Indo-west Pacific: Indian Ocean, off Nico­ bar Island; Indonesia: Java Sea, Bali Straits; north west & zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 663 Figure 12. Scalpellum stearnsi Pilsbry, 1890 (MNHN-IU-2019-4888). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 14 mm (a–d). Figure 12. Scalpellum stearnsi Pilsbry, 1890 (MNHN-IU-2019-4888). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 14 mm (a–d). Type genus. Meroscalpellum Zevina, 1978b: 1346. Type genus. Meroscalpellum Zevina, 1978b: 1346. Subfamily MEROSCALPELLINAE Zevina, 1978 Material examined. – Tanimbar Island: 2 specimens, MNHN-IU-2019-4903, Stn. CP 71, 08°38'S, 131°44'E, 477–480 m depth, 2 November 1991; 5 specimens, MNHN-IU-2019-4904, Stn. CC 21, 05°14'S, 133°00'E, 688–694 m depth, 25 October 1991 attached to glass rope sponge; 1 specimen, MNHN-IU-2019-4905, Stn. CC 40, 07°46'S, 132°31'E, 443–468 m depth, 28 October 1991; 5 specimens, MNHN-IU-2019-4906, Stn. CC 57, 08°19'S, 131°53'E, 603–620 m depth, 31 October 1991. Meroscalpellinae Zevina, 1978b: 1343. Diagnosis. Capitulum with 14 or 13 plates, reduced in differing stages or proportions; carina with two umbo positions; females considered rarer than hermaphrodites; males sac‑like, usually without plates, rarely with two or four reduced plates. Diagnosis. Capitulum with plates buried or obscure; plates reduced in size; tegum reduced, forming four-point­ ed star; scutum small; peduncle armed with distinct cir­ cles of large, calcareous scales. Type genus. Meroscalpellum Zevina, 1978b: 1346. Scalpellum persona Annandale, 1916a: 295, pl. IV, fig. 3; pl. V, figs 7, 8; pl. VI, figs 3–5. – Calman 1918b: 120. – Nilsson-Cantell 1934: 44, pl. 5, fig. 2. Alcockianum Zevina, 1978b: 1345. Description. Capitulum brownish, large, ovoid, inflated, with 13 capitular plates, including a vestigial rostrum, plates embedded and mostly concealed by thick, opaque membrane. Scutum small, widely separated from all re­ maining plates except tergum, margins not excavated or deeply concave; tergum reduced in form as four-pointed star, with two rays greatly and two rays slightly produced. Carina reduced in size, apex approaching terga, widely separated from remaining plates. Peduncle cylindrical, similar length to capitulum, with large calcareous scales arranged in alternating rows. Cirrus I with anterior ramus oval (8-segments), posterior ramus slender, long (12-seg­ ments); cirri II–VI slender, long, rami almost equal length; cirrus VI with caudal appendages; caudal appendages 1/3 length of cirrus VI, 15-segmented, tapering distally. Penis rather short, smooth, pointed. Maxilla bilobed, dense setae on margin. Maxillule relatively large, with broad, shallow excavation on lower margin occupying more than half mar­ gin, remainder of margin obliquely subtruncate; mandible with three main teeth in addition to inner angle, which is variously divided, broad as a whole. Measurements of five Diagnosis. Plates strongly reduced; partly obscured un­ der heavy cuticle; tergum double branched, depressed at base; scutum triangular; upper lateral triangular; mid‑lat­ eral very small, umbo apical; caudal appendages very long; hermaphrodites. Type species. Scalpellum alcockianum Annandale, 1905: 82; type locality: Gulf of Manaar and Andaman Sea. zse.pensoft.net – Jones 1992: 151–154, figs 4, 5. Alcockianum persona Zevina, 1978b: 1345. – 1981: 150–151, fig. 107. – Jones 1992: 151–154 figs 4 5 Scalpellum persona Annandale, 1916a: 295, pl. IV, fig. 3; pl. V, figs 7, 8; pl. VI, figs 3–5. – Calman 1918b: 120. – Nilsson-Cantell 1934: 44, pl. 5, fig. 2. Graviscalpellum persona Foster, 1980: 527, figs 1e, 2. Alcockianum persona Zevina, 1978b: 1345. – 1981: 150–151, fig. 107. – Jones 1992: 151–154, figs 4, 5. Graviscalpellum persona Foster, 1980: 527, figs 1e, 2. Genus Alcockianum Zevina, 1978 Alcockianum Zevina, 1978b: 1345. Alcockianum Zevina, 1978b: 1345. Alcockianum persona (Annandale, 1916) Figure 13 Scalpellum persona Annandale, 1916a: 295, pl. IV, fig. 3; pl. V, figs 7, 8; pl. VI, figs 3–5. – Calman 1918b: 120. – Nilsson-Cantell 1934: 44, pl. 5, fig. 2. Graviscalpellum persona Foster, 1980: 527, figs 1e, 2. – Jones 1992: 151–154, figs 4, 5. zse.pensoft.net 664 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 13. Alcockianum persona (Annandale, 1916) (MNHN-IU-2019-4903). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 9 mm (a–d). Figure 13. Alcockianum persona (Annandale, 1916) (MNHN-IU-2019-4903). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 9 mm (a–d). 69, taf. II, fig. 4. – Hiro 1937 b: 392. – Tarasov and Zevina 1957: 144, figs 46, 47. – Zevina 1969: 68. – 1970: 257–259, figs 5, 6. Scalpellum chitinosum Hoek, 1907: 73. Scalpellum curiosum Hoek, 1907: 49. Scalpellum japonicum biramosum Pilsbry, 1911: 68, fig. 4. – Weltner 1922: 69, taf. II, fig. 5. – Nilsson-Cantell 1938: 7. Scalpellum japonicum metapleurum Pilsbry, 1907b: 360. Annandaleum japonicum biramosum Chan et al., 2009b: 98–99, fig. 80. – Chan et al. 2010: 17–18, figs 2A, 14, 15. Annandaleum japonicum. – Newman & Ross, 1971: 122. – Zevina 1981: 166–167. – Jones et al. 2001: 251. – Young 2001: 465, fig. 8. – 2007: 23, fig. 22. specimens: height of capitulum 17.71–35.06 mm, width 12.91–28.06 mm, thickness 4.96–18.20 mm; length of pe­ duncle 13.42–28.67 mm, width 6.66–10.79 mm. Scalpellum chitinosum Hoek, 1907: 73. Scalpellum curiosum Hoek, 1907: 49. Scalpellum japonicum biramosum Pilsbry, 1911: 68, fig. 4. – Weltner 1922: 69, taf. II, fig. 5. – Nilsson-Cantell 1938: 7. Distribution. Indonesian Seas, eastern Australia, New Zea­ land; 109–915 m depth (Jones 1992). In this study, Alcock­ ianum persona was found at Tanimbar Island, Indonesia. Scalpellum japonicum metapleurum Pilsbry, 1907b: 360. Annandaleum japonicum biramosum Chan et al., 2009b: 98 99, fig. 80. – Chan et al. 2010: 17–18, figs 2A, 14, 15. – Chan et al. 2010: 17–18, figs 2A, 14, 15. Type лocality. Timor Sea (10°22'30"S, 120°7'30"E); 109–366 m depth: bottom temp. 13.1 °C (Jones 1992). Type лocality. Timor Sea (10°22'30"S, 120°7'30"E); 109–366 m depth: bottom temp. 13.1 °C (Jones 1992). Annandaleum japonicum. – Newman & Ross, 1971: 122. – Zevina 1981: 166–167. Alcockianum persona (Annandale, 1916) – Jones et al. 2001: 251. – Young 2001: 465, fig. 8. – 2007: 23, fig. 22. Genus Annandaleum Newman & Ross, 1971 Material examined. – Tanimbar Island: 4 specimens, MNHN-IU-2019-4932, Stn. CP 53, 08°18'S, 131°41'E, 1026–1053 m depth, 30 October 1991. Annandaleum Newman & Ross, 1971: 122. – Zevina 1978b: 1346. Diagnosis.Capitulum of female with 14 partially calcified plates; tergum in shape of inverted V; scutum with moder­ ately long apicolateral arm, 1/4 to 1/2 length of tergal mar­ gin; basal margin of scutum entire; upper latus pentagonal to triangular or subrectangular, with or without short de­ pending arm; carinolatus as high as or slightly higher than wide, umbo at basicarinal angle; inframedian latus higher than wide, vase-shaped, umbo submedial to basal; rostro­ latus wider than high, commonly less than twice height of inframedian latus; caudal appendage relatively long. Diagnosis. Capitulum with 14 partly calcified plates; ca­ rina with flat roof; apex of tergum curved towards carina; rostral latus trapezoidal in shape; peduncle short. Description. Capitulum yellowish, with 14 plates sepa­ rated by broad, chitinous spaces. Peduncle short, curved, scales large, not numerous. Scutum long, narrow; umbo apical, divided into two segments, occludent segment large, increasing in width from upper to lower part, oth­ er segment very narrow. Tergum divided into occludent and carinal segments, with triangular, chitinous portion between, umbo apical, distinctly recurved. Cirrus I un­ equal, anterior ramus oval, posterior ramus slender long; cirrus VI with caudal appendages. Maxillule slightly notched, two long and one short setae on upper side; mandible with three teeth excluding inferior angle; la­ brum cutting edge straight, very fine teeth on cutting edge. Measurements of the four specimens: height of ca­ Type species. Scalpellum subflavum Annandale, 1906. Type species. Scalpellum subflavum Annandale, 1906. Type species. Scalpellum subflavum Annandale, 1906. Type species. Scalpellum subflavum Annandale, 1906. Scalpellum japonicum Hoek, 1883: 67, pl. III, figs 9, 10. – Annandale 1906: 131, fig. 3. – Pilsbry 1911: 66, pl. II, figs 1–3. – Weltner 1922: Annandaleum japonicum (Hoek, 1883) Figure 14 zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 665 Figure 14. Annandaleum japonicum (Hoek, 1883) (MNHN-UI-2019-4932). а. left lateral view showing the capitulum and pedun­ cle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). Figure 14. Annandaleum japonicum (Hoek, 1883) (MNHN-UI-2019-4932). а. left lateral view showing the capitulum and pedun­ cle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4931, Stn. CP 54, 08°21'S, 131°43'E, 836–869 m depth, 30 October 1991, attached to gas­ tropod shell. pitulum 9.56–12.73 mm, width 4.67–7.13 mm, thickness 1.85–3.21 mm; length of peduncle 3.37–4.77 mm, width 1.83–3.27 mm. Distribution. Eastern Indian Ocean; Northwest and Western Central Pacific; Malay Archipelago; Japan; Tai­ wan; Indonesia; attached to shell of gastropod, gorgoni­ ans, rocks; 805–6,810 m depth (Jones and Hosie 2016). In this study, Annandaleum japonicum was found at Ta­ nimbar Island, Indonesia. Diagnosis. Capitulum regularly oval; carina simply bowed; tergum almost triangular; scutum subtriangular; upper latus large, perfectly calcified part irregularly tri­ angular, constricted above; rostral latus quadrangular; peduncle at least half as long as capitulum. Description. Capitulum compressed; 13 plates complete­ ly covered by fine, hairless membrane. Peduncle half length of capitulum, stout, cylindrical, armed with small, transversely elongated plates. Scutum subtriangular, lat­ eral margin excavated with tooth above excavation blunt, short, simple; tergum almost triangular, scutal margin ex­ cavated, but not very boldly, occludent margin slightly, regularly convex outwards. Carina simply bowed, umbo subterminal, in contact with terga above or just entering between them. Cirrus I unequal, anterior ramus oval, posterior ramus slender, long; cirri II–VI slender, long, rami almost equal lengths; cirrus VI with long, slender caudal appendages. Maxillule slightly notched, two ma­ jor setae on upper side; mandible with four teeth. Mea­ surements of specimen: height of capitulum 18.88 mm, width 11.41 mm, thickness 6.30 mm; length of peduncle 11.45 mm, width 4.89 mm. Type locality. Pacific, Northwest (off Nagoya, south of Japan); 1,017 m depth (Shalaeva and Boxshall 2014). Type locality. Pacific, Northwest (off Nagoya, south of Japan); 1,017 m depth (Shalaeva and Boxshall 2014). Annandaleum laccadivicum (Annandale, 1906) Figure 15 Scalpellum laccadivicum Annandale, 1906b: 393. – 1908: pl. I, figs 3, 4. – 1913: 235; 1916b: 129, pl. vii, fig. 6. – Calman 1918b: 124. – Hiro 1933: 31, text figs 7, 8, pl. I, figs 11–11b. – 1937b: 392. – Stubbings 1936: 26. – Nilsson-Cantell 1938: 25. Scalpellum subflavum Annandale, 1906b: 397. – Newman and Ross 1971: 122. Scalpellum polymorphum Hoek, 1907: 80, pl. 7, figs 9–11. – Weltner 1922: 72. Scalpellum molliculum Pilsbry, 1911: 68, pl. 10, figs 4, 5. Annandaleum laccadivicum Rosell, 1991: 16, fig 1a. – Zevina 1981: 170, fig. 121. – Jones et al. 2001: 251. – Chan et al. 2009b: 102–103. zse.pensoft.net 666 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 15. Annandaleum laccadivicum (Annandale, 1906) (MNHN-IU-2019-4931). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 5 mm (a–d). Figure 15. Annandaleum laccadivicum (Annandale, 1906) (MNHN-IU-2019-4931). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 5 mm (a–d). Distribution. Gulf of Oman, Arabian Sea, Sri Lanka, Japan (Chan et al. 2009b). In this study, Annandaleum laccadivicum was found at Tanimbar Island, Indonesia. ly formed, thin, smooth, translucent plates, eight formed like a Greek lambda. Description. Capitulum with 14 plates. Peduncle shorter than capitulum, cylindrical, rather slender, ex­ panded at base. Tergum lambda-shaped, with promi­ nent tooth on carinal margin just above point where apex of carina approaches it. Scutum shaped similarly, but occludent branch much stouter, with vertical ridge running nearer to lateral than occludent margin. Cirrus I unequal, anterior ramus oval, posterior ramus slender, long; cirri II–VI slender, long, with rami almost equal length; cirrus VI with long, slender caudal appendag­ es. Maxillule not notched, two major setae on upper side; mandible with four teeth including inner angle. Measurements of five specimens: height of capitulum 16.61–24.27 mm, width 8.73–14.49 mm, thickness 3.17–6.31 mm; length of peduncle 5.54–15.27 mm, width 3.73–6.26 mm. Type locality. Laccadive Sea, 2,077 m depth; on living shells of Dentalium (Annandale 1906). Annandaleum lambda (Annandale, 1910) Figure 16 Scalpellum lambda Annandale, 1910b: 115. – 1916a: pl. VII, figs 6, 6a, pl. VIII, figs 12–15. Scalpellum longius Annandale, 1913: 234. Scalpellum longius Annandale, 1913: 234. Annandaleum lambda. – Newman & Ross, 1971: 122. – Jones, 1992: 154, fig. 6. Material examined. – Tanimbar Island: 10 specimens, MNHN-IU-2019-4915, Stn. CP 52, 08°03'S, 131°48'E, 1244–1266 m depth, 30 October 1991; 1 specimen, MNHN-IU-2019-4913, Stn. CP 53, 08°18'S, 131°41'E, 1026–1053 m depth, 30 October 1991; 1 specimen, MNHN- IU-2019-14, Stn. CP 89, 08°39'S, 131°08'E, 1058–1084 m depth, 5 November 1991, attached to pumice. Distribution. Indo-Pacific from off Zanzibar, Indian Ocean, eastern Australia; Sumbawa, Indonesia; Malay Arch.; SW of Calatagan Pt, Philippines; S Japan (S of Honda I.), SW Pacific; attached to shells of bivalves; 234–2077 m depth (Jones and Hosie 2016). In this study, Annandaleum lambda was found at Tanimbar Is­ land, Indonesia. Diagnosis. Capitulum narrow; carinal margin strongly curved; laterally compressed; bearing fourteen imperfect­ zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 667 Figure 16. Annandaleum lambda (Annandale, 1910) (MNHN-IU-2019-4915). а. left lateral view showing the capitulum and pe­ duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 6 mm (a–d). Figure 16. Annandaleum lambda (Annandale, 1910) (MNHN-IU-2019-4915). а. left lateral view showing the capitulum and pe­ duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 6 mm (a–d). 549–552 m depth, 31 October 1991; 1 specimen, MNHN- IU-2019-4898, stn. CP 71, 08°38'S, 131°44'E, 477– 480 m depth, 2 November 1991; 6 specimens, MNHN- IU-2019-4901, stn. CP 72, 08°36'S, 131°33'E, 676–699 m depth, 2 November 1991. Type locality. Eastern Indian Ocean (13°54'15"N, 94°02'15"E), Investigator Stn. 372; 1,176 m depth (Jones 1992). Genus Litoscalpellum Newman & Ross, 1971 Diagnosis. Capitulum compressed, oval, notched above; surface covered with very short velvety pubescence, ar­ eas of valves defined by grooves, lines of growth strongly marked; carina flattened. Litoscalpellum Newman & Ross, 1971: 108. – Zevina 1978b: 1344. – Liu and Ren 1985: 196. – 2007: 228. Diagnosis. Capitulum with 14 plates, mostly not reduced; tergum with straight basal margin or shallow notch; scu­ tum with straight, smooth basal margin; upper latus trian­ gular or elongate, commonly with slightly hollowed-out basal margin, rarely with deep notch; inframedian latus narrow, triangular or quadrilateral, umbo apical; caudal appendages present. Description. Capitulum with 14 plates, cartilaginous thick. Terqum irregularly triangular, apex projecting above cari­ na, recurved. Scutum triangular, basal width more than half height, apex overlapping tergum. Carina evenly curved, apex projecting freely for short distance, touching or enter­ ing between terga, its base rounded, widely separated from carinal latera; roof strongly convex. Upper latus triangular with base convex. Rostrum triangular; inframedian latus small, triangular, base deeply embedded. Peduncle with large transverse plates, not overlapping (widely spaced). Cirrus I unequal, anterior ramus oval, posterior ramus slen­ der, long; cirrus VI with caudal appendages. Maxillule with oral edge sinuous, a large seta at its proximal third, another at its distal end; mandible with four teeth. Measurements of five specimens: height of capitulum 16.94–39.25 mm, width 11.39–28.28 mm, thickness 5.20–19.89 mm; length of peduncle 15.02–52.02 mm, width 5.63–16.87 mm. Type species. Litoscalpellum fissicarinatum Newman & Ross, 1971: 108, pl. XC, text-fig. 55. Type species. Litoscalpellum fissicarinatum Newman & Ross, 1971: 108, pl. XC, text-fig. 55. Litoscalpellum recurvirostrum (Hoek, 1883) Figure 18 Scalpellum recurvirostrum Hoek, 1883: 77–79, pl. III, figs 11, 12, pl. VIII, figs 9, 10. Arcoscalpellum recurvirostrum. – Newman & Ross, 1971: 79–80, fig. 39. Litoscalpellum recurvirostrum. – Zevina, 1974: 214. – 1981: 127, 128, fig. 88. – Shalaeva and Boxshall 2014: 13, fig. 6. Scalpellum recurvirostrum Hoek, 1883: 77–79, pl. III, figs 11, 12, pl. VIII, figs 9, 10. Distribution. Indian Ocean, Antarctic and Southern (South of the Kerguelen Islands); known depth range 195 to 274 m (Shalaeva and Boxshall 2014). In this study, Litoscalpellum recurvirostrum was found at Kei Islands and Tanimbar Island, Indonesia. Arcoscalpellum recurvirostrum. – Newman & Ross, 1971: 79–80, fig. 39. Litoscalpellum recurvirostrum. – Zevina, 1974: 214. – 1981: 127, 128, fig. 88. – Shalaeva and Boxshall 2014: 13, fig. 6. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4934, Stn. CP 05, 05°49'S, 132°18'E, 296– 299 m depth, 22 October 1991; 1 specimen, MNHN- IU-2019-4936, Stn. CP 09, 05°23'S, 132°29'E, 368–389 m depth, 23 October 1991. Type locality. Between Kerguelen and Heard Islands; 52°4'S, 71°22'E; depth: 270 m; bottom temperature: 1.8 °C; bottom: rocks (Hoek 1883). Type locality. Between Kerguelen and Heard Islands; 52°4'S, 71°22'E; depth: 270 m; bottom temperature: 1.8 °C; bottom: rocks (Hoek 1883). – Tanimbar Island: 4 specimens, MNHN-IU-2019-4935, Stn. CP 83, 09°23'S, 131°00'E, 285–297 m depth, 4 Novem­ ber 1991; 16 specimens, MNHN-IU-2019-4937, Stn. CP 69, 08°42'S, 131°53'E, 356–368 m depth, 2 November 1991. Litoscalpellum juddi (Calman, 1918) Figure 17 Figure 17 Scalpellum juddi Calman, 1918b: 116, text-figs 5–7. Scalpellum regulus Calman, 1918b: 113, text-fig. 4. Litoscalpellum juddi Zevina, 1981:135–136, fig. 94. Scalpellum juddi Calman, 1918b: 116, text-figs 5–7. Scalpellum regulus Calman, 1918b: 113, text-fig. 4. Litoscalpellum juddi Zevina, 1981:135–136, fig. 94. Scalpellum juddi Calman, 1918b: 116, text-figs 5–7. Scalpellum regulus Calman, 1918b: 113, text-fig. 4. Litoscalpellum juddi Zevina, 1981:135–136, fig. 94. Material examined. – Tanimbar Island: 5 specimens, MNHN-IU-2019-4896, stn. CC 56, 08°16'S, 131°59'E, Distribution. In this study, Litoscalpellum juddi was found at Tanimbar Island, Indonesia. zse.pensoft.net 668 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 17. Litoscalpellum juddi (Calman, 1918) (MNHN-UI-2019-4898). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 11 mm (a–d). Figure 17. Litoscalpellum juddi (Calman, 1918) (MNHN-UI-2019-4898). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 11 mm (a–d). Type locality. 11°0'S, 121°30'E (Java-Australia), 720 m depth (Calman 1918b). Cirrus I with rami unequal, anterior ramus oval, posterior ramus slender, long, segments very hairy. Maxilla bilobed; maxillule notched, two large setae above notch; mandible with three teeth, inferior angle pectinated. Measurements of five specimens: height of capitulum 16.15–26.05 mm, width 9.24–16.59 mm, thickness 4.45–8.18 mm; length of peduncle 7.25–20.90 mm, width 4.13–8.95 mm. zse.pensoft.net zse.pensoft.net Litoscalpellum walleni Newman & Ross, 1971: 116, pl. X E text-fig. 60. et al.: Deep-sea barnacles from the Karubar expedition (1991) 670 376 – Poltarukha 2013: 52–53, fig. 1. –Shalaeva and Boxshall 2014: 16, fig. 9. 376 – Poltarukha 2013: 52–53, fig. 1. –Shalaeva and Boxshall 2014: 16, fig. 9. angle serrate along basal margin with four spines. Mea­ surements of specimen: height of capitulum 14.56 mm, width 8.09 mm, thickness 2.50 mm; length of peduncle 7.52 mm, width 3.89 mm. angle serrate along basal margin with four spines. Mea­ surements of specimen: height of capitulum 14.56 mm, width 8.09 mm, thickness 2.50 mm; length of peduncle 7.52 mm, width 3.89 mm. Material examined. – Tanimbar Island: 2 specimens, MNHN-IU-2019-4922, Stn. CC 21, 05°14'S, 133°00'E, 688–694 m depth, 25 October 1991, attached to glass rope sponge. Distribution. Southeast Pacific Ocean (Newman and Ross 1971). In this study, Litoscalpellum walleni was found at Tanimbar Island, Indonesia. Distribution. Southeast Pacific Ocean (Newman and Ross 1971). In this study, Litoscalpellum walleni was found at Tanimbar Island, Indonesia. Diagnosis. Capitulum triangular; plates ornamented with radial ridges, covered by sparsely hirsute integument; ca­ rina with roof deeply grooved; scutum with apex over­ lapping tergum; inframedian latus triangular, very small. Type locality. northeast of Peter I Island, southeast Pa­ cific Ocean; 65°50'S, 88°56'W; depth: 4,502 m (Newman and Ross 1971). Type locality. northeast of Peter I Island, southeast Pa­ cific Ocean; 65°50'S, 88°56'W; depth: 4,502 m (Newman and Ross 1971). Desciption. Capitulum yellowish, with 13 fully calci­ fied plates. Peduncles short with scales slightly over­ lapping in the middle part. Scutum with pit for comple­ mental males, above shallow pit for adductor muscle. Carina wide in lower part, ribbed in upper part. Upper latus with straight sides; rostrum appearing externally as inverted triangle. Cirrus I unequal, anterior ramus oval, posterior ramus slender, long; cirrus VI with very short caudal appendages. Maxillule with notch between two or three stout setae at upper angle, group of more slender setae on cutting edge; mandible with three teeth excluding inferior angle; labrum cutting edge slightly concave, numerous pointed teeth on cutting edge. Measurements of two specimens: height of ca­ pitulum 12.44–13.88 mm, width 6.97–7.52 mm, thick­ ness 2.77–3.47 mm; length of peduncle 2.99–3.15 mm, width 2.94–3.24 mm. Subfamily ARCOSCALPELLINAE Zevina, 1978 Arcoscalpellinae Zevina, 1978b: 1346. –1981: 184. – Liu and Ren 2007: 238. Diagnosis. Capitulum with plates packed very closely; ca­ rina slightly convex; umbo of scutum and tergum apical. Diagnosis. Capitulum with plates packed very closely; ca­ rina slightly convex; umbo of scutum and tergum apical. Type genus. Arcoscalpellum Hoek, 1907: 57. Genus Amigdoscalpellum Zevina, 1978 Arcoscalpellum Hoek, 1907: 85 (in part). – Newman and Ross 1971: 42 (part). Arcoscalpellum Hoek, 1907: 85 (in part). – Newman and Ross 1971: 42 (part). Amigdoscalpellum Zevina, 1978b: 1349. – Foster 1980: 527. – Liu and Ren 1985: 206. – 2007: 251. Amigdoscalpellum Zevina, 1978b: 1349. – Foster 1980: 527. – Liu and Ren 1985: 206. – 2007: 251. Distribution. Indian Ocean, Eastern and Western; Pacif­ ic, Southwest and Western Central; Atlantic, Southeast and Eastern central; 110–2,397 m depth (Shalaeva and Boxshall 2014). In this study, Amigdoscalpellum costel­ latum was found at Tanimbar Island, Indonesia. Diagnosis. Capitulum of female or hermaphrodite with 13 or 14 completely calcified plates; scutum and tergum with umbones apical; carina evenly bent, umbo apical or sub­ apical; middle latera narrowly triangular, baton-like, small, umbo apical, not reaching upper latera; carinolatera typi­ cally with umbo orientated in middle part of carinal area, not extending beyond rim of capitulum; caudal appendag­ es usually uni-articulate, but possibly absent or extended to seven segments; basically females with dwarf males. Type locality. 1. Station 135: Island of Tristan da Cunha; depth: 110 m, 137 m, 183 m, 274 m, 1,006 m, 1,829 m, 2,012 m; bottom: rocky, shells. 2. Station 164a: off Syd­ ney, 34°13'S, 151°38'E; depth: 750 m; bottom: grey ooze. 3. Station 169: off East Cape, Auckland, 37°34'S, 179°22'E; depth: 1,280 m; bottom temperature: 4.2 °C; bottom: grey ooze (Hoek 1883). Type species. Scalpellum manum Zevina, 1973: 843, figs 1–7; type locality: Indian Ocean, 12°18'S, 112°43'E. Type species. Scalpellum manum Zevina, 1973: 843, figs 1–7; type locality: Indian Ocean, 12°18'S, 112°43'E. Scalpellum elongatum Hoek, 1883: 93, pl. IV, figs 8, 9. – Nilsson-Can­ tell 1928: 8, fig. 4. – 1931: 3, text-fig.1. – 1938: 7. non Scalpellum (Arcoscalpellum) elongatum. – Steenstrup, 1837: 409. Scalpellum (Scalpellum) elongatum. – Stubbings, 1936: 25, text-fig. 10. Scalpellum (Arcoscalpellum) costellatum Withers, 1935: 279. Arcoscalpellum buccinum Newman & Ross, 1971: 55, pl. VIII, figs 22, 23. Arcoscalpellum costellatum. – Foster, 1978: 56, pl. 7B, C, fig. 33. Amigdoscalpellum costellatum. – Foster, 1980: 527–529, fig. 3J. – Zevi­ na 1981: 270–271, fig. 204. – Jones et al. 1990: 5 – Jones 2012: 371, Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4921, Stn. CP 20, 05°15'S, 132°59'E, 769– 809 m depth, 25 October 1991. Litoscalpellum walleni Newman & Ross, 1971: 116, pl. X E text-fig. 60. Diagnosis. Capitulum plates separated, covered by fine membrane; carina slightly angularly bent; upper latus trapeziform; inframedian latus triangular; peduncle with scattered, calcareous scales. Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4927, Stn. CP 91, 08°44'S, 131°05'E, 884–891 m depth, 5 November 1991. Diagnosis. Capitulum with 14 plates; inframedian latus triangular, very small, narrow; carina with flat roof; ros­ trum minute; scutum lacking prominent apicolateral arm; maxilla subtriangular, slightly trilobate; maxilulle slight­ ly notched with teeth diverging laterally from cutting edge; caudal appendage multi-articulate, slightly shorter than first segment of cirrus VI. Description. Capitulum with 14 plates, lines of growth not distinct, plates separated by broad, membranous interspac­ es. Scutum elongate quadrangular, broader at base than in upper half; umbo apical, slightly projecting outwards. Tergum triangular; carina bowed; upper latus trapeziform; inframedian latus very small, triangular. Peduncle cylindri­ cal with numerous calcareous scales scattered over surface. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 669 Figure 18. Litoscalpellum recurvirostrum (Hoek, 1883) (MNHN-UI-2019-4934). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 6 mm (a–d). Figure 18. Litoscalpellum recurvirostrum (Hoek, 1883) (MNHN-UI-2019-4934). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 6 mm (a–d). Figure 19. Litoscalpellum walleni Newman & Ross, 1971 (MNHN-UI-2019-4927). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 4 mm (a–d). Figure 19. Litoscalpellum walleni Newman & Ross, 1971 (MNHN-UI-2019-4927). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 4 mm (a–d). Description. Capitulum elongate, higher than broad; plates ornamented with growth lines. Carina bowed, roof slightly convex, bounded by angles. Tergum trian­ gular; scutum with lateral margin concave; upper latus roughly quadrangular; carinal latus higher than wide; rostral latus quadrangular, slightly broader than high; rostrum minute, narrow, essentially rectangular. Cirrus I unequal, anterior ramus shorter, broader than posterior ramus; both rami densely covered with long setae. Man­ dible with four teeth including inferior angle, inferior zse.pensoft.net Pitriana, P. Scalpellum tenue Hoek, 1883: 119, pl. IV, figs 20, 21, pl. X, fig. 6. – Gruvel 1912: 345. – Nilsson-Cantell 1938: 8. non Scalpellum tenue Annandale, 1906a: 142. Amigdoscalpellum tenue. – Zevina 1981: 292–293, fig. 220 (2). – Sha­ laeva and Boxshall 2014: 17, fig. 10. Amigdoscalpellum tenue (Hoek, 1883) Figure 21 Figure 21 Scalpellum elongatum Hoek, 1883: 93, pl. IV, figs 8, 9. – Nilsson-Can­ tell 1928: 8, fig. 4. – 1931: 3, text-fig.1. – 1938: 7. non Scalpellum (Arcoscalpellum) elongatum. – Steenstrup, 1837: 409. Scalpellum (Scalpellum) elongatum. – Stubbings, 1936: 25, text-fig. 10. Scalpellum (Arcoscalpellum) costellatum Withers, 1935: 279. Arcoscalpellum buccinum Newman & Ross, 1971: 55, pl. VIII, figs 22, 23. Arcoscalpellum costellatum. – Foster, 1978: 56, pl. 7B, C, fig. 33. Amigdoscalpellum costellatum. – Foster, 1980: 527–529, fig. 3J. – Zevi­ na 1981: 270–271, fig. 204. – Jones et al. 1990: 5 – Jones 2012: 371, Scalpellum elongatum Hoek, 1883: 93, pl. IV, figs 8, 9. – Nilsson-Can­ tell 1928: 8, fig. 4. – 1931: 3, text-fig.1. – 1938: 7. Amigdoscalpellum tenue. – Zevina 1981: 292–293, fig. 220 (2). – Sha­ laeva and Boxshall 2014: 17, fig. 10. Amigdoscalpellum tenue. – Zevina 1981: 292–293, fig. 220 (2). – Sha­ laeva and Boxshall 2014: 17, fig. 10. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4921, Stn. CP 20, 05°15'S, 132°59'E, 769– 809 m depth, 25 October 1991. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 671 Figure 20. Amigdoscalpellum costellatum (Withers, 1935) (MNHN-IU-2019-4922). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 3 mm (a–d). Figure 20. Amigdoscalpellum costellatum (Withers, 1935) (MNHN-IU-2019-4922). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 3 mm (a–d). Figure 21. Amigdoscalpellum tenue (Hoek, 1883) (MNHN-IU-2019-4921). а. left lateral view showing the capitulum and pedun­ cle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral view showing the capitulum and peduncle. Scale bar: 2 mm (a–d). Figure 21. Amigdoscalpellum tenue (Hoek, 1883) (MNHN-IU-2019-4921). а. left lateral view showing the capitulum and pedun­ cle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral view showing the capitulum and peduncle. Scale bar: 2 mm (a–d). Diagnosis. Capitulum with 13 fully calcified plates; cari­ na slightly bowed, umbo at apex; upper latus trapeziform; inframedian latus small, narrow; carinal latus not project­ ing beyond carina; peduncle short, cylindrical. Description. Catherinum rossi (Rao & Newman, 1972) Figure 22 Figure 22 Arcoscalpellum rossi Lakshmana Rao & Newman, 1972: 82, fig. 7. Arcoscalpellum rossi Lakshmana Rao & Newman, 1972: 82, fig. 7. Catherinum rossi. – Zevina, 1978b: 1348. – Liu and Ren 1985: 2 Catherinum rossi. – Zevina, 1978b: 1348. – Liu and Ren 1985: 203, fig. 12, pl. 2, figs 11–14. – 2007: 250, fig. 108. – Chan et al. 2009b: 113, figs 92–94. Distribution. Indian Ocean, Antarctic and Southern (North East of Prince Edward Island); known depth 2,516 m (Sha­ laeva and Boxshall 2014). In this study, Amigdoscalpellum tenue was found at Kei Islands, Indonesia. Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4933, Stn. DW 02, 05°47'S, 132°13'E, 209–240 m depth, 22 October 1991. Type locality. 46°46'S, 45°31'E; depth: 2,475 m; bottom temperature: 1.5 °C; bottom: globigerina ooze (Hoek 1883). Diagnosis. Capitulum smooth, elongate, 14 fully calci­ fied plates; inframedia latus rectangular, not constricted; rostrum large, ovo-triangular; caudal appendages short. Amigdoscalpellum tenue (Hoek, 1883) Figure 21 Capitulum yellowish, elongate-oval shape; surface with distinct lines of growth. Carina large, sim­ ply bowed. Scutum with umbo at apex, slightly recurved, projecting slightly over tergum; tergum triangular, stout, zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 672 umbo sub-basicarinal, not extending beyond carinal mar­ gin; caudal appendages long. broad, apex recurved, scutal margin almost straight. Up­ per latus quadrangular, apex slightly projecting over scu­ tum. Rostral latus quadrangular, scutal and basal margins parallel. Carinal latus quadrangular, carinal margin almost straight. Cirrus I unequal, anterior ramus oval, posterior ramus more slender. Maxillule with notch between two or three stout setae at upper angle, a group of more slen­ der setae on cutting edge; mandible with three teeth ex­ cluding inferior angle; labrum with numerous blunt teeth on straight, cutting edge. Measurements of specimen: height of capitulum 7.26 mm, width 3.74 mm, thickness 1.40 mm; length of peduncle 3.00 mm, width 1.99 mm. Type species. Scalpellum recurvitergum Gruvel, 1902; type locality: Atlantic Ocean, SW of Azores. Genus Catherinum Zevina, 1978 Description. Capitulum long, narrow, sparsely covered with hairs, plates separated by narrow, chitinous inter­ spaces, marked with growth lines. Occludent margin strongly convex; carinal margin irregularly straight; apex slightly retroverted towards carinal side. Carina long, simply bowed; roof flat; parietes well developed towards distal half of plate. Tergum triangular, occludent margin short, convex, scutal and basal margins almost straight, Acroscalpellum Hoek, 1907: 85. – Newman and Ross 1971: 42 (part). – Liu and Ren 1985: 202. Catherinum Zevina, 1978b: 1348. Acroscalpellum Hoek, 1907: 85. – Newman and Ross 1971: 42 (part). – Liu and Ren 1985: 202. Catherinum Zevina, 1978b: 1348. Acroscalpellum Hoek, 1907: 85. – Newman and Ross 1971: 42 (part). – Liu and Ren 1985: 202. Catherinum Zevina, 1978b: 1348. Catherinum Zevina, 1978b: 1348. Diagnosis. Capitulum with 13 or 14 plates; inframedian latus very narrow, bacilliform, umbo sub-medial; umbo of upper latus apical or sub-apical; carinal latus broad, Figure 22. Catherinum rossi (Rao & Newman, 1972) (MNHN-IU-2019-4933). а. left lateral view showing the capitulum and pe­ duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 3 mm (a–d). Figure 22. Catherinum rossi (Rao & Newman, 1972) (MNHN-IU-2019-4933). а. left lateral view showing the capitulum and pe­ duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 3 mm (a–d). zse.pensoft.net 673 Zoosyst. Evol. 96 (2) 2020, 649–698 Type species. Scalpellum planum Hoek, 1883; type lo­ cality: 42°42'S, 134°10'E; depth: 4,755 m; bottom tem­ perature: 0.2 °C; bottom: red clay. carinal margin concave. Scutum with umbo apical, over­ lapping occludent margin of tergum. Upper latus triangu­ lar; carinal latus twice as long as broad; inframedian latus rectangular; rostral latus nearly rectangular in outline; rostrum large, elongate triangular, broad above, pointed below. Cirrus I unequal, anterior ramus oval, posterior ramus more slender; cirrus VI with caudal appendages. Maxillule not notched stout spine along the cutting edge; mandible with three teeth excluding inferior angle. Mea­ surements of specimen: height of capitulum 9.67 mm, width 5.52 mm, thickness 2.58 mm; length of peduncle 2.79 mm, width 2.29 mm. Type species. Scalpellum planum Hoek, 1883; type lo­ cality: 42°42'S, 134°10'E; depth: 4,755 m; bottom tem­ perature: 0.2 °C; bottom: red clay. carinal margin concave. Scutum with umbo apical, over­ lapping occludent margin of tergum. Upper latus triangu­ lar; carinal latus twice as long as broad; inframedian latus rectangular; rostral latus nearly rectangular in outline; rostrum large, elongate triangular, broad above, pointed below. Cirrus I unequal, anterior ramus oval, posterior ramus more slender; cirrus VI with caudal appendages. Maxillule not notched stout spine along the cutting edge; mandible with three teeth excluding inferior angle. Mea­ surements of specimen: height of capitulum 9.67 mm, width 5.52 mm, thickness 2.58 mm; length of peduncle 2.79 mm, width 2.29 mm. Planoscalpellum distinctum (Hoek, 1883) Figure 23 Scalpellum distinctum Hoek, 1883: 111–112, pl. VI, figs 10, 11. – 1907: ii Scalpellum distinctum Hoek, 1883: 111–112, pl. VI, figs 10, 11. – 1907: 83, pl. VII, fig. 12 – Nilsson-Cantell 1927: 750, text-fig. 4. 83, pl. VII, fig. 12 – Nilsson-Cantell 1927: 750, text-fig. 4. Planoscalpellum distinctum. – Zevina, 1981: 186–187. – Foster and Buckeridge 1995 a: 360–361, fig. 7(A–C) – Shalaeva and Boxshall 2014: 36, fig. 22. Distribution. East China Sea, Pacific Ocean, Taiwan (Chan et al. 2009b). In this study, Catherinum rossi was found at Tanimbar Island, Indonesia. Material examined. – Tanimbar Island: 2 specimens, MNHN-IU-2019-4928, Stn. DW 49, 08°00'S, 132°59'E, 206–210 m depth, 29 October 1991. Type locality. Stn. 3, Hess Guyot (17°53.2'N, 174°24.8'W), 1,692–1,735 m depth (Sigsbee beam trawl); Stn. 1, Allison Guyot (18°31.0'N, 179°36.0'W), 1,413– 1,645 m depth (otter trawl) (Rao and Newman 1972). Diagnosis. Capitulum with 13 plates separated from each other by distinct chitinous interspaces; scutum almost tri­ angular; upper latus hexagonal, large. Description. Capitulum flattened, elongate-oval shape; plates with distinct growth lines, separated from each other by rather broad chitinous interspaces. Scutum tri­ angular, occludent margin arched, umbo of scutum api­ cal, apex slightly turned upwards. Tergum triangular, occludent margin not very long. Carina simply bowed. Upper latus irregularly hexagonal, rostral latus irreg­ ularly quadrangular, infra-median latus in the form of wine-glass, carinal latus irregularly quadrangular. Pe­ Teloscalpellum ecaudatum (Calman, 1918) Figure 24 Scalpellum ecaudatum Calman, 1918b: 106, text-fig. 2. Teloscalpellum ecaudatum. – Zevina, 1981: 365, fig. 282. – Chan 2009: 55, figs 1E, 7. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4917, Stn. CP 35, 06°08'S, 132°45'E, 390– 502 m depth, 27 October 1991. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4918, Stn. CP 59, 08°20'S, 132°11'E, 399–405 m depth, 31 Oc­ tober 1991. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4917, Stn. CP 35, 06°08'S, 132°45'E, 390– 502 m depth, 27 October 1991. Distribution. Pacific, Western Central; known depth range 1,302 to 2,745 m (Shalaeva and Boxshall 2014). In this study, Planoscalpellum distinctum was found at Tanimbar Island, Indonesia. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4918, Stn. CP 59, 08°20'S, 132°11'E, 399–405 m depth, 31 Oc­ tober 1991. Type locality. 2°33'S, 144°4'E; depth: 1,926 m; bot­ tom temperature: 2.1 °C; bottom: Globigerina ooze (Hoek 1883). Type locality. 2°33'S, 144°4'E; depth: 1,926 m; bot­ tom temperature: 2.1 °C; bottom: Globigerina ooze (Hoek 1883). Diagnosis. Capitulum compressed, elongated; 14 plates, sculptured with radiating ribs; inframedian latus triangu­ lar; rostro-lateral plates very low; penis and caudal ap­ pendages absent. Type species. Scalpellum spicatum Zevina, 1975. Type species. Scalpellum spicatum Zevina, 1975. duncle short, with very small, numerous, calcareous scales. Cirrus I unequal, anterior ramus oval, posterior ramus more slender; cirrus VI with short caudal append­ ages. Maxillule notched, three stout spines on the up­ per side; mandible with three teeth, excluding inferior angle. Measurements of two specimens: height of ca­ pitulum 6.80–7.90 mm, width 3.68–4.36 mm, thickness 1.24–1.26 mm; length of peduncle 1.39–2.49 mm, width 1.80–2.04 mm. Genus Planoscalpellum Zevina, 1978 Arcoscalpellum Hoek, 1907: 85 (part). Planoscalpellum Zevina, 1978b: 1347. Diagnosis. Upper latus with umbo on scutal margin; in­ framedian latus with umbo apical or sub-basal; carinal latus with umbo on carinal part, may be protruding. Figure 23. Planoscalpellum distinctum (Hoek, 1883) (MNHN-IU-2019-4928). а. left lateral view showing the capitulum and pe­ duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). Figure 23. Planoscalpellum distinctum (Hoek, 1883) (MNHN-IU-2019-4928). а. left lateral view showing the capitulum and pe­ duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 674 Genus Teloscalpellum Zevina, 1978 Arcoscalpellum Hoek, 1907: 85 (sectio, part.). – Newman and Ross 1971: 42 (part.); Section V. – Pilsbry 1907: 47 (part.). Description. Capitulum with all plates strongly ribbed, lines of growth marked by fine, inconspicuous striation. Tergum with occludent margin straight, carinal margin convex, but straight concave near apex. Scutum with occludent margin convex, other margins straight, apex slightly overlapping tergum. Carina evenly curved with flat roof bordered by strong ridges; upper latus quadran­ gular; rostrum small, triangular, overlapped at sides by rostral latera; inframedian latus very narrow. Peduncle shorter than capitulum, covered with strong scales. Cir­ Teloscalpellum Zevina, 1978b: 1350. – Liu and Ren 1985: 212. – 2007: 257. Diagnosis. Capitulum with 13 or 14 plates; carinal lateral umbo at basi-carinal angle, angle not extending beyond carinal margin; inframedian latus triangular or rod-like- shaped, umbo apical or sub-apical; caudal appendage multi-segmented. Figure 24. Teloscalpellum ecaudatum (Calman, 1918) (MNHN-IU-2019-4917). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 5 mm (a–d). Figure 24. Teloscalpellum ecaudatum (Calman, 1918) (MNHN-IU-2019-4917). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 5 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 675 ty: 5°42'S, 132°25'E; depth: 236 m; bottom: mud; it was found attached to an arm of a Comatula or Pentacrinus. ty: 5°42'S, 132°25'E; depth: 236 m; bottom: mud; it was found attached to an arm of a Comatula or Pentacrinus. rus I with unequal rami (anterior ramus: 11 segmented, posterior ramus: 13 segmented). Maxilla globular with serrulate setae; maxillule not notched; mandible with three major teeth, lower margin with three to four den­ ticles. Measurements of two specimens: height of capit­ ulum 12.07–18.59 mm, width 6.17–11.47 mm, thickness 2.68–5.41 mm; length of peduncle 6.30–8.33 mm, width 3.21–5.41 mm. Trianguloscalpellum balanoides (Hoek, 1883) Figure 25 Scalpellum balanoides Hoek, 1883: 129, pl. V, fig. 15, pl. X, fig. 11, pl. XI, figs 1–3. – Broch 1922: 242, fig. 10 – Weltner 1922: 63, taf. II, fig. 7, taf. III, fig. 8. – Nilsson-Cantell 1931: 2. – Hiro 1937b: 42, fig. 33. Scalpellum gonionotum Pilsbry, 1907b: 360. – Pilsbry 1911: 65, pl. IX, figs 2–4. – Hiro 1937a: 43. Trianguloscalpellum balanoides. – Zevina, 1978b: 1349. – 1981: 294, fig. 221. – Liu and Ren 1985: 210, fig. 16, pl. XI, figs 15–17. – 2007: 256–257, fig. 111. – Rosell 1991: 20–22, fig. 2c, d. – Huang 1994: 517. – 2001: 318. – Jones et al. 2001: 253. –Young 2001: 464, fig. 7. – Jones 2007: 292. – Chan et al. 2009b: 116, figs 95, 96. – Shalaeva and Boxshall 2014: 40, fig. 26. Scalpellum balanoides Hoek, 1883: 129, pl. V, fig. 15, pl. X, fig. 11, pl. XI, figs 1–3. – Broch 1922: 242, fig. 10 – Weltner 1922: 63, taf. II, fig. 7, taf. III, fig. 8. – Nilsson-Cantell 1931: 2. – Hiro 1937b: 42, fig. 33. Scalpellum gonionotum Pilsbry, 1907b: 360. – Pilsbry 1911: 65, pl. IX, figs 2–4. – Hiro 1937a: 43. Trianguloscalpellum balanoides. – Zevina, 1978b: 1349. – 1981: 294, fig. 221. – Liu and Ren 1985: 210, fig. 16, pl. XI, figs 15–17. – 2007: 256–257, fig. 111. – Rosell 1991: 20–22, fig. 2c, d. – Huang 1994: 517. – 2001: 318. – Jones et al. 2001: 253. –Young 2001: 464, fig. 7. – Jones 2007: 292. – Chan et al. 2009b: 116, figs 95, 96. – Shalaeva and Boxshall 2014: 40, fig. 26. Distribution. Java Sea, Indonesia; Philippines (Chan 2009). In this study, Teloscalpellum ecaudatum was found at Kei Islands and Tanimbar Island, Indonesia. Type locality. Java Sea; 7°35'S, 114°30'30"E; 132–315 m depth (Calman 1918b). Type locality. Java Sea; 7°35'S, 114°30'30"E; 132–315 m depth (Calman 1918b). Genus Trianguloscalpellum Zevina, 1978b Carina very short, simply, not very strongly, bowed, umbo at apex. Upper latus tri­ angular, carinal margin absent. Rostral latus quadrangu­ lar, umbo at apex of angle formed by rostral and scutal margins. Infra-median latus elongate, triangular, umbo apical. Carinal latus very large, convex, umbo at top of carinal margin. Cirrus I unequal, posterior ramus more slender than anterior ramus; cirrus VI with caudal ap­ pendages. Maxillule distinctly notched, two stout spines on upper side; mandible with three teeth excluding inferi­ or angle. Measurements of specimen: height of capitulum 8.20 mm, width 4.40 mm, thickness 1.75 mm; length of peduncle 5.06 mm, width 2.10 mm. Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4908, Stn. CP 91, 08°44'S, 131°05'E, 884–891 m depth, 5 November 1991. Diagnosis. Capitulum with 14 plates covered by mem­ brane, covered by very long hairs; carina simply bowed, umbo at apex, roof flat; upper latus triangular; rostrum very narrow stripe distinctly visible at surface. Description. Capitulum small, long hairs covering plates. Scutum elongate, convex, more than twice as long as broad. Tergum large, elongate rhomboid, umbo at apex. Carina simply bowed, roof flat. Upper latus al­ most triangular, carinal-basal margin arched. Rostrum small, linear-shaped. Rostral latus quadrangular, basal and scutal margins parallel. Infra-median latus triangu­ lar, umbo apical. Carinal latus with irregular shape, umbo near apex. Peduncle short. Cirrus I with rami unequal, an­ terior ramus oval, posterior ramus more slender; cirrus VI with caudal appendages. Maxillule not notched, two stout spines on upper side; mandible with three teeth excluding inferior angle. Measurements of the specimen: height of capitulum 12.18 mm, width 6.50 mm, thickness 3.74 mm; length of peduncle 4.10 mm, width 3.43 mm. Description. Capitulum small, long hairs covering plates. Scutum elongate, convex, more than twice as long as broad. Tergum large, elongate rhomboid, umbo at apex. Carina simply bowed, roof flat. Upper latus al­ most triangular, carinal-basal margin arched. Rostrum small, linear-shaped. Rostral latus quadrangular, basal and scutal margins parallel. Infra-median latus triangu­ lar, umbo apical. Carinal latus with irregular shape, umbo near apex. Peduncle short. Cirrus I with rami unequal, an­ terior ramus oval, posterior ramus more slender; cirrus VI with caudal appendages. Maxillule not notched, two stout spines on upper side; mandible with three teeth excluding inferior angle. Measurements of the specimen: height of capitulum 12.18 mm, width 6.50 mm, thickness 3.74 mm; length of peduncle 4.10 mm, width 3.43 mm. Distribution. Genus Trianguloscalpellum Zevina, 1978b Arcoscalpellum Hoek, 1907: 85 (part). – Pilsbry 1907a: 47. – Newman and Ross 1971: 42 (part). Trianguloscalpellum Zevina, 1978b: 1349. – Liu and Ren 1985: 205. Arcoscalpellum Hoek, 1907: 85 (part). – Pilsbry 1907a: 47. – Newman and Ross 1971: 42 (part). Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4923, Stn. CP 16, 05°17'S, 132°50'E, 330– 350 m depth, 24 October 1991, attached to crinoid. – Tanimbar Island: 2 specimens, MNHN- IU-2019-4924, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 November 1991. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4923, Stn. CP 16, 05°17'S, 132°50'E, 330– 350 m depth, 24 October 1991, attached to crinoid. Trianguloscalpellum Zevina, 1978b: 1349. – Liu and Ren 1985: 205. – Tanimbar Island: 2 specimens, MNHN- IU-2019-4924, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 November 1991. Diagnosis. Capitulum of female or hermaphrodite with 13 or 14 completely calcified plates; tergum and scutum with umbones apical; middle latera triangular, sometimes quadrangular, with apical umbo reaching upper latera; ca­ rinolatera triangular or subtriangular with apical umbo; caudal appendages distinctly long. Diagnosis. Capitulum with 13 fully calcified plates, cov­ ered by thin membrane; carina and extremely short, sim­ ply bowed without distinct roof; umbo of carina at apex; upper latus triangular; carinal latus almost as high as ca­ rina, umbo apical, carinal latus very large, almost similar size to tergum. Type species. Scalpellum balanoides Hoek, 1883: 129, pl. V, fig. 15, pl. X, fig. 11, pl. XI, figs 1–3; type locali­ Figure 25. Trianguloscalpellum balanoides (Hoek, 1883) (MNHN-IU-2019-4923). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). Figure 25. Trianguloscalpellum balanoides (Hoek, 1883) (MNHN-IU-2019-4923). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 676 figs 1F, 9A–H. – Jones 2012: 371, 376. – Shalaeva and Boxshall 2014: 44, fig. 29. Description. Capitulum flat, narrow at base, same breadth as peduncle. Scutum about twice as long as broad; occlu­ dent margin almost straight, umbo at apex. Tergum trian­ gular, the same size as scutum. Genus Trianguloscalpellum Zevina, 1978b Pacific, Western Central; South and East China Sea; South of Sumatra, Banda Sea, Indonesia; Vietnam; Philippines; Taiwan; South of Japan; attached to crinoids, hydroids; 220–1,097 m depth (Jones et al. 2001; Chan et al. 2009b; Shalaeva and Boxshall 2014). In this study, Trianguloscalpellum balanoides was found at Kei Islands and Tanimbar Island, Indonesia. Type locality. 5°42'S, 132°25'E; depth: 232 m; bottom: mud; attached to arm of a Comatula or Pentacrinus (Hoek 1883). Distribution. Borneo, Indonesia; Pacific, Western Cen­ tral and Southeast; Australia; Taiwan; Antarctica; at­ tached to gastropod shells, rocks; 1,502–1,965 m depth (Chan et al. 2010; Jones 2012; Shalaeva and Boxshall 2014). In this study, Trianguloscalpellum hirsutum was found at Tanimbar Island, Indonesia. Trianguloscalpellum hirsutum (Hoek, 1883) Figure 26 Scalpellum hirsutum Hoek, 1883: 88, pl. IV, fig. 19. – Gruvel 1905: 66, fig. 74. – Pilsbry 1907a: 25. Scalpellum hirsutum Hoek, 1883: 88, pl. IV, fig. 19. – Gruvel 1905: 66, fig. 74. – Pilsbry 1907a: 25. Arcoscalpellum hirsutum. – Newman & Ross, 1971: 62–64, fig. 28. Trianguloscalpellum hirsutum. – Zevina, 1981: 309, fig. 233. – Chan et al. 2009b: 119–121, figs 100–102. – Chan et al. 2010: 13, Arcoscalpellum hirsutum. – Newman & Ross, 1971: 62–64, fig. 28. Type locality. Pacific, Western Central (Moluccas Sea); 0°48'S, 120°58'E; depth: 252 m; bottom temperature: 2.4 °C; bottom: rock (Hoek 1883). Trianguloscalpellum hirsutum. – Zevina, 1981: 309, fig. 233. – Chan et al. 2009b: 119–121, figs 100–102. – Chan et al. 2010: 13, Figure 26. Trianguloscalpellum hirsutum (Hoek, 1883) (MNHN-IU-2019-4908). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 5 mm (a–d). Figure 26. Trianguloscalpellum hirsutum (Hoek, 1883) (MNHN-IU-2019-4908). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 5 mm (a–d). zse.pensoft.net 677 Zoosyst. Evol. 96 (2) 2020, 649–698 Genus Verum Zevina, 1978 Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4930, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 November 1991. Arcoscalpellum Pilsbry, 1907: 47 (part). – Newman and Ross 1971: 42 (part). Verum Zevina, 1978b: 1348. Arcoscalpellum Pilsbry, 1907: 47 (part). – Newman and Ross 1971: 42 (part). Verum Zevina, 1978b: 1348. Arcoscalpellum Pilsbry, 1907: 47 (part). – Newman and Ross 1971: 42 (part). Verum Zevina, 1978b: 1348. Diagnosis. Capitulum with 13 plates closely locked to­ gether, surface smooth with slightly prominent ridges; carina simply bowed with flat roof; upper latus trapezi­ form; inframedian latus elongate, narrow; carinal latus with umbo projecting beyond carina. Verum Zevina, 1978b: 1348. Diagnosis. Capitulum of female or hermaphrodite, with 13–14 completely calcified plates; tergum with carinal margin straight or slightly to moderately recurving; scutum with or without short apico-lateral arm, umbo apical; carina with apical or subapical umbo, evenly curved or angular­ ly flexed; upper latera sub-triangular or pentagonal, umbo apical or subapical; middle latera narrow, triangular or quadrangular, umbo basal or sub-basal; carinolatera short, umbo basal to sub-basocarinal, usually not or slightly pro­ jecting beyond rim of capitulum; caudal appendages short. Description. Capitulum elongate-oval, flat with distinct ridges and furrow. Scutum slightly convex, umbo apical, oc­ cludent margin arched. Tergum triangular, umbo apical, dis­ tinctly recurved. Rostral latus irregularly quadrangular; infra median latus narrow, umbo near base; carinal latus quadran­ gular. Peduncle slightly conical, short, scales near capitulum larger than those near base. Cirrus I unequal, anterior ramus oval, posterior ramus more slender; cirri II to VI very long, rami subequal, segments elongate; cirrus VI with caudal ap­ pendages. Maxillule not notched, large spine on upper side, cutting edge almost straight; mandible with three large teeth excluding inferior angle. Measurements of specimen: height of capitulum 10.33 mm, width 6.09 mm, thickness 2.08 mm; length of peduncle 4.62 mm, width 2.24 mm. Type species. Scalpellum zenkevichi Zevina, 1972: 44, fig. 3; type locality: Pacific Ocean, 24°27'S, 70°42'E. Scalpellum australicum Hoek, 1883: 118, pl. V, fig. 11. – Weltner 1922: 63. – Nilsson-Cantell 1938: 7. Verum australicum. – Zevina, 1981: 223–224, fig. 160. – Jones et al. 1990: 5. – Jones 1992: 156–160, figs 7–8. – Jones 2012: 371, 376. – Shalaeva and Boxshall 2014: 52, fig. 36. Scalpellum novae-zelandiae Hoek, 1883: 124, pl. V, figs 7, 8. – 1907: 100, pl. VIII, figs 4, 4a; 1913: 14. – Weltner 1897: 249. – 1922: 71. – Annandale 1905: 83. – 1906b: 389. – 1908: pl. 5, fig. 7. – 1913: 231. – Broch 1927a: 540, fig. 512. Scalpellum Novae-Zealandiae. – Gruvel, 1905: 88, fig. 99. Scalpellum (Scalpellum) novae-zelandiae. – Calman, 1918b: 123. Arcoscalpellum novaezelandiae. – Foster, 1978: 65, pl. 8D, fig. 39. Verum carinatum (Hoek, 1883) Figure 28 Scalpellum carinatum Hoek, 1883: 76, pl. III, figs 7, 8. – Pilsbry 1907a: 53, fig. 18. – Gruvel 1920: 20. – Barnard 1925: 3. – Broch 1953: 7. – Weisbord 1977: 243–244, pl. 27, figs 3, 4. Scalpellum imperfectum Pilsbry, 1907a: 75, fig. 30, pl. IV, figs 15–18. – Annandale 1913: 233. – Barnard 1924: 47. – MacDonald 1929: 537. – Broch 1953: 9. – Stubbings 1961: 11, text-fig. 2. – 1967: 234. Meroscalpellum imperfectum. – Newman & Ross, 1971: 119, text-fig. 62. Verum carinatum. – Zevina, 1981: 225–226, fig. 163. – Shalaeva and Boxshall 2014: 53, fig. 37. Scalpellum carinatum Hoek, 1883: 76, pl. III, figs 7, 8. – Pilsbry 1907a: 53, fig. 18. – Gruvel 1920: 20. – Barnard 1925: 3. – Broch 1953: 7. – Weisbord 1977: 243–244, pl. 27, figs 3, 4. Scalpellum imperfectum Pilsbry, 1907a: 75, fig. 30, pl. IV, figs 15–18. – Annandale 1913: 233. – Barnard 1924: 47. – MacDonald 1929: 537. – Broch 1953: 9. – Stubbings 1961: 11, text-fig. 2. – 1967: 234. Meroscalpellum imperfectum. – Newman & Ross, 1971: 119, text-fig. 62. Verum carinatum. – Zevina, 1981: 225–226, fig. 163. – Shalaeva and Boxshall 2014: 53, fig. 37. Distribution. Atlantic, excluding polar areas; Pacific, Southeast. Known depth range 600 to 2,400 m (Shalaeva and Boxshall 2014). In this study, Verum carinatum was found at Kei Islands, Indonesia. Boxshall 2014: 53, fig. 37. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4920, Stn. DW 18, 05°18'S, 133°01'E, 205– 212 m depth, 24 October 1991. Type locality. Station 135, near the Island of Tristan da Cunha; depth: 1,800 m; bottom: rock, shells (Hoek 1883). Verum australicum (Hoek, 1883) Figure 27 Figure 27 Scalpellum australicum Hoek, 1883: 118, pl. V, fig. 11. – Weltner 1922: 63. – Nilsson-Cantell 1938: 7. Verum australicum. – Zevina, 1981: 223–224, fig. 160. – Jones et al. 1990: 5. – Jones 1992: 156–160, figs 7–8. – Jones 2012: 371, 376. – Shalaeva and Boxshall 2014: 52, fig. 36. Scalpellum australicum Hoek, 1883: 118, pl. V, fig. 11. – Weltner 1922: 63. – Nilsson-Cantell 1938: 7. Distribution. Indian Ocean, Western; Pacific Western Central and Southeast; Zanzibar; known depth range 463– 2,561 m (Shalaeva and Boxshall 2014). In this study, Ver­ um australicum was found at Tanimbar Island, Indonesia. Verum australicum. – Zevina, 1981: 223–224, fig. 160. – Jones et al. 1990: 5. – Jones 1992: 156–160, figs 7–8. – Jones 2012: 371, 376. – Shalaeva and Boxshall 2014: 52, fig. 36. Figure 27. Verum australicum (Hoek, 1883) (MNHN-IU-2019-4930). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 3 mm (a–d). Figure 27. Verum australicum (Hoek, 1883) (MNHN-IU-2019-4930). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 3 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 678 Type locality. between New Guinea and Australia (12°08'S, 145°10'E): Challenger station 184: depth: 2,561 m: bottom temperature: 1.8 °C; substrate grey ooze (Hoek 1883). latus flat, irregular pentagonal; rostrum narrow, elon­ gated; rostral lateral convex with rostral margin short; inframedian latus wine-glass-shaped; carinal latus flat, large. Peduncle short, calcareous scales distinct. Cirrus I unequal, anterior ramus broader than posterior ramus; cirri II to VI long, rami equal; cirrus VI with caudal appendages. Maxillule not notched, two large spines on upper side, cutting edge almost straight; mandible with three large teeth excluding inferior angle. Mea­ surements of specimen: height of capitulum 9.14 mm, width 4.53 mm, thickness 1.74 mm; length of peduncle 2.54 mm and width 2.20 mm. Verum novaezelandiae (Hoek, 1883) Diagnosis. Capitulum with 14 plates separated by chitin­ ous interspaces, surface smooth; carina bowed, angularly bent, with flat roof; upper latus irregular pentagonal; in­ framedian latus large, wine-glass-shaped. Figure 29 Description. Capitulum elongate, plates covered by thin, chitinous membrane. Scutum elongated, apex pointed, occludent margin very convex. Tergum flat, triangular, apex very recurved, occludent margin very arched. Carina with umbo at top of flat roof. Upper rcoscalpellum novaezelandiae. – Foster, 1978: 65, pl. 8D, fig. 39. Figure 28. Verum carinatum (Hoek, 1883) (MNHN-IU-2019-4920). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 3 mm (a–d). Figure 28. Verum carinatum (Hoek, 1883) (MNHN-IU-2019-4920). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 3 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 679 Figure 29. Verum novaezelandiae (Hoek, 1883) (MNHN-IU-2019-4919). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 3 mm (a–d). Figure 29. Verum novaezelandiae (Hoek, 1883) (MNHN-IU-2019-4919). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale bar: 3 mm (a–d). non Scalpellum Novae-Zelandiae. – Gruvel, 1902: 54, pl. 2, figs 12, 13, 15. – Gruvel 1912: 346 (= V. parazelandiae Young, 1998b). Verum novaezelandiae. – Zevina, 1978b: 1348. – Zevina 1981: 228, fig. 165. – Chan et al. 2009b: 17, figs 1I, 12A–H, 13A–D. – Chan et al. 2010: 17, figs 11–13. – Shalaeva and Boxshall 2014: 53, fig. 39. 13.46 mm, width 6.98 mm, thickness 2.99 mm; length of peduncle 6.02 mm, width 3.23 mm. non Scalpellum Novae-Zelandiae. – Gruvel, 1902: 54, pl. 2, figs 12, 13, 15. – Gruvel 1912: 346 (= V. parazelandiae Young, 1998b). Verum novaezelandiae. – Zevina, 1978b: 1348. – Zevina 1981: 228, fig. 165. – Chan et al. 2009b: 17, figs 1I, 12A–H, 13A–D. – Chan et al. 2010: 17, figs 11–13. – Shalaeva and Boxshall 2014: 53, fig. 39. Distribution. Suborder VERRUCOMORPHA, Pilsbry 1916 Description. Capitulum flatted, elongate, plates separat­ ed by narrow, chitinous interspaces. Scutum quadrangu­ lar, umbo apical, occludent margin arched. Tergum tri­ angular with umbo slightly produced apex. Carina with bowed, flat roof, increasing little in width from upper to lower. Upper latus irregularly trapeziform; rostral latus with umbo at top of rostral margin; infra-median latus irregular, quadrangular, elongate, narrow; carina latus quadrangular. Cirrus I unequal, anterior ramus broad­ er than posterior ramus; cirrus VI with short caudal ap­ pendages. Maxillule slightly notched, two large spines on upper side; mandible with three large teeth excluding inferior angle; labrum slightly convex, with numerous teeth. Measurements of specimen: height of capitulum Verrucomorpha Pilsbry, 1916: 14. – Newman et al. 1969: 281. – New­ man and Ross 1971: 135. – Newman 1987: 8. – 1996: 501. – Bucker­ idge 1994: 89. – Young 1998: 74. Verrucomorpha Pilsbry, 1916: 14. – Newman et al. 1969: 281. – New­ man and Ross 1971: 135. – Newman 1987: 8. – 1996: 501. – Bucker­ idge 1994: 89. – Young 1998: 74. Diagnosis. Shell asymmetrical with four plates (carina, ros­ trum, tergum and scutum), base membranous or calcareous. Verum novaezelandiae (Hoek, 1883) Indo-west Pacific: Eastern Africa, Gulf of Aden, eastwards to New Zealand; Philippines; Taiwan; at­ tached to sunken wood, gorgonians, glassy spicule of hex­ actinellid sponges; 822–4850 m depth (Shalaeva and Box­ shall 2014; Jones and Hosie 2016). In this study, Verum novaezelandiae was found at Tanimbar Island, Indonesia. Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4919, Stn. CP 87, 08°47'S, 130°49'E, 1017–1024 m depth, 5 November 1991. Diagnosis. Capitulum with 13 plates covered by very thin, chitinous membrane; carina simply bowed, flat roof with umbo apical; upper latus quadrangular or rather pentagonal, lower edge being truncated; inframedian la­ tus elongate, quadrangular; carinal latus large, elongate, umbo near base; peduncle short. Type locality. 37°34'S, 179°22'E; depth: 1,260 m; bottom temperature: 4.2 °C; bottom: grey ooze (Hoek 1883). Family VERRUCIDAE Darwin, 1854 Verrucidae Darwin, 1854: 495. – Gruvel 1905: 169. – Withers 1935: 323. – Newman et al. 1969: 281. – Newman and Ross 1971: 135. – Buckeridge 1994: 89. – Young 1998: 74. Verrucidae Darwin, 1854: 495. – Gruvel 1905: 169. – Withers 1935: 323. – Newman et al. 1969: 281. – Newman and Ross 1971: 135. – Buckeridge 1994: 89. – Young 1998: 74. zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 680 Diagnosis. Scutum and tergum without depressor mus­ cles; movable only on one side; other side immovable, united with rostrum and carina. face with numerous articular ridges. Movable tergum large, quadrangular; surface with strongly developed, curved ax­ ial articular ridge. Carina and rostrum irregular quadrangu­ lar, with carina higher, rostrum broader. Fixed tergum with two parts: (1) triangular portion very narrow at apex, slight­ ly broader in its inferior (2) flat and broad part at a rear por­ tion of shell. Fixed scutum pointed with distinctly beaked apex; composed of broader, nearly flat, triangular portion and narrower inflected portion, only widening towards its inferior. Base of shell elongatedly oval-shaped. Cirrus I with rami very unequal (anterior ramus: 12-segmented, posterior ramus: 28-segmented); cirrus VI with caudal ap­ pendages. Maxilla bilobed, fringed with setae, except on the notch; maxillule widely notched, horizontally elongat­ ed, two large spines above notch, numerous dense setae at notch; mandible with three teeth excluding inferior angle; labrum slightly concave, conical teeth on cutting margin. Genus Alteriverruca Pilsbry, 1916 Verruca Section D Altiverruca Pilsbry, 1916: 40. Altiverruca Pilsbry, 1916: 40 – Newman et al. 1969: 282. – Newman and Ross 1971: 135. – Ren 1984b: 172. – Buckeridge 1994: 92. – Young 1998: 77. Altiverruca navicula (Hoek, 1913) Figure 30 Distribution. Pacific Ocean (Chan et al. 2010). In this study, Altiverruca navicula was found at Tanimbar Is­ land, Indonesia. Verruca navicula Hoek, 1913: 134, figs 4–6. – Nilsson-Cantell 1927: 778, figs a–f. Altiverruca navicula. – Buckeridge 1994: 100, fig. 5. – Chan et al. 2009b: 280, figs 245–247. – Chan et al. 2010: 29, figs 2E, 21, 22. Verruca navicula Hoek, 1913: 134, figs 4–6. – Nilsson-Cantell 1927: 778, figs a–f. Type locality. (1) 9°3.4'S, 119°56.7'E; depth 959 m; bot­ tom: globigerina ooze. (2) 3°37.7'S, 131°26.4'E; depth 924 m; bottom: fine grey mud (Hoek 1913). Altiverruca navicula. – Buckeridge 1994: 100, fig. 5. – Chan et al. 2009b: 280, figs 245–247. – Chan et al. 2010: 29, figs 2E, 21, 22. Material examined. – Tanimbar Island: 3 specimens, MNHN-IU-2019-4941, Stn. CP 91, 08°44'S, 131°05'E, 884–891 m depth, 5 November 1991, attached to pedun­ cle of Alcockianum persona (Annandale, 1916). Diagnosis. Verrucids with erect form; bases of plates not inflected. Operculum close to vertical, myophore absent. Diagnosis. Verrucids with erect form; bases of plates not inflected. Operculum close to vertical, myophore absent. Type species. Verruca hoeki Pilsbry, 1907. Newmaniverruca albatrossiana (Pilsbry, 1912) Figure 31 Operculata tribe Symetrica Gruvel, 1905: 189. Balanomorpha Pilsbry, 1916: 47 (Balanidae sensu Darwin, 1854). Operculata tribe Symetrica Gruvel, 1905: 189. Balanomorpha Pilsbry, 1916: 47 (Balanidae sensu Darwin, 1854). Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47. – Buckeridge 1994: 91, fig. 1a–f. – Buckeridge 1997: 129. Verruca grex Hoek, 1913: 142, pl. XI, figs 7–13, pl. XIII, figs 11–13. Verruca (Euverruca) albatrossiana. – Broch, 1931: 45. Verruca (Verruca) albatrossiana. – Ren, 1984: 168, fig. 2, pl. 1 (7–11). Newmaniverruca albatrossiana. – Young, 1998: 77. – Chan 2009: 74, figs 2G, 22A–I. Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47. – Buckeridge 1994: 91, fig. 1a–f. – Buckeridge 1997: 129. Verruca grex Hoek, 1913: 142, pl. XI, figs 7–13, pl. XIII, figs 11–13. Verruca (Euverruca) albatrossiana. – Broch, 1931: 45. Verruca (Verruca) albatrossiana. – Ren, 1984: 168, fig. 2, pl. 1 (7–11). N i lb t i Y 1998 77 Ch 2009 74 Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47. – Buckeridge 1994: 91, fig. 1a–f. – Buckeridge 1997: 129. Diagnosis. Peduncle absent; shells bilaterally symmetri­ cal on either side of rostrocarinal axis; shell with parietal plates of carina, rostrum and one to three pairs of lateral plates, all separate from one another, variously fused or totally concrescent; base membranous or calcareous; her­ maphroditic or with dwarf males. erruca grex Hoek, 1913: 142, pl. XI, figs 7–13, pl. XIII, figs 11–13 Verruca (Euverruca) albatrossiana. – Broch, 1931: 45. Verruca (Verruca) albatrossiana. – Ren, 1984: 168, fig. 2, pl. 1 (7–11). Newmaniverruca albatrossiana. – Young, 1998: 77. – Chan 2009: 74, Newmaniverruca albatrossiana. – Young, 1998: 77. – Chan 2009: 74, figs 2G, 22A–I. Material examined. – Kei Islands: 1 specimen, MNHN- IU-2019-4942, Stn. CC 21, 05°14'S, 133°00'E, 688–694 m depth, 25 October 1991, attached to glass rope sponge. Family BATHYLASMATIDAE Newman & Ross, 1971 Bathylasmatidae Newman & Ross, 1971: 138 (part.). – Newman and Ross 1976: 37, 45 (part.). Bathylasmatidae Newman & Ross, 1971: 138 (part.). – Newman and Ross 1976: 37, 45 (part.). Bathylasmatidae Newman & Ross, 1971: 138 (part.). – Newman and Ross 1976: 37, 45 (part.). Bathylasmatinae Newman & Ross, 1976: 37, 45. – Buckeridge 1983: 68. – Newman 1996: 502. – Buckeridge 1999: 522. – Jones 2000: 231, tables 1, 2. Diagnosis. Rostrum and fixed scutum with unusual length; rostrum low, fixed scutum larger than fixed tergum. Bathylasmatinae Newman & Ross, 1976: 37, 45. – Buckeridge 1983: 68. – Newman 1996: 502. – Buckeridge 1999: 522. – Jones 2000: 231, tables 1, 2. Description. Movable plates parallel to base, wall of parietal vertically ribbed; fixed scutum without internal pit. Movable scutum with crescentic ridge and longi­ tudinal striations; movable tergum with articular ribs and diagonal rib. Apices of fixed scutum and tergum contiguous. Carina occupying carino-rostral wall, api­ ces marginal. Cirrus I with rami unequal and serrulate setae; cirrus VI with caudal appendages. Maxilla glob­ ular, with fringing setae; maxillule notched, two large setae on upper side; mandible with three teeth exclud­ ing inferior angle. Diagnosis. Shell with four or six plates; wall solid or per­ meated by single row of chitin-filled longitudinal canals; radii absent; one or both rami of cirri I and cirri II some­ times antenniform; labrum without notch in crest. Genus Newmaniverruca Young, 1998 Verruca Section B: Verruca, Group of Verruca alba Pilsbry, 1916: 25 (part.). Verruca Section B: Verruca, Group of Verruca calotheca Pilsbry, 1916: 30 (part.). Verruca (Verruca): Foster, 1979: 68 (part.). – Zevina 1987: 1812 (part. – Buckeridge 1994: 90 (part.). Newmaniverruca Young, 1998: 77. Verruca Section B: Verruca, Group of Verruca alba Pilsbry, 1916: 25 (part.). Verruca Section B: Verruca, Group of Verruca calotheca Pilsbry, 1916: 30 (part.). Diagnosis. Shell not depressed; carina and rostrum inter­ locking with single rib from each plate; movable plates large, scutum with four articular ribs, tergum with six articu­ lar ribs, growth lines very distinct; caudal appendages long. Newmaniverruca Young, 1998: 77. Description. Shell yellowish. Movable scutum elongately triangular, apex distinctly beaked, projecting freely; sur­ Diagnosis. Shell with four plates (carina, rostrum, scu­ tum and tergum); operculum parallel to base. Description. Shell yellowish. Movable scutum elongately triangular, apex distinctly beaked, projecting freely; sur­ Figure 30. Altiverruca navicula (Hoek, 1913) (MNHN-IU-2019-4941). а. left lateral view; b. right lateral view; c. upper lateral view; d. lower lateral view. Scale bar: 7 mm (a–d). Figure 30. Altiverruca navicula (Hoek, 1913) (MNHN-IU-2019-4941). а. left lateral view; b. right lateral view; c. upper lateral view; d. lower lateral view. Scale bar: 7 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 681 Type locality. East of Luzon, Philippines (Pilsbry 1912). Type species. Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47; type locality: East of Luzon, Philippines. Hexelasminae Newman & Ross, 1976: 37, 46. Hexelasminae Newman & Ross, 1976: 37, 46. Diagnosis. Shell with six plates, infiltrated by chitin-filled tubes; basis calcareous; scuta parallel to basis; tergum with distinct spur; cirrus II resembling cirrus I more than cirrus III. Distribution. Indo-west Pacific: NW Australia; South China Sea; China; Philippines; attached to cidaroid spines (e.g. Stereocidaris indica philippinensis Mortensen, 1928), 345–620 m depth (Jones and Hosie 2016). In this study, Newmaniverruca albatrossiana was found at Kei Islands, Indonesia. Type genus. Hexalasma Hoek, 1913. Figure 32 Figure 32 Hexelasma arafurae Hoek, 1913: 251, pl. 25, figs 12–16. – Utinomi 1965: 11. – Newman and Ross 1971: 155. – 1976: 46. – Foster 1981: fig. 6F–H. – Jones 2000: 246, fig. 51, tables 28–31. Aaptolasma arafura. – Foster, 1978: 79. Type genus. Hexalasma Hoek, 1913. Figure 31. Newmaniverruca albatrossiana (Pilsbry, 1912) (MNHN-IU-2019-4942). а. left lateral view; b. right lateral view; c. up­ per lateral view; d. lower lateral view. Scale bar: 3 mm (a–d). Figure 31. Newmaniverruca albatrossiana (Pilsbry, 1912) (MNHN-IU-2019-4942). а. left lateral view; b. right lateral view; c. up­ per lateral view; d. lower lateral view. Scale bar: 3 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 682 Genus Hexelasma Hoek, 1913 apex beaked, carinal margin rounded, growth-ridges less distinct than on scutum. Cirrus I with unequal rami (an­ terior ramus: 8-segmented; posterior ramus: 12-segment­ ed), dense long setae on surface areas. Cirrus II with equal rami, dense long setae. Cirri IV–VI with equal rami with numerous segments; segments almost without exception furnished with two pairs very long, stiff, needle-like spines along inner faces. Measurements of specimen: basal length of shell 14.32 mm, orifice length 8.00 mm, carinal height 12.39 mm, orifice width 6.49 mm, basal width 13.76 mm. Hexelasma Hoek, 1913: 244 (part.). – Utinomi 1965: 13. – Jones 2000: 240, figs 51, 64, tables 28–30. Aaptolasma Newman & Ross, 1971: 158. Diagnosis. Parietes solid; radii absent; plates with a ca­ rina, paired carinolatera and latera and a rostrum without any sign of fusion; caudal appendages absent. Type species. Hexelasma velutinum Hoek, 1913: 246 (part.); type locality: Indonesia, 6°08'S, 121°19'E (Sibo­ ga Station 105). Distribution. Arafura Sea, Indonesia; 205–560 m depth (Jones and Hosie 2016). In this study, Hexelasma arafu­ rae was found at Kei Islands, Indonesia. Hexelasma arafurae Hoek, 1913 Figure 32 Type locality. Indonesia; Siboga stat. 262; 5°54'S, 132°49'E; 560 m depth; bottom: solid bluish-grey mud (Hoek 1913). Superfamily BALANOIDEA Leach, 1817 Balanidae Leach, 1817: 68 – Pilsbry 1916: 48 – Newman et al. 1969: 284 – Newman and Ross 1976: 38, 49 – Newman 1993: 408. Balaninae – Darwin, 1854: 175. Balanoidea – Newman, 1996: 502. Material examined. – Kei Islands: 2 specimens, MNHN- IU-2019-4851, Stn. DW 18, 05°18'S, 133°01'E, 205– 212 m depth, 24 October 1991. Diagnosis. Shell wall composed of four or six plates (rostrum, carina and one to three pairs of laterals); pa­ rietes solid or tubiferous, when tubiferous rarely sec­ ondarily filled; radii solid or tubiferous. Basis com­ monly calcareous, solid or permeated by tubes, rarely membranous; when basis calcareous, internal surfaces of compartments commonly with uniform ribs; when calcareous commonly forming complex interdigitations with wall. Opercular plates occlude aperture; articu­ lations between the pairs generally shallow or fused. Cirrus I with rami subequal or grossly unequal; cirri II Diagnosis. Shell with very wide orifice; alae broadly-tri­ angular with summits oblique; scutum folded longitudi­ nally, articular ridge slightly prominent, deep pit for ad­ ductor muscle; tergum beaked, with prominent articular ridge, spur very broadly rounded; inner side of scutum and tergum orange-coloured. Description. Shell yellowish, conical, with six plates. Or­ ifice diamond-shaped; scutum triangular elongated with protruding growth-ridges; tergum smaller than scutum, Figure 32. Hexelasma arafurae Hoek, 1913 (MNHN-IU-2019-4851). а. view from above; b. right lateral view. Scale bar: 4 mm (a, b). exelasma arafurae Hoek, 1913 (MNHN-IU-2019-4851). а. view from above; b. right lateral view. Scale bar: 4 mm (a, b). Figure 32. Hexelasma arafurae Hoek, 1913 (MNHN-IU-2019-4851). а. view from above; b. right lateral view. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 683 and III with rami never antenniform; cirrus III resem­ bling II more than IV; caudal appendages absent; penis with basidorsal point. Labrum thin, never bullate; crest with pronounced medial incision; mandible quadri- or quinquedentoid; second and following teeth with one or more subsidiary cusps; fifth tooth often vestigial; inferi­ or angle commonly molariform. Diagnosis. Shell firm, strong, with six thick compart­ ments; parietes with or without pores; radii solid with summits parallel to basal margin of parietes and dentic­ ulated sutural margins; basis calcareous, elongated along carino-rostral axis, boat-shaped; orifice smooth, not dentated; scutum with simple growth ridges; penis with basidorsal point; attached to gorgonians or antipatharians. Type species. Conopea elongata Say, 1822; type locality: eastern Florida, USA. Type species. Conopea elongata Say, 1822; type locality: eastern Florida, USA. Conopea cymbiformis (Darwin, 1854) Figure 33 Diagnosis. Shell with four or sixplates; parietes solid, rarely tubiferous; tubes uniformly or irregularly arranged; radii solid; basis commonly calcareous, rarely tubiferous. Balanus cymbiformis Darwin, 1854: 221, pl. 3, fig. 5a, b. – Broch 1931: 85, fig. 29a, b. – Nilsson-Cantell 1938: 55, pl. 2, fig. 3. – Utinomi 1962: 219, fig. 2. – Dong et al. 1982: 103. Balanus proripiens Hoek, 1913: 228, pl. 24, figs 1–3, pl. 24, figs 1–3. – Nilsson-Cantell 1921: 331, fig. 70c, d. Pyrgoma jedani Hoek, 1913: 262, pl. 27, figs 3–8. Conopea cymbiformis. – Newman & Ross, 1976: 55. Subfamily ARCHAEOBALANINAE Newman & Ross, 1976 Pyrgoma jedani Hoek, 1913: 262, pl. 27, figs 3–8. Conopea cymbiformis. – Newman & Ross, 1976: 55. Archaeobalaninae Newman & Ross, 1976: 38, 49. Material examined. – Kei Islands: 9 specimens, MNHN-IU-2019-4823, Stn. DW 30, 05°39'S, 132°56'E, 111–118 m depth, 26 October 1991; 1 specimen, MNHN- IU-2019-4824, Stn. DW 22, 05°22'S, 133°01'E, 82 m depth, 25 October 1991. Diagnosis. Shell with four or six plates; parietes solid or tubiferous; when tubiferous, tubes uniformly arranged in single row; interlaminate figures simple; basis calcareous or membranous, when membranous wall solid. Type genus. Archaeobalanus Menesini, 1971: 19. Type genus. Archaeobalanus Menesini, 1971: 19. Diagnosis. Shell irregularly conical; orifice oval with swollen border; basis irregularly cup-formed; scutum and tergum triangular, not calcified together; spur of tergum feebly developed. Conopea cymbiformis (Darwin, 1854) Archaeobalanidae Newman & Ross, 1976: 38, 49. Genus Conopea Say, 1822 Conopea Say, 1822: 323. Description. Shell yellowish with orange rust-brown in proximal areas. Carina, carinolatera and latera with pale orange-brown and rust red-brown longitudinal stripes, latter may have oblique white spots. Radii with pale or­ ange-brown and rust red-brown horizontal striation. Oper­ Conoplea Gray, 1825: 98, 103. Balaninus Costa, 1839: 181. Balanus Section B Darwin, 1854: 216. Patella-Balanus Hoek, 1913: 160, 162, 221. Figure 33. Conopea cymbiformis (Darwin, 1854) (MNHN-IU-2019-4823). а. left lateral view; b. right lateral view; c. view from above; d. basal view. Scale bar: 3 mm (a–d). Figure 33. Conopea cymbiformis (Darwin, 1854) (MNHN-IU-2019-4823). а. left lateral view; b. right lateral view; c. view from above; d. basal view. Scale bar: 3 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 684 Conopea navicula. – Newman & Ross, 1976: 55. cular plates with scutum pink-brown, transparent; tergum transparent white. Shell may appear longer and lower, due to elongation of carina and rostrum or low and comparative­ ly shorter, due to development of rostrum alone or more up­ right and comparatively higher, with neither carina nor ros­ trum elongated. Cirrus I with unequal rami (anterior ramus: 7-segmented; posterior ramus: 12-segmented). Cirri II–VI with equal rami, numerous segments. Penis very long, del­ icate hairs scattered over surface, a few more disposed near tip. Labrum deeply notched, two small teeth on each side of notch. Mandibles with five teeth, inferior angle not distinct­ ly separated from fifth; distance between tips of first and second teeth slightly more than that between those of sec­ ond and third teeth; third tooth larger; fourth and fifth small­ er than others. Maxillule with straight edge and numerous large setae. Measurements of specimen: basal length of shell 7.73 mm, orifice length 4.60 mm, carinal height 8.70 mm, orifice width 3.88 mm, basal width 4.94 mm. Material examined. – Tanimbar Island: 12 specimens, MNHN-IU-2019-4821, Stn. CP 82, 09°32'S, 131°02'E, 215–219 m depth, 4 November 1991 attached to gorgo­ nian; 4 specimens, MNHN-IU-2019-4822, Stn. DW 49, 08°00'S, 132°59'E, 206–210 m depth, 29 October 1991, attached to gorgonian. Diagnosis. Shell with parietes and basis not porose; cari­ no-lateral compartments very narrow, almost same width from top to bottom; radii with smooth sutural edges; scu­ tum externally striated longitudinally. Description. Specimens covered with coenosarc of coral, except orifice. Balanus navicula Darwin, 1854: 221, pl. 3, fig. 6a–d. – Hoek 1913: 223, pl. 22, fig. 26, pl. 23, figs 1–3. – Stubbings 1936: 48. – Utinomi 1962: 74, fig 1. – Dong et al. 1982: 103. Acasta spinitergum Foster, 1982: 209, fig. 4d. Genus Conopea Say, 1822 Easily recognisable species due to narrow carino-lateral plate, which is nearly same width at top as bottom; scutum externally longitudinally striated; pari­ etal plates studded with calcareous points. Parietal plates pearly white, solid, superficially appearing to possess longitudinal tubes, growth lines horizontal. Alae moder­ ately developed. Basis calcareous. Size small. Rostrum well developed, concave, lying at angle of ~ 45°. Later­ als very well developed. Carino lateral parietes thin, ra­ dii and alae well developed. Carina tall, about half width of rostrum. External surfaces of all parietes with very small, calcareous studs, regularly spaced, arranged along horizontal growth lines. Opercular plates sunk down into orifice. Cirrus I with unequal rami (anterior ramus: 5-segmented; posterior ramus: 7-segmented). Cirrus II with unequal rami (anterior ramus: 6-segmented; pos­ terior ramus: 9-segmented). Cirri III–VI with subequal rami more slender, longer, with segments more elongate. Penis very long, tapering towards tip, bearing few, very minute hairs. Maxilulle with straight edge with numer­ ous large setae. Mandibles with five teeth and inferior angle. Measurements of four specimens: basal length of Distribution. Indo-west Pacific: Indian Ocean; Gulf of Aden, India, east to Fiji and NW to Indonesia, N Aus­ tralia, Malay Arch.; China; Philippines; S Japan; Fiji Is; attached to coenosarc of gorgonians or antipatharians; lit­ toral–453 m depth (Jones and Hosie 2016). In this study, Conopea cymbiformis was found at Kei Islands, Indonesia. Type locality. Near Madras, India; attached to a gorgo­ nian (Darwin 1854). Solidobalanus auricoma (Hoek, 1913) Figure 35 Figure 35 Type locality. Madras, India; attached to gorgonian (Dar­ win 1854). Balanus (Solidobalanus) auricoma Hoek, 1913: 198, pl. XVIII, figs 20– 22, pl. XIX, figs 1–7. – Broch 1922: 323, fig. 62. – 1931: 71. – Foster 1978: 100, fig. 60. – Rosell 1981: 303. Solidobalanus (Solidobalanus) auricoma. – Newman & Ross, 1976: 50. Solidobalanus auricoma. – Foster ,1981: 364, fig. 2G. – Rossel 1991: 38. Genus Solidobalanus Hoek, 1913 Solido-Balanus Hoek, 1913: 159, 192. Balanus (Solidobalanus). – Pilsbry, 1916: 220. Solidobalanus. – Newman & Ross, 1976: 23, 50. Solido-Balanus Hoek, 1913: 159, 192. Material examined. – Tanimbar Island: 2 specimens, MNHN-IU-2019-4842, Stn. CP 45, 07°54'S, 132°47'E, 302–305 m depth, 29 October 1991, shell only; 10 spec­ imens, MNHN-IU-2019-4841, Stn. DW 49, 08°00'S, 132°59'E, 206–210 m depth, 29 October 1991, attached to sea urchin spines; 1 specimen, MNHN-IU-2019-4838, Stn. CP 82, 09°32'S, 131°02'E, 215–219 m depth, 4 No­ vember 1991, attached to antipatharian; 7 specimens, MNHN-IU-2019-4847, Stn. CP 85, 09°22'S, 131°14'E, 240–245 m depth, 4 November 1991, attached to spines of sea urchin; 63 specimens, MNHN-IU-2019-4850, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 Novem­ ber 1991, attached to spines of sea-urchins. Diagnosis. Shell parietes solid, six plates; radii solid, well developed, with denticulate sutural edges; basis cal­ careous, solid; complemental male, when present, in pit of rostral plate of hermaphrodite. Type species. Balanus auricoma Hoek, 1913: 198, pl. XVIII, figs 20–22, pl. XIX, figs 1–7; type locality: Ter­ nate, Indonesia (Siboga station 136). Conopea navicula (Darwin, 1854) Figure 34 Balanus navicula Darwin, 1854: 221, pl. 3, fig. 6a–d. – Hoek 1913: 223, pl. 22, fig. 26, pl. 23, figs 1–3. – Stubbings 1936: 48. – Utinomi 1962: 74, fig 1. – Dong et al. 1982: 103. Acasta spinitergum Foster, 1982: 209, fig. 4d. Balanus navicula Darwin, 1854: 221, pl. 3, fig. 6a–d. – Hoek 1913: 223, pl. 22, fig. 26, pl. 23, figs 1–3. – Stubbings 1936: 48. – Utinomi 1962: 74, fig 1. – Dong et al. 1982: 103. Acasta spinitergum Foster, 1982: 209, fig. 4d. Acasta spinitergum Foster, 1982: 209, fig. 4d. Figure 34. Conopea navicula (Darwin, 1854) (MNHN-IU-2019-4822). a. left lateral view; b. orifice view. Scale bar: 1 mm (a, b). onopea navicula (Darwin, 1854) (MNHN-IU-2019-4822). a. left lateral view; b. orifice view. Scale bar: 1 mm (a, b). zse.pensoft.net 685 Zoosyst. Evol. 96 (2) 2020, 649–698 shallow furrow without infolded sides; conspicuous teeth on anterior margins of fourth cirri absent. shallow furrow without infolded sides; conspicuous teeth on anterior margins of fourth cirri absent. shell 2.23–4.22 mm, orifice length 1.09–2.02 mm, carinal height 2.04–3.09 mm, orifice width 0.94–1.59 mm, basal width 1.79–2.80 mm. shell 2.23–4.22 mm, orifice length 1.09–2.02 mm, carinal height 2.04–3.09 mm, orifice width 0.94–1.59 mm, basal width 1.79–2.80 mm. Type species. Balanus auricoma Hoek, 1913: 198, pl. XVIII, figs 20–22, pl. XIX, figs 1–7; type locality: Ter­ nate, Indonesia (Siboga station 136). Type species. Balanus auricoma Hoek, 1913: 198, pl. XVIII, figs 20–22, pl. XIX, figs 1–7; type locality: Ter­ nate, Indonesia (Siboga station 136). Distribution. Indo-west Pacific, from Gulfs of Aden and Persia, India, Malaysia, Indonesia, Gulf of Siam, to southern Japan; 45–220 m depth (Jones and Hosie 2016). In this study, Conopea navicula was found at Tanimbar Island, Indonesia. Solidobalanus pseudauricoma (Broch, 1931) Figure 36 Balanus (Solidobalanus) pseudauricoma Broch, 1931. – Utinomi 1949: 97, fig. 4. Diagnosis. Shell flatly-conical, colour reddish basally, whitish apically; radii narrow, summits slightly oblique; opercular plates with golden hairs along occludent mar­ gins; scutum with articular ridge slightly prominent, ad­ ductor ridge absent; tergum narrow, scutal margin dis­ tinctly dentated. Solidobalanus (Solidobalanus) pseudauricoma. – Newman & Ross, 1976: 51. – Jones 2007: 294. Material examined. – Kei Islands: 46 specimens, MNHN-IU-2019-4818, Stn. CP 25, 05°30'S, 132°52'E, 336–346 m depth, 26 October 1991, 30 specimens at­ tached to gorgonian, 16 specimens attached to cor­ al; 6 specimens, MNHN-IU-2019-4825, Stn. CP 27, 05°33'S, 132°51'E, 304–314 m depth, 27 October 1991; 1 specimen, MNHN-IU-2019-4826, Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22 October 1991, attached to crinoid; 13 specimens, MNHN-IU-2019-4827, Stn. CP 16, 05°17'S, 132°50'E, 330–350 m depth, 24 October 1991; 2 specimens, MNHN-IU-2019-4828, Stn. DW 22, 05°22'S, 133°01'E, 82 m depth, 25 October 1991, 1 spec­ imen attached to gorgonian stem; 8 specimens, MNHN- IU-2019-4830, Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22 October 1991, 5 specimens attached to gor­ gonian, 1 specimen attached to crinoid. Material examined. – Kei Islands: 46 specimens, MNHN-IU-2019-4818, Stn. CP 25, 05°30'S, 132°52'E, 336–346 m depth, 26 October 1991, 30 specimens at­ tached to gorgonian, 16 specimens attached to cor­ al; 6 specimens, MNHN-IU-2019-4825, Stn. CP 27, 05°33'S, 132°51'E, 304–314 m depth, 27 October 1991; 1 specimen, MNHN-IU-2019-4826, Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22 October 1991, attached to crinoid; 13 specimens, MNHN-IU-2019-4827, Stn. CP 16, 05°17'S, 132°50'E, 330–350 m depth, 24 October 1991; 2 specimens, MNHN-IU-2019-4828, Stn. DW 22, 05°22'S, 133°01'E, 82 m depth, 25 October 1991, 1 spec­ imen attached to gorgonian stem; 8 specimens, MNHN- IU-2019-4830, Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22 October 1991, 5 specimens attached to gor­ gonian, 1 specimen attached to crinoid. Description. Shell with plates ribbed longitudinally. Shell colour brownish-pink to dull rose-pink, ribs tend­ ing to white, colour often faded with specimens appear­ ing uniform white. Parietes of carinolatera very narrow, with single, conspicuous, longitudinal ridge. Scutum with occludent margin straight, surface indistinctly ridged, pit for adductor muscle scarcely visible. Tergum short, narrow, scutal margin straight, unusually distinctly dentated, carinal margin short, convex, depressor muscle crests moderately well developed. Opercular plates with long, golden setae fringing occludent margins, especial­ ly distally. Subgenus Solidobalanus Hoek, 1913 p – Kei Islands: 1 specimen, MNHN-IU-2019-4849, Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22 October 1991, attached to lateral plate of Chirona te­ nuis; 1 specimen, MNHN-IU-2019-4844, Stn. CP 12, 05°23'S, 132°37'E, 413–436 m depth, 23 October 1991, attached to antipatharian; 6 specimens, MNHN- IU-2019-4848, Stn. CP 16, 05°17'S, 132°50'E, 330– 350 m depth, 24 October 1991, attached to dead gorgo­ nian, spines of sea-urchins, arm of crinoid; 3 specimens, MNHN-IU-2019-4840, Stn. CP 35, 06°08'S, 132°45'E, 390–502 m depth, 27 October 1991; 1 specimen, p – Kei Islands: 1 specimen, MNHN-IU-2019-4849, Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22 October 1991, attached to lateral plate of Chirona te­ nuis; 1 specimen, MNHN-IU-2019-4844, Stn. CP 12, 05°23'S, 132°37'E, 413–436 m depth, 23 October 1991, attached to antipatharian; 6 specimens, MNHN- IU-2019-4848, Stn. CP 16, 05°17'S, 132°50'E, 330– 350 m depth, 24 October 1991, attached to dead gorgo­ nian, spines of sea-urchins, arm of crinoid; 3 specimens, MNHN-IU-2019-4840, Stn. CP 35, 06°08'S, 132°45'E, 390–502 m depth, 27 October 1991; 1 specimen, Solido-Balanus Hoek, 1913: 159, 192. Balanus (Solidobalanus). – Pilsbry, 1916: 220. Solidobalanus. – Newman & Ross, 1976: 23, 50. Solido-Balanus Hoek, 1913: 159, 192. Balanus (Solidobalanus). – Pilsbry, 1916: 220. Solidobalanus. – Newman & Ross, 1976: 23, 50. Solidobalanus. – Newman & Ross, 1976: 23, 50. Diagnosis. Parietes and radii rather thick, smooth, with­ out pores; basis flat, calcareous; rostrum not very elongat­ ed; radii with strongly septate sutural edges; tergal spur narrow; crests for lateral depressor muscles of scutum absent; adductor ridge weak or absent; tergum flat or with Figure 35. Solidobalanus auricoma (Hoek, 1913) (MNHN-IU-2019-4850). a. upper lateral view from above; b. left lateral view; c. right lateral view; d. basal view. Scale bar: 2 mm (a–d). Figure 35. Solidobalanus auricoma (Hoek, 1913) (MNHN-IU-2019-4850). a. upper lateral view from above; b. left lateral view; c. right lateral view; d. basal view. Scale bar: 2 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 686 MNHN-IU-2019-4843, Stn. CP 36, 06°05'S, 132°44'E, 210–268 m depth, 27 October 1991. Striatobalanus amaryllis (Darwin, 1854) Figure 37 Figure 37 Figure 37 Distribution. Manado Bay (Indonesia); Japan (Broch 1931–1932). In this study, Solidobalanus pseudauricoma was found at Kei Islands and Tanimbar Island, Indonesia. Distribution. Manado Bay (Indonesia); Japan (Broch 1931–1932). In this study, Solidobalanus pseudauricoma was found at Kei Islands and Tanimbar Island, Indonesia. alanus amaryllis var. (a), var. (b) Darwin, 1854: 279, pl. 7, fig. 6a–c , p ,i g , p ,i g y 217. – Nilsson-Cantell 1925: 329. – Hiro 1936: 624. – 1939c: 243. Type locality. Manado Bay, Indonesia (1°31'N, 124°47'E), 500 m depth; Japan (32°25'N, 128°33'E), 400 m depth (Broch 1931–1932). – Stubbings 1936: 174. – Utinomi 1962: 216. – 1969: 88. Balanus amaryllis dissimilis Lanchester, 1902: 369, pl. XXXIV, figs 3–3C, with var. clarovittata Lanchester, 1902: 370. Balanus amaryllis var. a (= Balanus roseus Lamarck, 1818). – Gruvel 1905: 250, with var. b (niveus). – Gruvel, 1905: 250 (= Balanus amaryllis var. b Darwin, 1854: 279)). Balanus amaryllis var. a (= Balanus roseus Lamarck, 1818). – Gruvel 1905: 250, with var. b (niveus). – Gruvel, 1905: 250 (= Balanus amaryllis var. b Darwin, 1854: 279)). Remarks. In the type description, Broch (1931–1932) commented that the specimens were white, without stripes. However, several of the specimens collected by KARUBAR had a pale pink tinge and one specimen (from station DW22) had pale brownish-pink parietes with small, narrow ellipsoidal whitish spots, the latter ori­ entated longitudinally, thus producing a reticulated effect. Radii whitish with pink tinge along distal borders. Balanus (Chirona) amaryllis. – Nilsson-Cantell 1921: 329, pl. 3, fig. 9. – Rosell 1981: 302. Balanus (Chirona) amaryllis. – Nilsson-Cantell 1921: 329, pl. 3, fig. 9. – Rosell 1981: 302. Balanus amaryllis forma euamaryllis. – Broch, 1922: 321. Balanus amaryllis forma euamaryllis. – Broch, 1922: 321. Balanus amaryllis forma laevis Broch, 1931: 67, fig. 24. Balanus amaryllis forma laevis Broch, 1931: 67, fig. 24. Balanus sp. Dong & Mao, 1956: 290, fig. 8. Chirona (Striatobalanus) amaryllis. – Newman & Ross, 1976: 50 Chirona (Striatobalanus) amaryllis. – Newman & Ross, 1976: 50. Chirona amaryllis. – Foster, 1981: 350. Solidobalanus pseudauricoma (Broch, 1931) Figure 36 Cirri I–II with rami slightly unequal, covered with setae; cirri III–VI longer, more slender, dense setae on inner face. Mandible with four teeth, second to fourth with accessory cusps, lower angle molariform with three blunt cusps in series, lower edge with row of stiff setae. Measurements of five specimens: basal length of shell 6.11–8.18 mm, orifice length 4.18–5.83 mm, carinal height 4.11–6.30 mm, orifice width 2.58–3.19 mm, basal width 4.51–5.96 mm. – Tanimbar Island: 11 specimens, MNHN- IU-2019-4839, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 November 1991, several specimens attached to spines of sea urchins (associated with Solidobalanus au­ ricoma (Hoek, 1913)). – Tanimbar Island: 11 specimens, MNHN- IU-2019-4839, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 November 1991, several specimens attached to spines of sea urchins (associated with Solidobalanus au­ ricoma (Hoek, 1913)). Diagnosis. Shell with smooth, glossy white plates, co­ loured stripes absent; internal plates with thick, solid, fine­ ly ribbed longitudinally; base non-porous, radially ribbed. Distribution. Banda Sea (Moluccas, Indonesia); SW Australia; New Zealand; New Caledonia; Philippines to southern Japan; Malaysian water; Gulf of Oman, Persia. 27–502 m depth (Jones and Hosie 2016). In this study, Solidobalanus auricoma was found at Kei Islands and Tanimbar Island, Indonesia. Description. Shell plates white, stripes absent. Sever­ al specimens with pale pink tinge, one with pale brown­ ish-pink parietes with small, narrow ellipsoidal whitish spots, latter orientated longitudinally producing reticulated effect. Radii whitish, pink tinge along distal borders. Scutal growth lines without longitudinal striations; articular ridge absent; pit for adductor muscle small, round. Tergum with Type locality. Ternate anchorage; 27 m depth; bottom: mud and stone; numerous specimens on the surface of pieces of rock (Hoek 1913). Figure 36. Solidobalanus pseudauricoma (Broch, 1931) (MNHN-IU-2019-4818). a. left lateral view; b. orifice view; c. basal view; d. right lateral view. Scale bar: 4 mm (a–d). Figure 36. Solidobalanus pseudauricoma (Broch, 1931) (MNHN-IU-2019-4818). a. left lateral view; b. orifice view; c. basal view; d. right lateral view. Scale bar: 4 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 687 Chirona (Striatobalanus). – Newman & Ross, 1976: 23, 50 (as subgenus). Striatobalanus. – Jones, 2004: 150. Chirona (Striatobalanus). – Newman & Ross, 1976: 23, 50 (as subgenus). Striatobalanus. – Jones, 2004: 150. shallow, wide furrow running from apex to base. Cirrus I with unequal (anterior ramus: 7-segmented; posterior ramus: 15-segmented). Solidobalanus pseudauricoma (Broch, 1931) Figure 36 Cirrus II with rami subequal (an­ terior ramus: 11-segmented; posterior ramus: 12-segment­ ed). Cirri I and II with very dense, long setae on surface areas. Cirri III–VI with rami slightly subequal, rounded. Penis sturdy, not long. Labrum with very shallow notch, three or four irregularly arranged, blunt teeth on each side. Maxillule with distinct, narrow notch with two large se­ tae on upper side. Mandibles with five teeth, second and third bifid and fifth is rudimentary. Measurements of five specimens: basal length of shell 6.23–12.08 mm, orifice length 4.63–9.62 mm, carinal height 3.48–13.22 mm, ori­ fice width 3.24–6.33 mm, basal width 5.66–9.56 mm. shallow, wide furrow running from apex to base. Cirrus I with unequal (anterior ramus: 7-segmented; posterior ramus: 15-segmented). Cirrus II with rami subequal (an­ terior ramus: 11-segmented; posterior ramus: 12-segment­ ed). Cirri I and II with very dense, long setae on surface areas. Cirri III–VI with rami slightly subequal, rounded. Penis sturdy, not long. Labrum with very shallow notch, three or four irregularly arranged, blunt teeth on each side. Maxillule with distinct, narrow notch with two large se­ tae on upper side. Mandibles with five teeth, second and third bifid and fifth is rudimentary. Measurements of five specimens: basal length of shell 6.23–12.08 mm, orifice length 4.63–9.62 mm, carinal height 3.48–13.22 mm, ori­ fice width 3.24–6.33 mm, basal width 5.66–9.56 mm. Diagnosis. Parietes and radii without pores; radii usual­ ly narrow; scutum striated longitudinally with adductor ridge; tergum furrow deep; cirrus I with rami very un­ equal in length. Type species. Balanus amaryllis var. (a), var. (b) Darwin, 1854: 279, pl. 7, fig. 6a–c. Type species. Balanus amaryllis var. (a), var. (b) Darwin, 1854: 279, pl. 7, fig. 6a–c. Type locality. Unknown. Type locality. Unknown. Genus Striatobalanus Hoek, 1913 – Nilsson-Cantell 1921: 302. Balanus tenuis. – Dong et al.. 1982: 97, fig. A–C. Description. Shell conical; tips of rostrum and cari­ na slightly curved inwards. Orifice large, pentagonal, toothed. Colour yellowish-white, with slightly darker longitudinal lines on main parts of plates. Radii with very oblique summits, broadest a little distance from the orifice, narrower towards basis. Alae broader than radii, summits rounded. Specimen without scutum, ter­ gum and soft parts. Measurements of specimen: basal length of shell 16.76 mm, orifice length 9.13 mm, cari­ nal height 9.89 mm, orifice width 7.24 mm, basal width 15.14 mm. Chirona tenuis. – Zevina et al.. 1992: 74, fig. 50. Striatobalanus tenuis. – Jones. 2004: 152. – Liu and Ren 2007: 363, fig. 161. – Chan 2009: 74, fig. 2H and fig. 23A–G. Material examined. – Kei Islands: 5 specimens, MNHN- IU-2019-4820, Stn. CC 10, 05°21'S, 132°30'E, 329– 389 m depth, 23 October 1991; 1 specimen (shell only), MNHN-IU-2019-4835, Stn. CP 16, 05°17'S, 132°50'E, 330–350 m depth, 24 October 1991. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4832, Stn. DW 49, 08°00'S, 132°59'E, 206–2010 m depth, 29 Oc­ tober 1991; 1 specimen (shell only), MNHN-IU-2019-4833, Stn. CP 65, 09°14'S, 132°27'E, 174–176 m depth, 1 Novem­ ber 1991; 4 specimens (shell only), MNHN-IU-2019-4834, Stn. CP 79, 09°16'S, 131°22'E, 239–250 m, 3 November 1991; 1 specimen (only shell), MNHN-IU-2019-4813, Stn. CP 85, 09°22'S, 131°14'E, 240–245 m, 4 November 1991, attached to shell of gastropod; 2 specimens, MNHN- IU-2019-4819, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 November 1991, attached to gastropod shells. Distribution. Arafura Sea, Indonesia. Indo-west Pa­ cific: South and East Africa, Indian Ocean; Australia (N); Singapore; Malay Arch.; Cambodia; Gulf of Thai­ land; Mouth of Bassac; Vietnam (Bay of Nhatrang; Lien Chien, Tourane; Codor Is); Hong Kong; S China Sea; China; Philippines; Taiwan; Japan (S); attached to rocks, gastropod shells, rusting iron, fouling species; sublittoral, 5–500 m depth (Jones and Hosie 2016). In this study, Striatobalanus amaryllis was found at Ta­ nimbar Island, Indonesia. Diagnosis. Shell colour whitish to yellowish; sur­ face smooth, glossy; orifice pentagonal shaped, deep­ ly toothed; radii narrow, summits very oblique, slightly concave; basis solid; scutum with longitudinal striations; tergum with short, rather broad spur. Type locality. Unknown. Genus Striatobalanus Hoek, 1913 Balanus amaryllis. – Dong et al., 1982: 95. Balanus amaryllis euamaryllis Dong et al., 1982: 96, fig. A–C. Balanus Section F (part.) Darwin, 1854: 277. Chirona Gray, 1825: 37. – Pilsbry, 1916: 204. Balanus (Striato-Balanus) Hoek, 1913: 159, 179. Balanus Section F (part.) Darwin, 1854: 277. Chirona Gray, 1825: 37. – Pilsbry, 1916: 204. Balanus (Striato-Balanus) Hoek, 1913: 159, 179. Chirona amaryllis: Zevina et al., 1992: 72, fig. 49. Striatobalanus amaryllis. – Jones, 2004: 150. – Chan et al. 2009b: 222, 190–192. Figure 37. Striatobalanus amaryllis (Darwin, 1854) (MNHN-IU-2019-4814). a. orifice view; b. right lateral view. Scale bar: 3 mm (a, b). triatobalanus amaryllis (Darwin, 1854) (MNHN-IU-2019-4814). a. orifice view; b. right lateral view. Scale bar: 3 mm (a, b zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 688 1916: 216. – Barnard 1924: 74. – Nilsson-Cantell 1925: 34, fig. 13, pl. 1, figs 5, 6. – 1927: 785. – 1938: 46. – Broch 1931: 70. – Hiro 1937b: 439, fig. 24. – Utinomi 1962: 216. – 1968: 174. – 1969: 88, fig. 6. – Utinomi and Kikuchi 1966: 6. 1916: 216. – Barnard 1924: 74. – Nilsson-Cantell 1925: 34, fig. 13, pl. 1, figs 5, 6. – 1927: 785. – 1938: 46. – Broch 1931: 70. – Hiro 1937b: 439, fig. 24. – Utinomi 1962: 216. – 1968: 174. – 1969: 88, fig. 6. – Utinomi and Kikuchi 1966: 6. 1916: 216. – Barnard 1924: 74. – Nilsson-Cantell 1925: 34, fig. 13, pl. 1, figs 5, 6. – 1927: 785. – 1938: 46. – Broch 1931: 70. – Hiro 1937b: 439, fig. 24. – Utinomi 1962: 216. – 1968: 174. – 1969: 88, fig. 6. – Utinomi and Kikuchi 1966: 6. Material examined. – Tanimbar Island: 1 specimen (only shell), MNHN-IU-2019-4814, Stn. CP 65, 09°14'S, 132°27'E, 174–176 m depth, 1 November 1991, attached to broken sea urchin shell. Balanus albus Hoek, 1913: 185, pl. 16, figs 12, 13, pl. 17, figs 1–6. – Stubbings 1936: 41, fig. 18. Diagnosis. Shell conical; orifice large, pentagonal, toothed; shell colour pinkish, lighter transparent stripes run longitudinally over surface; radii very narrow, reddish, upper margins very oblique; alae broader, lighter colour. Chirona (Striatobalanus) tenuis. – Newman & Ross, 1976: 50. – Rosell 1989: 33, pl. 10g. – 1991: 38. Balanus (Chirona) tenuis. – Ren & Liu, 1978: 161, fig. 22, pl. 7, fi il ll figs 6–10. Subfamily MEGATREMAOTINAE Holthuis, 1982 striations; tergum slightly beaked, exhibiting traces of lon­ gitudinal striations, spur short, slightly narrow, depressor crests distinctly developed. Cirri I to VI with rami slight­ ly equal in length, transparent and orange-coloured. Penis rather long, sparse, minute hairs on the surface, tapering to­ wards tip, distinctly hairy at tip. Maxillule with edge slight­ ly straight, all the setae similar sized. Mandibles with five teeth, fourth and fifth small. Measurements of five speci­ mens basal length of shell 18.92–28.30 mm, orifice length 13.78–16.29 mm, carinal height 18.02–20.24 mm, orifice width 10.15–13.70 mm, basal width 20.51–28.03 mm. striations; tergum slightly beaked, exhibiting traces of lon­ gitudinal striations, spur short, slightly narrow, depressor crests distinctly developed. Cirri I to VI with rami slight­ ly equal in length, transparent and orange-coloured. Penis rather long, sparse, minute hairs on the surface, tapering to­ wards tip, distinctly hairy at tip. Maxillule with edge slight­ ly straight, all the setae similar sized. Mandibles with five teeth, fourth and fifth small. Measurements of five speci­ mens basal length of shell 18.92–28.30 mm, orifice length 13.78–16.29 mm, carinal height 18.02–20.24 mm, orifice width 10.15–13.70 mm, basal width 20.51–28.03 mm. Bosciinae Newman & Ross, 1976: 59. Megatrematinae Holthuis, 1982: 319. Bosciinae Newman & Ross, 1976: 59. Bosciinae Newman & Ross, 1976: 59. Megatrematinae Holthuis, 1982: 319. Megatrematinae Holthuis, 1982: 319. Megatrematinae Holthuis, 1982: 319. Diagnosis. Wall of four plates wholly concrescent; oper­ cular valves normal; tergum with weakly developed lat­ eral depressor muscle crests or crests lacking; when shell concrescent, sheath with paired sulci. Type genus. Megatrema Sowerby, 1823, by original des­ ignation (Holthuis 1982). Type genus. Megatrema Sowerby, 1823, by original des­ ignation (Holthuis 1982). Distribution. Arafura Sea, Indonesia; Indo-west Pacif­ ic: S Africa; Persian Gulf; Indian Ocean; Australia (N); Singapore; Malay Arch.; Vietnam; Hong Kong; S Chi­ na Sea; E China Sea; Philippines; Taiwan; Japan (S); W Pacific; attached to crabs, gastropod, bivalve, shells sol­ itary coral, bark of coconut, gorgonians, antipatharians, stones; 7–551 m depth (Jones and Hosie 2016). In this study, Striatobalanus tenuis was found at Kei Islands and Tanimbar Island, Indonesia. Family PYRGOMATIDAE Gray, 1825 Balanidae Leach, 1817: 68 (part.). – Darwin 1854: 33 (part.). Balaninae. – Nilsson-Cantell, 1921: 306 (part.). Pyrgomatidae Gray, 1825: 102. – Newman 1996: 503. Pyrgomatinae. – Ross & Newman, 1973: 149. Type locality. Pliocene (Piacenzian Stage); Gournes, Iraklion nomarchia, Island of Crete, Greece, 35°06'N, 25°47'E; host coral unknown (Ross and Pitombo 2002). Pyrgomatinae. – Ross & Newman, 1973: 149. Tribe PYRGOMINI Ross & Pitombo, 2002 Pyrgominini Ross & Pitombo, 2002: 58. Pyrgominini Ross & Pitombo, 2002: 58. Type genus. Pyrgomina Baluk & Radwanski, 1967. Type genus. Pyrgomina Baluk & Radwanski, 1967. Striatobalanus tenuis (Hoek, 1883) Figure 38 Figure 38 Description. Shell with six plates, conical; orifice large, distinctly toothed; radii well-developed; basis thin, ribbed, solid. Scutum triangular, not elongated, with longitudinal Balanus tenuis Hoek, 1883: 154, pl. 13, figs 29–33. – 1913: 190, pl. 17, figs 14–19, pl. 18, fig. 1. – Gruvel 1905: 247, fig. 275. – Pilsbry Figure 38. Striatobalanus tenuis (Hoek, 1883) (MNHN-IU-2019-4820). a. orifice view; b. left lateral view; c. right lateral view; d. basal view. Scale bar: 10 mm (a–d). Figure 38. Striatobalanus tenuis (Hoek, 1883) (MNHN-IU-2019-4820). a. orifice view; b. left lateral view; c. right lateral view; d. basal view. Scale bar: 10 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 689 Subfamily MEGATREMAOTINAE Holthuis, 1982 Genus Pyrgomina Baluk & Radwanski, 1967 Type locality. West of Mindoro, Philippines; 6°8'N, 121°19'E; depth: 275 m, at coral-bottom (Hoek 1913). Pyrgomina Baluk & Radwanski, 1967b: 691, pl. 1–2. Type species. Pyrgomina seguenzai Baluk & Radwanski, 1967a: 485. Pyrgomina sp. Diagnosis. Wall of four plates wholly concrescent; pa­ rietes solid or tubiferous; when tubiferous, tubes occur between outer lamina and sheath or between external ribs of wall; interlaminate figures complex, essentially arbo­ rescent; radii solid; basis calcareous, rarely tubiferous, membranous in Pyrgopsella. Material examined. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4817, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 November 1991, attached to coral. Figure 39. Pyrgomina sp. (MNHN-UI-2019-4817). a. orifice view; b. left lateral view; c. right lateral view. Scale bar: 2 mm (a–c). Figure 39. Pyrgomina sp. (MNHN-UI-2019-4817). a. orifice view; b. left lateral view; c. right lateral view Pyrgomina sp. (MNHN-UI-2019-4817). a. orifice view; b. left lateral view; c. right lateral view. Scale bar: 2 mm (a–c zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 690 Diagnosis. Shell purple-pinkish, tall, conical; oriface oval; base permeated by pores, expanded out of the coral; tergum and scutum subtriangular. Diagnosis. Shell purple-pinkish, tall, conical; oriface oval; base permeated by pores, expanded out of the coral; tergum and scutum subtriangular. lower side only; alae not cleft; basis with single tubif­ erous; scutum with conspicuous adductor ridge; tergum with well-developed depressor muscle crests, growth lines in tergum spur display an obvious change in di­ rection; second maxilla with smooth anterior margin of distal lobe, acuminate setae with enlarged, modified tips. Cirrus III with inner face of endopod with pinnate setae, rarely bifurcate (complex) setae. Description. Shell stands exserted on the coral, externally furnished with ribs, lower part rounded with radiating ribs; oriface oval, small and narrow. Scutum triangular, basal margin curved; tergum triangular with narrow spur. Cirrus I with rami unequal (anterior ramus: 10-segmented; pos­ terior ramus: 15-segmented); posterior ramus of cirrus IV without spines. Maxilla globular; maxillule not notched, eight large setae on straight cutting edge; mandible with five major teeth. Measurements of specimen: basal length of shell 7.46 mm, orifice length 2.42 mm, carinal height 10.45 mm, orifice width 1.41 mm, basal width 6.15 mm. Type species. Balanus amphitrite Darwin, 1854: 240 (part.), pl. 5, fig. 2a–d, i–k, m–o; type locality: Natal, South Africa. Type species. Balanus amphitrite Darwin, 1854: 240 (part.), pl. 5, fig. 2a–d, i–k, m–o; type locality: Natal, South Africa. Amphibalanus amphitrite (Darwin, 1854) Figure 40 Distribution. Pyrgomina sp. was found at Tanimbar Is­ land, Indonesia. Balanus amphitrite Darwin, 1854: 240 (part.), pl. 5, fig. 2a-d, i-k, m-o. – Weltner 1897: 264. – Hoek 1913: 167. – Pilsbry 1916: 89. – Zevina et al. 1992: 89, fig. 61. – Puspasari et al. 2001: 7. Balanus amphitrite Darwin, 1854: 240 (part.), pl. 5, fig. 2a-d, i-k, m-o. – Weltner 1897: 264. – Hoek 1913: 167. – Pilsbry 1916: 89. – Zevina et al. 1992: 89, fig. 61. – Puspasari et al. 2001: 7. Balanus amphitrite var. (1) communis Darwin, 1854: 240, pl. 5, fig. 2e, h, l. Balanus amphitrite communis. – Nilsson-Cantell, 1921: 311, fig. 64. Subfamily AMPHIBALANINAE Pitombo, 2004 Balanus amphitrite amphitrite. – Harding, 1962: 274, pl. 1a–g, pl. 2a–k. – Dong et al. 1982: 90, fig. A-E. – Rosell 1981: 302. – Dong et al. 1982: 90, fig. A-E. – Rosell 1981: 302. Balanus amphitrite var. Hawaiiensis. – Stubbings, 1963b: 15. – Dong et al. 1982: 90, fig. A-E. – Rosell 1981: 302. Amphibalaninae Pitombo, 2004: 263. Balanus amphitrite var. Hawaiiensis. – Stubbings, 1963b: 15. Amphibalanus amphitrite. – Pitombo, 2004: 263, 274, figs 2A, B, 7A, Amphibalanus amphitrite. – Pitombo, 2004: 263, 274, figs 2A, B, 7A, Amphibalanus amphitrite. – Pitombo, 2004: 263, 274, figs 2A, B, 7A, B, 8C. – Chan et al. 2009b: 241. – Chen et al. 2014: 1071. – Pochai et al. 2017: 27, fig. 9. – Pitriana et al. 2020: 42, fig. 21. Amphibalanus amphitrite. Pitombo, 2004: 263, 274, figs 2A, B, 7A, B, 8C. – Chan et al. 2009b: 241. – Chen et al. 2014: 1071. – Pochai et al. 2017: 27, fig. 9. – Pitriana et al. 2020: 42, fig. 21. Diagnosis. Shell with four or six plates; parietal tubes with one or more rows, commonly transverse septa; radii with transverse teeth on sutural edge with denticles on lower side only; alae not cleft; basis with single tubif­ erous; scutum with conspicuous adductor ridge; tergum with well-developed depressor muscle crests, growth lines in tergum spur display an obvious change in direc­ tion; second maxilla with smooth anterior margin of dis­ tal lobe, acuminate setae with enlarged, modified tips. Material examined. – Tanimbar Island: 4 specimens, MNHN-IU-2019-4815, Stn. CP 52, 08°03'S, 131°48'E, 1244–1266 m depth, 30 October 1991. Diagnosis. Primary parietal tubes with transverse septa; exterior of shell with longitudinal purple striations, hori­ zontal striations absent; tergum short with wide spur; cirri III–VI with erect teeth below posterior angles of distal; cirrus III without complex setae. Family BALANIDAE Leach, 1817 Balanus amphitrite var. (1) communis Darwin, 1854: 240, pl. 5, fig. 2e, h, l. Balanus amphitrite communis. – Nilsson-Cantell, 1921: 311, fig. 64. Balanidae Leach, 1817: 68. – Gray 1825: 104. – Darwin 1854: 33 (part.). – Pilsbry 1916: 48. – Nilsson-Cantel 1921: 306. – Newman and Ross 1976: 59. – Foster 1978: 95. – Buckeridge 1983: 103. – Newman 1996: 503. – Pitombo 2004: 262. – Chan et al. 2009b: 229. Balanus amphitrite forma hawaiiensis Broch, 1922: 314, fig. 56 (part.). Balanus amphitrite hawaiiensis. – Hiro, 1937c: 432, figs 20, 21. Balanus amphitrite cochinensis Nilsson-Cantell, 1938b: 43, fig. 11a–e. Balanus amphitrite var. fluminensis Oliveira, 1941: 21, pl. 4, fig. 4, pl. 5, figs 1, 2, pl. 8, figs 1–5. Diagnosis. Shell with four or six plates; parietes tubif­ erous; tubes arranged in single uniform row formed be­ tween inner and outer lamina, supplementary tubes may form basally; interlaminate figures complex, arborescent; radii solid or tubiferous, more-or-less developed; basis calcareous, commonly tubiferous; caudal appendages ab­ sent; penis commonly with basidorsal point. Balanus amphitrite var. aeratus Oliveira, 1941: 22, pl. 4, fig. 5, pl. 9, i Balanus amphitrite var. aeratus Oliveira, 1941: 22, pl. 4, fig. 5, pl. 9, figs 1–4. figs 1–4. Balanus amphitrite herzi Rogers, 1949: 28, pl. 1, figs 6, 12–15. Balanus amphitrite franciscanus Rogers, 1949: 29, pl. 1, figs 5, 7, 16–19. Balanus amphitrite var. columnarius Tarasov & Zevina, 1957: 179, 184, fig. 68 a–e. Balanus amphitrite denticulata Henry, 1959: 192, pl. 1, fig. 5, pl. 3, i Balanus amphitrite denticulata Henry, 1959: 192, pl. 1, fig. 5, pl. 3, fig. 7, upper row right. Balanus amphitrite amphitrite. – Harding, 1962: 274, pl. 1a–g, pl. 2a–k. zse.pensoft.net Genus Amphibalanus Pitombo, 2004 Amphibalanus Pitombo, 2004: 263. Description. Shell six-plated, conical, round; external­ ly smooth, white with groups of well-spaced, dark pur­ ple vertical stripes; horizontal striations on shell surface absent. Interior of paries with single row of tubes; radii solid, wide; alae with summits moderately oblique; basis Diagnosis. Shell with four or six plates; parietal tubes with one or more rows, commonly transverse septa; radii with transverse teeth on sutural edge, with denticles on zse.pensoft.net zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 691 Figure 40. Amphibalanus amphitrite (Darwin, 1854) (MNHN-IU-2019-4815). a. orifice view; b. right lateral view; c. basal view. Scale bar: 4 mm (a–c). Figure 40. Amphibalanus amphitrite (Darwin, 1854) (MNHN-IU-2019-4815). a. orifice view; b. right lateral view; c. basal view. Scale bar: 4 mm (a–c). Remarks. Known as an important fouling species of ships and marine installations. The suggestion of an­ ti-fouling paint on the bases of the specimens examined suggests that these specimens were probably knocked off the ship during trawling operations, explaining the great depth at which these specimens were collected, as the normal depth range is 0–9 m. porose, calcareous. Scutum externally striped, occludent margin toothed, lateral depressor muscle pit small. Ter­ gum with spur wider than long, less than its own width from basi-scutal angle. Cirrus I with unequal rami (ante­ rior ramus: 10-segmented; posterior ramus: 16-segment­ ed). Cirrus II with slightly equal rami (anterior ramus: 10-segemented; posterior ramus: 12-segmented). Cirrus III with rami subequal (anterior ramus: 12-segmented; posterior ramus: 13-segmented. Cirri IV–VI with rami subequal, longer, more slender. Penis moderately long, tapering distally, sparse, minute hairs on surface, distinct­ ly hairy distally. Labrum deeply notched, numerous teeth on each side. Maxilla without notch below upper pair of spines, nine spines between upper and lower pairs, few short spines on inferior angle; lower pair of spines usually on slight prominence, which may be sometimes moder­ ately strong or strong. Mandible with five teeth, inferi­ or angle with second tooth bifid. Measurements of four specimens: basal length of shell 5.27–12.10 mm, orifice length 3.81–7.89 mm, carinal height 3.64–5.65 mm, or­ ifice width 2.72–5.22 mm, basal width 4.62–12.03 mm. porose, calcareous. Scutum externally striped, occludent margin toothed, lateral depressor muscle pit small. Ter­ gum with spur wider than long, less than its own width from basi-scutal angle. Cirrus I with unequal rami (ante­ rior ramus: 10-segmented; posterior ramus: 16-segment­ ed). Genus Amphibalanus Pitombo, 2004 Cirrus II with slightly equal rami (anterior ramus: 10-segemented; posterior ramus: 12-segmented). Cirrus III with rami subequal (anterior ramus: 12-segmented; posterior ramus: 13-segmented. Cirri IV–VI with rami subequal, longer, more slender. Penis moderately long, tapering distally, sparse, minute hairs on surface, distinct­ ly hairy distally. Labrum deeply notched, numerous teeth on each side. Maxilla without notch below upper pair of spines, nine spines between upper and lower pairs, few short spines on inferior angle; lower pair of spines usually on slight prominence, which may be sometimes moder­ ately strong or strong. Mandible with five teeth, inferi­ or angle with second tooth bifid. Measurements of four specimens: basal length of shell 5.27–12.10 mm, orifice length 3.81–7.89 mm, carinal height 3.64–5.65 mm, or­ ifice width 2.72–5.22 mm, basal width 4.62–12.03 mm. Type locality. Natal, on a piece of bamboo (Darwin 1854). Discussion Prior to the Karubar expedition, 24 species of barnacles had been collected from the Kei Islands and Aru Island by the Siboga expedition (Hoek 1913). Other pertinent refer­ ence works to the barnacles from these islands are Jones et al. (2001) and Jones and Hosie (2016), who recorded 15 species from the Kei Islands and Aru Island. In addition to the works of Hoek (1913), Jones et al. (2001) and Jones and Hosie (2016), Broch (1931–1932) reported on 67 species of barnacles collected by the Dan­ ish expedition to the Kei Islands (1922) and deposited in the Zoological Museum of Copenhagen University. In his report, only four species, Euscalpellum rostratum (Dar­ win, 1851), Lepas (Anatifa) anatifera Linnaeus, 1758, Conchoderma virgatum Spengler, 1789 and Acasta den­ tifer (Broch, 1922), were explicitly collected in the Kei Is­ lands. The other barnacle species recorded were collected at other places along the route of this expedition, such as Lampung Bay, Krakatau, Java Sea, Sunda Strait, Makas­ sar Strait, Tual, Banda Neira, Ambon and Saparua Bay. Distribution. Cosmopolitan in tropical and subtropical waters. Bermuda & SE USA to Brazil; England and West Europe to South coast of Africa Red, Black and Medi­ terranean Seas; Suez Canal; SE Africa; Indian Ocean; Australia; Singapore; Malaysia; Réam (Cambodia); Gulf of Siam; Vietnam; Condor Island; Tang Trien (South An­ nam); Cauda Nhatrang; Hongay, Tonkin; S China Sea; Hong Kong; China; Bohai Sea; Taiwan; Philippines; Japan; South Honsyu, Kyusyu & Ryukyu Island; Vlad­ ivostok; Hawaii; central California to SW Mexico; foul­ ing species; lower littoral to sublittoral (Jones and Hosie 2016). Recently, also recorded from Ambon Island and Saparua Island, Indonesia (Pitriana et al. 2020). In this study, Amphibalanus amphitrite was found at Tanimbar Island, Indonesia. The lists of Hoek (1913), Broch (1931–1932), Jones et al. (2001) and Jones and Hosie (2016) record a total of 25 species from the Kei Islands, Aru Island and Tanimbar Island. The results currently recorded herein reveal that 40 species are now recorded from these Islands. Of the 40 species herein, ten are recorded in previ­ ous studies: Megalasma striatum Hoek, 1883 (listed in Hoek (1913)); Scalpellum stearnsi Pilsbry, 1890 (listed in Hoek (1913)); Annandaleum japonicum (Hoek, 1883) Type locality. Natal, on a piece of bamboo (Darwin 1854). Type locality. Natal, on a piece of bamboo (Darwin 1854). zse.pensoft.net zse.pensoft.net 692 Pitriana, P. Discussion et al.: Deep-sea barnacles from the Karubar expedition (1991) The results of the studies of barnacles from the Phil­ ippines and eastern Indonesian waters reconfirm the In­ do-Malayan region as the epicentre of marine biodiversity. (listed in Hoek (1913)); Annandaleum laccadivicum (An­ nandale, 1906) (listed in Hoek (1913)); Regioscalpellum moluccanum (Hoek, 1883) (listed in Hoek (1913)); Al­ tiverruca navicula (Hoek, 1913) (listed in Hoek (1913) and Buckeridge (1994)); Newmaniverruca albatrossiana (Pilsbry, 1912) (listed in Hoek (1913) and Buckeridge (1994)); Hexelasma arafurae Hoek, 1913 (listed in Hoek (1913) and Jones and Hosie (2016)); Solidobalanus au­ ricoma (Hoek, 1913) (listed in Hoek (1913) and Jones et al. (2001)); Striatobalanus tenuis (Hoek, 1883) (listed in Hoek (1913) and Jones and Hosie (2016)).The remaining 30 species can, therefore, be considered as new records for the Kei Islands and Tanimbar Island. Acknowledgements The first author would like to thank Prof. Frank Riedel from the Freie Universität Berlin for his general support and Bani Wahyudi and Abdul Basit for their support and comments on picture editing. Thanks to Bernhard Schuri­ an for supporting the morphological work at the Museum für Naturkunde Berlin. We would like to thank Dr. Martin Husemann, Prof. Benny Kwok Kan Chan and anonymous reviewer for valuable comments on the manuscript.i The present study and previous works on the barna­ cles of the Kei Islands, Aru Island and Tanimbar Island, especially the works of Hoek (1883, 1907, 1913), Broch (1922, 1931–1932), Buckeridge (1994, 1997), Jones et al. (2001) and Jones and Hosie (2016), enrich our knowl­ edge of the barnacle fauna of these islands. This study demonstrates once more the value of museum collections as a resource in biodiversity science. The first author would also like to thank Prof. Keiji Baba, Dr. Enrique Macpherson, Ferran Palero, PhD, Pau­ la Martin-Lefèvre and Sébastien Soubzmaigne for the great time working in the Crustacean Laboratory of the Muséum National d’Histoire Naturelle (MNHN), Paris and to Thomas von Rintelen (MfN Berlin) for providing funds for the research visit to Paris. The result of this study also strengthens the state­ ment of Hoeksema (2007) that the Indo-Malayan re­ gion (which extends from East Indonesia to the Philip­ pines and the Solomon Islands) is a centre of maximum marine biodiversity. Darwin (1854) demonstrated that this area had greater species richness than elsewhere in the world at the time. He named it the East Indian Archipelago (including the Philippines, Borneo, New Guinea, Sumatra, Java, Malacca and the eastern coast of India) and categorised it as his third province of bar­ nacles. In this province, he found 37 barnacle species, the largest number known at that time, compared with the other provinces. The second author would like to acknowledge Dr Alain Crosnier (retired, MNHN) for his generous support to enable her study of the KARUBAR cirripede collec­ tion in Paris. This study was funded by the Ministry of Research, Technology and Higher Education, the Republic of Indo­ nesia within the Program for Research and Innovation in Science and Technology (RISET-Pro), World Bank Loan No. 8245-ID. zse.pensoft.net References Regarding the biodiversity of barnacles, the Indo-Ma­ layan region as the centre of benthic biodiversity has not been replaced by other areas. In recent times, many studies and expeditions have been conducted in this area, revealing many more species of barnacles. For example, three expeditions have been undertaken within Philippine waters from 1976 until 1985 through MUSORSTOM Cruises and the collections the U.P. Marine Biological Laboratory at Puerto Galera, Oriental Mindoro (Rosell 1991; Chan 2009). Overall, the three of scientific cruis­ es of MUSORSTOM collected 78 species of barnacles, 43 of which are new records and 12 species are new to science (Rosell 1991). Through the Philippine Panglao expedition (2005), Chan (2009) has also increased the number of barnacles from the Philippines, reporting 20 barnacle species with two new to science. Agassiz A (1879) Preliminary report on the Echini of the Exploring Ex­ pedition of H.M.S “Challenger”. Sir C. Wyville Thomson Chief of Civilian Staff. Proceedings of the American Academy of Arts and Sciences, new series 6: 190–212. https://doi.org/10.2307/25138537 Agassiz L (1842) Nomenclator Zoologicus: Continens Nomina System­ atica Generum Animalium tam Viventum quam Fossilium, Secun­ dum Ordinem Alphabeticum Disposita, Adjectis Auctoribus, Libris, in Quibus Reperiuntur, anno Editionis, Etymologia et Familiis, ad quas Pertinent, in Singulis Classibus. Soloduri: Jent et Gassmann, 1564 pp. https://doi.org/10.5962/bhl.title.49761 Annandale N (1905) Malaysian Barnacles in the Indian Museum, with a list of the Indian Pedunculata. Memoirs of the Asiatic Society of Bengal 1: 73–84. Annandale N (1906a) Report on the Cirripedia collected by Professor Herdman at Ceylon, in 1902. Report to the Government of Ceylon on the pearl oyster fisheries of the Gulf of Manaar 5: 137–150. Similar to the Philippine waters, eastern Indonesian waters also have a high diversity of barnacles. Recently, it has been revealed that the Moluccan Islands in east­ ern Indonesia have 97 species of barnacles, 23 of which are new records and two species are still awaiting their species descriptions (Pitriana et al. 2020). Furthermore, this number will increase with the results of the study of the barnacles from Karubar expedition (1991) that have revealed 40 species of barnacles. Annandale N (1906b) Natural history notes from the R.I.M.S. ‘‘Inves­ tigator’’, Captain I.H. Heming, R.N., commanding. Series III, No 12. Preliminary report on the Indian Stalked Barnacles. The An­ nals and Magazine of Natural History 7(17): 389–400. https://doi. org/10.1080/00222930608562544 zootaxa.4319.3.8 Barnard KH (1924) Contributions to the Crustacean Fauna of South Africa. Cirripedia. Annals of the South African Museum 20(1): 1–103. Burmeister H (1834) Beiträge zur Naturgeschichte der Rankenfusser (Cir­ ripedia). 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Videnskabelige Med­ delelser Naturhistorisk Forening i København 132: 79–94. delelser Naturhistorisk Forening i København 132: 79–94. Rosell NC (1991) Crustacea Cirripedia Thoracica: MUSORSTOM 3 Philippines collection. In: Crosnier A (Ed.) Résultats des Cam­ pagnes MUSORSTOM (Vol. 9). Mémoires du Muséum national d’Histoire naturelle (A) 152: 9–61. Utinomi H (1970) Studies on the cirripedian fauna of Japan 9. Distri­ butional survey of the thoracic cirripedes in the southeastern part of the Japanese Sea. Publications of the Seto Marine Laboratory 17(5): 339–372. [figs. 57–68. https://doi.org/10.4282/sosj.19.57 Weltner W (1897) Verzeichnis der bisher beschriebenen recenten Cir­ ripedien-Arten. Mit Angabe der im Berliner Museum vorhandenen species und ihrer fundorte. Archiv für Naturgeschichte, Jahrbücher 63(1): 227–280. Say T (1822) An account of some marine shells of the United States. Journal of the Academy Natural Sciences Philadelphia 2(2): 221– 248. [302–325.] Shalaeva K, Boxshall G (2014) An illustrated catalogue of the scalpellid barnacles (Crustacea: Cirripedia: Scalpellidae) collected during the HMS “Challenger” expedition and deposited in the Natural History Museum, London. Zootaxa 3804(1): 1–63. https://doi.org/10.11646/ zootaxa.3804.1.1 Weltner W (1922) Cirripedia der Deutschen Tiefsee-Exped. Wissen­ schaftliche Ergebnisse der Deutschen Tiefsee- Expedition auf dem Dampfer ‘Valdivia’ 1895–1899 23(2): 61–112. zootaxa.3804.1.1 Withers TH (1935) Catalogue of Fossil Cirripedia in the Department of Geology (Vol. 2). Cretaceous. British Museum (Natural History), London, 534 pp. Sowerby GB (1823) The genera of recent and fossil shells, for the use of students in Conchology and Geology, with original plates by James Sowerby (nos. i–xvii), continued by J. de C. Sowerby (nos. xviii– xlii). GB Sowerby, London. [No pagination, 270 plates] [1821–1834] WoRMS Editorial Board (2020) World Register of Marine Species. https://www.marinespecies.org at VLIZ. [Accessed 2020-06-07] Steenstrup J (1837) Om Forverdenens Dyrarter af de tvende Familier Anatiferidae (Gray) og Pollicipedidae (Gray). Krøyer’s Naturhis­ toriske Tidsskrift 1: 396–415. Young PS (1998) Cirripedia (Crustacea) from the “Campagne Biacores” in the Azores region, including a generic revision of Verrucidae. Zoosystema 20: 31–92. Young PS (2001) Redescription of Scalpellopsis striatociliata Broch, 1922, with a discussion on its phylogenetic position and the pe­ duncular plate pattern in scalpellids. Journal of Crustacean Biology 21(2): 456–468. https://doi.org/10.1163/20021975-99990147 Stubbings HG (1936) Cirripedia. The John Murray Expedition 1933– 34. Scientific Reports, Zoology 4(1): 1–70. Stubbings HG (1961) Cirripedia Thoracica from tropical West Africa. Scientific results of the Danish Expedition to the coasts of tropical West Africa, 1945–1946. Atlantide Reports 6: 7–41. Young PS (2007) The Scalpellomorpha (Crustacea, Cirripedia), with a list of extant species (except the Calanticidae). Galathea Report 21: 7–73. Stubbings HG (1963a) Cirripedia from South Vietnam. Videnskabelige Meddelelser Naturhistorisk Forening i København 125: 327–335. Stubbings HG (1963b) Cirripedia of the tropical South Atlantic coast of Yusa Y, Yasuda N, Yamamoto T, Watanabe HK, Higashiji T, Kaneko A, Nishida K, Høeg JT (2018) Direct growth measurements of two deep-sea scalpellid barnacles, Scalpellum stearnsii and Graviscal­ pellum pedunculatum. Zoological Study 57: 1–29. Africa. Expédition Océanographique belge dans les eaux cȏtières afric­ Africa. Expédition Océanographique belge dans les eaux cȏtières afric­ aines de l‘Atlantique sud (1948–1949). Research Science 3(10): 1–39. aines de l‘Atlantique sud (1948–1949). Research Science 3(10): 1–39. Stubbings HG (1967) The cirriped fauna of tropical west Africa. Bul­ Stubbings HG (1967) The cirriped fauna of tropical west Africa. Bul­ letin of the British Museum (Natural History), Zoology 15(6): 229– Zevina GB (1969) Cirripedia Thoracica. In: Kort VG (Ed.) The Biology of the Pacific Ocean. Deep Sea Bottom Fauna (Vol. 2). Nauka, Mos­ cow, 66–68. [in Russian] letin of the British Museum (Natural History), Zoology 15(6): 229– si.03629236.60.1 1–72.] https://doi.org/10.5134/175604 Ross A, Newman WA (1973) Revision of the coral-inhabiting barnacles (Cirripedia: Balanidae). Transactions of the San Diego Natural His­ tory Society 17(12): 136–173. [figs. 1–25.] Utinomi H, Kikuchi T (1966) Fauna and flora of the sea around the Amakusa Marine Biological Laboratory. Amakusa Marine Biology Laboratory, Kyushu University 6: 1–12. [fig. 1.] Ross A, Pitombo FB (2002) Notes on the coral-inhabiting Megatremati­ nae and the description of a new tribe, new genus and three new spe­ cies (Cirripedia: Sessilia: Pyrgomatidae. Sessile Organisms 19(2): 57–68. https://doi.org/10.4282/sosj.19.57 Weisbord NE (1977) Scalpellid barnacles (Cirriredia) of Florida and surrounding waters. Bulletin of American Paleontology 72(299): 235–311. 319. [pl. 1.] https://doi.org/10.5962/bhl.part.27518 319. [pl. 1.] https://doi.org/10.5962/bhl.part.27518 Tarasov NE, Zevina ZB (1957) Cirripedia Thoracica of the Seas of Tarasov NE, Zevina ZB (1957) Cirripedia Thoracica of the Seas of USSR. Fauna Russian Natural Science 69, 6(1): 1–267. [In Russian] Tarasov NE, Zevina ZB (1957) Cirripedia Thoracica of the Seas of USSR. Fauna Russian Natural Science 69, 6(1): 1–267. [In Russian] Utinomi H (1949) Further notes on cirripeds from the Ogasawara Is­ lands. Pacific Science 3(1): 93–99. https://doi.org/10.5134/174435 Tarasov NE, Zevina ZB (1957) Cirripedia Thoracica of the Seas of USSR. Fauna Russian Natural Science 69, 6(1): 1–267. [In Russian] Utinomi H (1949) Further notes on cirripeds from the Ogasawara Is­ USSR. Fauna Russian Natural Science 69, 6(1): 1–267. [In Russian] Zevina GB (1970) Cirripedia Thoracica from North West Pacific. Trudy Instituta Oceanologii Akademii Nauk SSSR 86: 252–276. [in Russian] USSR. Fauna Russian Natural Science 69, 6(1): 1–267. [In Russian] Utinomi H (1949) Further notes on cirripeds from the Ogasawara Is­ lands. Pacific Science 3(1): 93–99. https://doi.org/10.5134/174435 Utinomi H (1962) Studies on the cirripedian fauna of Japan. VIII Thoracic cirripeds from Western Kyusu. Publications of the Seto Marine Laboratory 10(2): 211–239. [figs. 1–12.] https://doi. org/10.5134/175313 Utinomi H (1949) Further notes on cirripeds from the Ogasawara Is­ lands. Pacific Science 3(1): 93–99. https://doi.org/10.5134/174435 Utinomi H (1962) Studies on the cirripedian fauna of Japan. VIII Thoracic cirripeds from Western Kyusu. Publications of the Seto Marine Laboratory 10(2): 211–239. [figs. 1–12.] https://doi. org/10.5134/175313 Zevina GB (1972) Benthic Lepadomorpha (Cirripedia) from the southeast Pacific. Crustaceana 22: 39–63. https://doi. org/10.1163/156854072X00660 Supplementary material 1 Zevina GB (1978a) A new classification of the family Scalpellidae Pils­ bry (Cirripedia, Thoracica). Part 1. Subfamilies Lithotryinae, Calan­ ticinae, Pollicipinae, Scalpellinae, Brochiinae and Scalpellopsinae. Zoologicheskii Zhurnal 57 (7): 998–1007. [in Russian] org/10.5134/175313 Zevina GB (1974) The Cirripedia Thoracica of the Kerguelen Islands. Crus­ taceana 27: 209–215. https://doi.org/10.1163/156854074X00433 Utinomi H (1965) A giant Antarctic barnacle Hexelasma antarcticum Borradaile (Cirripedia Thoracica). Scientific Reports of the Japanese Antarctic Research Expedition 1956–1962, Series E, 25: 1–15. [figs. 1–4. [pls. 1–2]] Zevina GB (1975) Cirripedia Thoracica of the American Caribbean. Zevina GB (1975) Cirripedia Thoracica of the American Caribbean. Trudy Instituta Oceanologii Akademii, Nauk USSR 100: 233–258. [in Russian] zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 698 Tables S1–S39 Authors: Pipit Pitriana, Diana S. Jones, Laure Corbari, Kristina von Rintelen Authors: Pipit Pitriana, Diana S. Jones, Laure Corbari, Kristina von Rintelen Zevina GB (1978b) A new classification of the Scalpellidiae (Cir­ ripedia, Thoracica). Part 2. Subfamilies Arcoscalpellinae and Meroscalpellinae. Zoologicheskii Zhurnal 57(9): 1343–1352. [in Russian] Data type: Checklist table of species and morphometric for each species Data type: Checklist table of species and morphometric for each species Explanation note: Table S1. Checklist of barnacle spe­ cies collected during the Karubar expedition (1991) and deposited in MNHN, Paris. Tables S2–39. Mor­ phometric/measurements for each species. Zevina GB (1981) Barnacles of the Suborder Lepadomorpha (Cirripe­ dia, Thoracica) of the world oceans. I: Family Scalpellidae. Fauna SSSR 127: 1–398. [in Russian] Explanation note: Table S1. Checklist of barnacle spe­ cies collected during the Karubar expedition (1991) and deposited in MNHN, Paris. Tables S2–39. Mor­ phometric/measurements for each species. Zevina GB (1982) Barnacles of the suborder Lepadomorpha (Cirripe­ dia, Thoracica) of the world ocean. Part 2. Guides to the fauna of the USSR-Zoologicheskii Institut Akademy Nauk SSSR, Leningrad, 223 pp. [162 figs. [in Russian]] Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us­ ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us­ ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us­ ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Zevina GB (1987) Deep-sea Verrucomorpha (Cirripedia, Thoracica) of the Pacific. 1. The North Pacific. Zoologicheskii Zhurnal 66(12): 1812–1821. [in Russian] Zevina GB, Zvyagintsev AY, Negashev SE (1992) Usonogie raki po­ berezh’ya V’etnama i ikh rol’v obrastanii [Barnacles of the Vietnam Coast and their role in encrustation]. Vladivostok, Dal’nauka, 142 pp. [figs. 69, tables 8. zse.pensoft.net Tables S1–S39 [in Russian]] g ( ) Link: https://doi.org/10.3897/zse.96.55733.suppl1 Zullo VA, Newman WA (1964) Thoracic Cirripedia from a South East Pacific Guyot. Pacific Science 18: 355–372. zse.pensoft.net
https://openalex.org/W2526236365
https://link.springer.com/content/pdf/10.1007%2Fs12560-016-9262-4.pdf
English
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Contamination by Norovirus and Adenovirus on Environmental Surfaces and in Hands of Conscripts in Two Finnish Garrisons
Food and environmental virology
2,016
cc-by
7,838
Satu Oristo1 • Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 • Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 • Leena Maunula1 This study increased our understanding of the possible transmission of viruses via contaminated environment and hands. p g y p In addition to the swab samples, 14 fecal samples were obtained for viral analysis. In total, norovirus was present in 9.0 % of the surface swabs in 2013, whereas enteric adenovirus was present in 0.0 % and non-enteric aden- ovirus in 9.4 %. In the same year, 2.6 % of the hand swabs contained norovirus, 2.6 % enteric adenovirus, and 40.3 % non-enteric adenovirus. Norovirus GI.6 was continually detected on the surfaces of garrison A, and identical virus was detected in some of the fecal samples. In garrison B, two slightly different norovirus GII.4 strains were present on the surfaces. The questionnaires revealed no recent Keywords Adenovirus  Environmental contamination  Gastroenteritis  Norovirus  Questionnaire  Surface swab Satu Oristo1 • Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 • Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 • Leena Maunula1 Satu Oristo1 • Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 • Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 • Leena Maunula1 Satu Oristo1 • Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 • Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 • Leena Maunula1 Received: 13 July 2016 / Accepted: 21 September 2016 / Published online: 30 September 2016  The Author(s) 2016. This article is published with open access at Springerlink.com Abstract This study investigated the presence of norovirus and adenovirus, especially enteric adenovirus, on the environmental surfaces (n = 481) and military conscripts’ hands (n = 109) in two Finnish garrisons (A and B) in 2013 and 2014. A questionnaire study was conducted to reveal possible correlations between viral findings on the conscripts’ hands and their acute gastroenteritis symptoms. In addition to the swab samples, 14 fecal samples were obtained for viral analysis. In total, norovirus was present in 9.0 % of the surface swabs in 2013, whereas enteric adenovirus was present in 0.0 % and non-enteric aden- ovirus in 9.4 %. In the same year, 2.6 % of the hand swabs contained norovirus, 2.6 % enteric adenovirus, and 40.3 % non-enteric adenovirus. Norovirus GI.6 was continually detected on the surfaces of garrison A, and identical virus was detected in some of the fecal samples. In garrison B, two slightly different norovirus GII.4 strains were present on the surfaces. The questionnaires revealed no recent Abstract This study investigated the presence of norovirus and adenovirus, especially enteric adenovirus, on the environmental surfaces (n = 481) and military conscripts’ hands (n = 109) in two Finnish garrisons (A and B) in 2013 and 2014. A questionnaire study was conducted to reveal possible correlations between viral findings on the conscripts’ hands and their acute gastroenteritis symptoms. acute gastroenteritis cases in garrison A, but in garrison B, where the norovirus-positive hand swabs were collected, 30.6 % of the conscripts reported of recent symptoms. In 2014, norovirus was rarely detected, but adenovirus was again frequently present, both on the surfaces and hands. Taken together, our results suggest that gastroenteritis outbreaks occurred in 2013, but not in 2014. Due to the low number of hand swabs positive for enteric viruses, no conclusions about associations between viral findings and gastroenteritis symptoms could be drawn. Food Environ Virol (2017) 9:62–71 DOI 10.1007/s12560-016-9262-4 Food Environ Virol (2017) 9:62–71 DOI 10.1007/s12560-016-9262-4 ORIGINAL PAPER & Satu Oristo satu.oristo@helsinki.fi Introduction Norovirus (NoV) is the leading cause of acute viral gas- troenteritis in all age groups, as it has been reported to be responsible for almost 20 % of all acute gastroenteritis (AGE) cases worldwide (Ahmed et al. 2014). Several NoV genotypes are recognized among the three genogroups (GI, GII, GIV) that infect humans. Each genotype possesses a characteristic set of epidemiological and clinical features (Matthews et al. 2012; Kirby et al. 2014). Clinical mani- festations of NoV infection are typically vomiting, abdominal cramps, and diarrhea, but viral shedding can also be asymptomatic (Teunis et al. 2015). The infectious dose of NoV is low (Atmar et al. 2008; Teunis et al. 2008) and the virus exploits several transmission routes. It spreads efficiently, especially in semi-closed settings; during a NoV outbreak in a scout camp setting, it was estimated that 14 secondary cases occurred per every Electronic supplementary material The online version of this article (doi:10.1007/s12560-016-9262-4) contains supplementary material, which is available to authorized users. & Satu Oristo satu.oristo@helsinki.fi 1 Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Agnes Sjo¨bergin katu 2, 00790 Helsinki, Finland 2 Finnish Food Safety Authority Evira, Mustialankatu 3, 00790 Helsinki, Finland 3 Centre for Military Medicine, Tukholmankatu 8 A, 00290 Helsinki, Finland 4 Finnish Defence Command Logistics Division, Fabianinkatu 2, 00130 Helsinki, Finland 123 Food Environ Virol (2017) 9:62–71 63 primary case, when enhanced hygienic measures were not practiced (Heijne et al. 2009). 2014), but swab samples from frequently touched objects in the conscripts’ living quarters were also included (Table 1; Figs. 1, 2). primary case, when enhanced hygienic measures were not practiced (Heijne et al. 2009). Over 60 adenovirus (AdV) types are recognized to date (Robinson et al. 2013), and in addition to respiratory dis- ease, different AdV types are capable of causing menin- gitis, eye infections, and gastroenteritis (Lynch et al. 2011). Respiratory AdV infections have affected the armed forces so severely in the past that an efficient vaccine against the most common types of AdV responsible for respiratory disease (types 4 and 7) is routinely used in the US Armed Forces (Radin et al. 2014). AdV types 40 and 41 are known as enteric AdVs (eAdVs), as they are the most common types associated with gastroenteritis (Lynch et al. 2011). Although clinical gastroenteritis due to eAdV usually only occurs in children and immunocompromised people (Lynch et al. Introduction 2011), they are so common in the general population that they have been proposed as viral markers of fecal contamination of water (Rusin˜ol et al. 2014). Surface Swab Sampling Our sampling scheme included the collection and analysis of only swab samples, but after the surface and hand swab sampling period was finished in 2013, we obtained 11 anonymous fecal samples from conscripts who had suf- fered from gastroenteritis in garrison A between March 5, and May 8, 2013 (Table 1). These samples were collected by the health care personnel of garrison A. In garrison B, no fecal samples were collected in 2013 but three were obtained in 2014. In March–May 2013, we collected 132 surface swabs in garrison A, and 135 surface swabs in garrison B, during six visits to each garrison (Table 1). In addition, 214 surface swabs were collected during 11 visits to garrison B in January–February 2014. The swabbing was performed as previously described by Ro¨nnqvist et al. (2013). Briefly, a 25 cm2 surface area (or the whole object in case it was smaller) was swabbed with a polyester or microfiber swab moistened in phosphate-buffered saline (PBS). The swabs were taken from surfaces that are often touched, e.g., door handles, flushing buttons, vending machines, and electronic devices. Most of the sampling sites within both garrisons were in the lavatories (76.4 % in 2013 and 74.3 % in Questionnaires All the conscripts (n = 109) who participated in the hand swab study filled in a questionnaire, in which they reported when they had last experienced AGE symptoms (diarrhea and either abdominal pain, vomiting, or both) and whether they had been in contact with other conscripts or non- military persons who had AGE symptoms within the pre- vious 6 days. Although the participants for the hand swab study were selected in the sick bay waiting area in 2013, their reason for visiting there on the sampling date was not enquired. The hand swabs and questionnaires were col- lected anonymously. Hand Swab Sampling We collected 28 hand swabs during two of the visits to garrison A in April 2013, and 49 hand swabs during three of the visits to garrison B in April–May 2013 (Table 1). The conscripts who participated in the hand swab study were randomly selected in the sick bay waiting area. Of the garrison A and B conscripts, 8/28 (28.6 %) and 25/49 (51.0 %), respectively, resided in the barracks from where the surface swabs were taken. In January–February 2014, 32 hand swabs were collected during two of the visits to garrison B. In contrast to the hand swab study performed in 2013, the garrison B conscripts who participated in 2014 were all residing in the sampled barracks. Hand swabbing was performed similarly to the surface swabbing but using only the microfiber swab. Both palms were swabbed for at least 1 min. Both NoV and AdV infections are problematic for the armed forces because these are capable of causing a remarkable reduction in the operational efficiency of the affected units. The aim of this study therefore was to characterize the contamination by NoV and AdV, espe- cially eAdV, on environmental surfaces and army con- scripts’ hands in military garrison settings. Hand swabbing was coupled with a questionnaire to reveal any correlation between viral findings on conscripts’ hands and their AGE symptoms, or other signs of a possible AGE outbreak. In 2013, the sampling was performed in March–May, when NoV outbreaks typically occur (Kroneman et al. 2008). In 2014, the sampling was done earlier, in January–February, in order to follow the possible transmission of NoV among the new conscripts during their first training period. Table 1 A summary of the different samples collected in garrisons A and B during the study period Swab and Fecal Sample Preparation A known amount of either murine norovirus (MuNoV) strain MNV-1 (kindly gifted by Professor Herbert W. Virgin, Washington University, St. Louis, MO, USA) or 3 3 12 64 Food Environ Virol (2017) 9:62–71 Sample category Year 2013 2014 Garrison A Garrison B Garrison B Surface swabs No. of swabs per sampled building Health center 22 22 6 Barracks 110 107 208 Cafe 0 6 0 Total 132 135 214 Sampling period (no. of visits) Mar.12–May 14 (6) Apr.10–May 22 (6) Jan.3–Feb.2 (11) Hand swabs No. of hand swabs 28 49 32 Sampling period (no. of visits) Apr.16–Apr.23 (2) Apr.17–May 8 (3) Jan.29–Feb.2 (2) Fecal samples No. of fecal samples 11 0 3 Sampling perioda Mar.5–May 14 – Jan.30–Feb.4 a The 11 fecal samples collected in 2013 were available for viral analysis only after the surface and hand swabbing period was finished in May 2013 a The 11 fecal samples collected in 2013 were available for viral analysis only after the surface and hand swabbing period was finished in May 2013 a The 11 fecal samples collected in 2013 were available for viral analysis only after the surface and hand swabbing period was finished in May 2013 70 50 30 10 10 30 50 70 Lavatory, door handle (n=44) Lavatory, flushing button (n=32) Door handle or light switch (n=17) Game controller (n=12) Soft drinks vending machine (n=11) Lavatory, water tap (n=5) Lavatory, bowl (n=3) Remote controller (n=3) Snacks vending machine (n=2) Telephone (n=2) Basement water tap (n=1) NoV GI % AdV % Fig. 1 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison A in 2013. All NoV findings represented genogroup I (NoV GI) Fig. 1 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison A in 2013. All NoV findings represented genogroup I (NoV GI) 60 40 20 0 20 40 60 Lavatory, door handle (n=62) Lavatory, flushing button (n=49) Lavatory, bowl (n=7) Soft drinks vending machine (n=6) Computer keyboard and mouse (n=6) Door handle or light switch (n=2) Lavatory, water tap (n=2) Wardrobe (n=1) NoV GII % AdV % Fig. 2 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison B in 2013. Real-Time Reverse Transcription PCR and PCR Protocols The swab and fecal samples were screened for NoV GI and GII by real-time reverse transcription PCR (rRT-PCR), whereas real-time PCR (rPCR) was used for screening AdV. All primers and probes used in this study are pre- sented in Online Resource 1. NoV GII detection was per- formed as previously described (Ro¨nnqvist et al. 2013), and the same protocol was used for NoV GI, except 0.9 lM of each GI-specific primer and 0.3 lM of GI-specific probe were used. MuNoV and MeV were analyzed by a similar method to NoV GII but with virus-specific primers and probes. The QuantiTect Probe PCR kit (QIAGEN) was used both for the detection of all AdVs and then for the detection of eAdV in the AdV-positive samples. The 20 ll AdV (or eAdV) reaction mix consisted of 10 ll of 2 x QuantiTect Probe PCR Master Mix, 1.0 lM of reverse and forward primers, 0.2 lM of probe, 0.6 ll of PCR-grade H2O, and 5 ll of template. Initial activation was performed at 95 C for 15 min, followed by 45 cycles of 94 C for 15 s, 55 C for 45 s, and 72 C for 45 s. Both rRT-PCR and rPCR reactions were performed using the Rotor-Gene 3000 thermal cycler (QIAGEN). All viral findings were immediately reported to the respective garrisons’ personnel. Swab and Fecal Sample Preparation All NoV findings represented genogroup II (NoV GII) 60 40 20 0 20 40 60 Lavatory, door handle (n=62) Lavatory, flushing button (n=49) Lavatory, bowl (n=7) Soft drinks vending machine (n=6) Computer keyboard and mouse (n=6) Door handle or light switch (n=2) Lavatory, water tap (n=2) Wardrobe (n=1) NoV GII % AdV % Fig. 2 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison B in 2013. All NoV findings represented genogroup II (NoV GII) 12 Food Environ Virol (2017) 9:62–71 65 mengovirus (MeV) strain MC0 (kindly gifted by Professor Albert Bosch, University of Barcelona, Spain) was added directly on the surface and hand swabs to act as a process control. Approximately every 12th swab sample was spiked with 1.0 9 105 PCR units (PCR-u) of MuNoV in 2013, so that at least one spiked sample was included in each nucleic acid extraction batch. In 2014, every 6th swab sample was spiked either with 2.0 9 104 or 2.0 9 105 PCR-u of MeV. The viral particles were eluted from the swabs by a semi-direct lysis method, and the nucleic acids were extracted as previously described (Ro¨nnqvist et al. 2013). 10 % fecal suspensions were prepared in sterile 1 x PBS, and nucleic acids were extracted with the QiaAmp Mini Viral RNA kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. and sequenced according to the Sanger sequencing method in the Institute of Biotechnology, University of Helsinki, Finland. NoV and AdV Detection on the Environmental Surfaces of Garrisons A and B in 2013 In total, NoV was present in 9.0 % of the surface swabs collected in garrisons A and B in 2013, whereas eAdV was present in 0.0 % and non-eAdV in 9.4 %. p NoV GI was detected in garrison A in 9.1 % (12/132) of the surface swabs (Table 2). Most of the NoV-positive samples were collected from the lavatories (Fig. 1), but the difference between NoV findings for every garrison A lavatory (10.7 %; 9/84) and other surface (6.3 %; 3/48) was not significant. One of the sampled lavatory surfaces tested positive for NoV GI in two consecutive visits 4 weeks apart. None of the AdV findings on the surfaces of garrison A were confirmed as eAdV. Non-eAdV findings on the garrison A surfaces (6.1 %; 8/132) were similarly distributed between the lavatories and the other environ- mental surfaces (7.1 %; 6/84 vs. 4.2 %; 2/48) as for NoV, but none of the swabs were positive for both viruses. Non- eAdV was once detected twice on the same lavatory sur- face in two consecutive visits 1 week apart. Data Analyses Raw sequence data were analyzed using BioEdit software version 7.0.5.3 (http://www.mbio.ncsu.edu/BioEdit/bioe dit.html) and sequence identities calculated using the Clustal Omega software version 1.2.1 (http://www.ebi.ac. uk/Tools/msa/clustalo/). The sequences were genotyped using the RIVM norovirus genotyping tool (Kroneman et al. 2011) (http://www.rivm.nl/mpf/norovirus/typingtool) and NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). IBM SPSS Statistics software version 22 (IBM Corp., Armonk, NY, USA) and OpenEpi version 3.03a (http:// openepi.com/Menu/OE_Menu.htm) were used for statisti- cal analyses of the results. P values \ 0.05 were consid- ered to be statistically significant. Reverse Transcription PCR Protocols NoVs were detected in garrison B on three sampling visits, but in contrast to garrison A, all strains belonged to the GII genogroup (8.9 %; 12/135) (Table 3), and all the NoV-positive swabs were collected from the lavatories (Fig. 2). One of the sampled lavatory surfaces tested pos- itive for NoV GII in two consecutive visits 1 week apart. As in garrison A, none of the swabs were positive for eAdV or both NoV and AdV. Non-eAdV was again a frequent finding (12.6 %; 17/135), both on the lavatory (10.8 %; The samples that were positive for NoVs by rRT-PCR were subjected to reverse transcription PCR (RT-PCR) reac- tions, performed with the QIAGEN One-Step RT-PCR kit reagents (QIAGEN). Four different primer pairs that tar- geted the polymerase (ORF1) and/or the capsid (ORF2) region were used (Online Resource 1). Amplified products were visualized on 1.5 % SeaKem LE (Lonza, Basel, Switzerland) agarose gel with ethidium bromide staining, 12 3 3 Food Environ Virol (2017) 9:62–71 66 Table 2 Viral findings on the surface and hand swabs collected in garrison A during the study period Sampling date Garrison A Surface swabs (%) Hand swabs (%) Norovirusa Adenovirusb Norovirus Adenovirus Year 2013 Mar.12 8/30 (26.7) 3/30 (10.0) – – Apr.9 2/20 (10.0) 0/20 (0.0) – – Apr.16 1/21 (4.8) 4/21 (19.0) 0/16 (0.0) 7/16 (43.8) Apr.23 0/18 (0.0) 1/18 (5.6) 0/12 (0.0) 7/12 (58.3)c May 7 1/21 (4.8) 0/21 (0.0) – – May 14 0/22 (0.0) 0/22 (0.0) – – Total 12/132 (9.1) 8/132 (6.1) 0/28 (0.0) 14/28 (50.0) a All detected noroviruses on the surfaces of this garrison belonged to genogroup I; of these norovirus- positive samples, three were confirmed as genotype GI.6 by sequencing b None of the adenoviruses detected on the surfaces were confirmed as adenovirus type 40/41 c Two adenovirus strains detected on the hand swabs represented adenovirus type 40/41 Table 3 Viral findings on the surface and hand swabs in garrison B during the study period Sampling date Garrison B Surface swabs (%) Hand swabs (%) Norovirusa Adenovirusb Norovirusa Adenovirusb Year 2013 Apr. these, 2.6 % (2/77) contained NoV, 2.6 % (2/77) eAdV and 40.3 % (31/77) non-eAdV. The hand swabs of garrison A were all negative for NoV (Table 2). eAdV was, however, detected in 7.1 % (2/28) and non-eAdV in 42.9 % (12/28) of the hand swab sam- ples. Two of the hand swabs collected in garrison B (4.1 %; 2/49) were positive for NoV GII (Table 3). Non- eAdVs were present in 38.8 % (19/49) of the hand swabs. NoV GII and non-eAdV were detected in the same hand swab sample on one occasion. Table 3 Viral findings on the surface and hand swabs in garrison B during the study period Table 2 Viral findings on the surface and hand swabs collected in garrison A during the study period Reverse Transcription PCR Protocols 10 6/24 (25.0) 3/24 (12.5) – – Apr.17 5/25 (20.0) 2/25 (8.0) 1/27 (3.7) 8/27 (29.6) Apr.24 0/21 (0.0) 7/21 (33.3) 0/13 (0.0) 7/13 (53.8) May 8 0/21 (0.0) 1/21 (4.8) 1/9 (11.1) 4/9 (44.4) May 15 1/22 (4.5) 0/22 (0.0) – – May 22 0/22 (0.0) 4/22 (18.2) – – Total 12/135 (8.9) 17/135 (12.6) 2/49 (4.1) 19/49 (38.8) Year 2014 Jan.3 0/21 (0.0) 2/21 (9.5) – – Jan.9 1/21 (4.8) 2/21 (9.5) – – Jan.13 and Jan.16 0/44 (0.0) 1/44 (2.3) – – Jan.21 and Jan.23 0/44 (0.0) 2/44 (4.5) – – Jan.27, Jan.29, and Feb.2 0/64 (0.0) 2/64 (3.1) 0/32 (0.0) 6/32 (18.8) Feb.5 and Feb.7 0/20 (0.0) 2/20 (10.0) – – Total 1/214 (0.5) 11/214 (5.1) 0/32 (0.0) 6/32 (18.8) a All detected noroviruses on the surfaces and hands in this garrison belonged to genogroup II; of these norovirus-positive samples, five were confirmed as genotype GII.4 by sequencing b None of the detected adenoviruses were confirmed as adenovirus type 40/41 a All detected noroviruses on the surfaces and hands in this garrison belonged to genogroup II; of these norovirus-positive samples, five were confirmed as genotype GII.4 by sequencing b None of the detected adenoviruses were confirmed as adenovirus type 40/41 13/120) and the other surfaces (26.7 %; 4/15). Three of the surfaces were non-eAdV-positive in two consecutive visits (twice the computer keyboard and once a door knob in the sick bay). these, 2.6 % (2/77) contained NoV, 2.6 % (2/77) eAdV and 40.3 % (31/77) non-eAdV. NoV and AdV Findings in the Hand Swab Samples in Garrisons A and B in 2013 We collected a total of 77 hand swabs during two of the visits to garrison A and three of the visits to garrison B. Of 123 Food Environ Virol (2017) 9:62–71 67 NoV and AdV Detection in the Fecal Samples Collected in Garrison A in 2013 obtained from eight garrison A samples (Table 4). Regardless of the sample type (fecal or surface), all the GI.Pb-GI.6 sequences from six samples were 100 % identical. The two NoV GII-positive fecal samples repre- sented different genotypes: sample F1 was a recombinant between the pandemic variants GII.P4-New Orleans-2009 and GII.4-Sydney-2012, while sample F4 represented genotype GII.7. After we had finished analyzing the swab samples in 2013, we obtained 11 fecal samples for NoV and AdV analysis (Table 1). Three (33.3 %) of the nine fecal samples col- lected in the sick bay in the beginning of March 2013 were found to be positive for NoV GI, and two (22.2 %) were positive for NoV GII. All these NoV-positive fecal samples were collected less than a week before the first surface swabbing visit in March 12, 2013, when the number of NoV-positive surface samples (26.7 %; 8/30) was highest. The two fecal samples that were collected later, on March 13 and May 14, were NoV negative. All 11 samples were AdV-negative. Partial NoV capsid sequences (ORF1/2 junction) were obtained from five garrison B samples (Table 4). The capsid sequences of the surface samples S4, S6, and S7 were 100.0 % identical with each other, and also with the short sequence obtained from the hand swab sample H1. This variant was identified as the NoV GII.4-Sydney-2012 by the RIVM norovirus genotyping tool (Kroneman et al. 2011). The capsid region of the other detected NoV GII.4- Sydney-2012 variant (S5) was 97.2 % identical with the surface samples S4, S6, and S7 but 100 % identical with the capsid region of the garrison A fecal sample F1. The exact variants of the samples F1 and S5 were not identified by the RIVM norovirus genotyping tool (Kroneman et al. 2011), but according to the NCBI BLAST, they were 100 % identical with the capsid region of a recombinant strain New Orleans 2009/Sydney 2012 (GenBank acces- sion no. KF378731) that was detected in Italy in 2013 (Martella et al. 2013). NoV and AdV Findings in January–February 2014 (Garrison B Only) One lavatory surface tested positive for NoV GII (0.5 %; 1/214) in 2014 (Table 3). AdVs, all non-eAdVs, were detected in 4.4 % (7/159) of the lavatory surfaces and 7.2 % (4/55) of the other surfaces (in total 5.1 %; 11/214). None of the hand swabs were positive for NoVs or eAdVs, but non-eAdV was detected in 18.8 % (6/32). The three fecal samples collected in 2014 were negative for NoVs and AdVs. Detection of the Process Control Viruses In 2013, all 28 conscripts in garrison A reported themselves as healthy (i.e., no AGE symptoms within 6 days at the time of the hand swabbing), but 28.6 % (8/28) of them had been in contact with another conscript who had AGE symptoms within the previous 6 days (Table 5). In con- trast, 30.6 % (15/49) of the conscripts in garrison B in 2013 had suffered from AGE symptoms within 6 days before hand swabbing, and 63.3 % (31/49) of them had been in contact with another conscript who had suffered from AGE symptoms recently. Also, the conscripts in garrison B in 2013 had more contacts with non-military persons suffer- ing from AGE symptoms than the conscripts in garrison A (10.7 vs. 40.8 %; P = 0.005, Mid-P exact test) in the same year. Recent AGE symptoms were rarer among the gar- rison B conscripts in 2014 when compared to their coun- terparts in 2013 (30.6 vs. 9.4 %; P = 0.025, Mid-P exact test). The conscripts’ contacts with other people (military or non-military) suffering from recent AGE symptoms did not differ significantly between years 2013 and 2014 in garrison B. The lower limit of an acceptable result for the process control virus detection by rRT-PCR was decided to be a Ct value \ 40. In all expect four occasions, the positive control virus gave a positive result. We were not able to reanalyze the samples that remained negative for the pro- cess control viruses because no sample material remained after the initial nucleic acid extraction. The majority of the samples (94.1 %; data not shown) that were positive for NoVs and/or AdVs were, however, not the ones that were spiked with the process control viruses. Sequence Analysis These sequences were not submitted to the GenBank database d Th ORF1/2 j ti f th l S3 d H1 198 b d 127 b ti l Table 4 Genotypes of the sequenced samples a Genotype and variant, if available, according to the ORF1 sequence obtained with primers RegA and MJV12 c Genotype according to the ORF2 sequence obtained with primers GI.6RR/FF. These sequences were not submitted to the GenBank database d The ORF1/2 junction sequences of the samples S3 and H1 were 198 bp and 127 bp, respectively Wadl et al. 2010; Fankem et al. 2014). In contrast, NoV contamination on the surfaces was rare in January–Febru- ary 2014. These results seem to reflect the overall NoV situation in Finland during our study periods, because in March–May 2013, the health authorities of Finland repor- ted twice as many laboratory-confirmed NoV cases as they did in January–February 2014 (THL 2015). Although the detection of viral genome does not necessarily indicate the presence of infectious virus, NoV is known to be relatively stable on environmental surfaces (D´ Souza et al. 2006), so transmission of viruses via fomites may have occurred. Also, Boxman et al. (2011) showed that even when there was no evidence of an ongoing NoV outbreak, surface contamination by NoV correlated with the food producing facility’s NoV outbreak history. on his hands also had AGE symptoms within 6 days before the hand swabbing, whereas the other conscript who gave a NoV-positive hand swab, although not having AGE symptoms, had to be given intravenous fluids at the time of swabbing to treat dehydration. The conscripts that had eAdV on their hands did not report of recent AGE symp- toms. Non-eAdV findings were not correlated with AGE symptoms, as expected. ype and variant, if available, according to the ORF1 sequence obtained with primers RegA and MJV12 Sequence Analysis Of the total number of samples that were NoV-positive by rRT-PCR in 2013 and 2014 (n = 32; 25 surface swabs, two hand swabs, and five fecal samples), 17 surface and one hand swab sample collected in 2013, and one surface swab sample collected in 2014 did not show a right-sized product in any of the conventional RT-PCR-tests that targeted different regions of the genome, so these samples were not subjected to sequencing. Due to the low number of NoV- or eAdV-positive hand swabs, reliable statistical analysis between these findings, and the occurrence of AGE symptoms could not be per- formed. However, one of the conscripts that had NoV GII Partial NoV sequences from either the polymerase (ORF1) and/or capsid regions (ORF1/2 junction) were 12 123 3 Food Environ Virol (2017) 9:62–71 68 Table 4 Genotypes of the sequenced samples Garrison Sampling date (Year 2013) Sample code Sample type Genotype Genbank accession no. ORF1a ORF1/2b ORF2c ORF1 ORF1/2 A Mar.5 F1 Fecal GII.P4-New Orleans-2009 GII.4-Sydney- 2012 KT943510 Identical to KT943512 Mar.6 F2 Fecal GI.Pb GI.6 GI.6 Identical to KT943508 KT943509 Mar.6 F3 Fecal GI.Pb GI.6 NA Identical to KT943508 Identical to KT943509 Mar.7 F4 Fecal NA GII.7 NA NA KT943513 Mar.8 F5 Fecal GI.Pb GI.6 NA Identical to KT943508 Identical to KT943509 Mar.12 S1 Surface swab GI.Pb GI.6 GI.6 KT943508 Identical to KT943509 Mar.12 S2 Surface swab NA NA GI.6 NA NA Apr.9 S3d Surface swab NA GI.6 GI.6 NA Identical to KT943509 B Apr.10 S4 Surface swab NA GII.4-Sydney- 2012 NA NA KT943511 Apr.10 S5 Surface swab NA GII.4-Sydney- 2012 NA NA KT943512 Apr.17 S6 Surface swab NA GII.4-Sydney- 2012 NA NA Identical to KT943511 Apr.17 S7 Surface swab NA GII.4-Sydney- 2012 NA NA identical to KT943511 Apr.17 H1d Hand swab NA GII.4-Sydney- 2012 NA NA Identical to KT943512 a Genotype and variant, if available, according to the ORF1 sequence obtained with primers RegA and MJV12 b Genotype and variant, if available, according to the ORF1/2 junction sequence obtained either with primers JJVMF/G1SKR (samples F2, F3, F5, S1, S3) or QNIF2D/G2SKR (samples F1, F4, S4 – S7, H1) c Genotype according to the ORF2 sequence obtained with primers GI.6RR/FF. c Genotype according to the ORF2 sequence obtained with primers GI.6RR/FF. These sequences were not submitted to the GenBank database d The ORF1/2 junction sequences of the samples S3 and H1 were 198 bp and 127 bp, respectively b Genotype and variant, if available, according to the ORF1/2 junction sequence obtained either with primers JJ F5, S1, S3) or QNIF2D/G2SKR (samples F1, F4, S4 – S7, H1) Discussion P value 2 is calculated between the questionnaire results collected in garrison B in 2013 and 2014 * P values \ 0 05 were considered statistically significant a P values were calculated with mid-P exact test. P value 1 is calculated between the questionnaire results collected in garrisons A and B in 2013. P value 2 is calculated between the questionnaire results collected in garrison B in 2013 and 2014 * P values \ 0.05 were considered statistically significant Although NoV was detected on the surfaces of both garrisons for several weeks in 2013, NoV contamination on the same surface during two consecutive visits happened only twice. This implies that the surfaces had been cleaned and these surfaces were then recontaminated, either by new cases or by prolonged shedding of NoV by the recovered or asymptomatic cases. The spread of NoV via lavatory sur- faces is a known risk. However, the frequent presence of NoV and AdV on several other environmental surfaces on the same premises suggest inadequate hygiene practices. Virus transmission via hands or fomites was therefore also possible in other facilities, recreational or otherwise. It has been reported that viral contamination can spread via contaminated cleaning equipment (Fankem et al. 2014), but it seems that the cleaning procedures in the two garrisons we studied were adequate for inactivating and removing NoVs from surfaces. in early March. Their suspicions were later supported by the detection of NoV in five of the fecal samples. Also, it was the same genotype (GI.6-GI.Pb) that was detected on the surfaces—including a surface in the sick bay—and in three of the fecal samples. Somewhat surprisingly, the questionnaires collected in this garrison did not indicate that gastroenteritis cases had occurred recently. However, these questionnaires and the hand swabs were collected several weeks later than most of the positive surface samples. Moreover, most of the conscripts who participated in the hand swab and questionnaire study were residing in living quarters which were not swab sampled. In contrast, the questionnaires collected in garrison B clearly indicated that gastroenteritis cases had occurred during the study period: almost one-third (30.6 %) of the conscripts had AGE symptoms within 6 days before the hand swabbing, and the majority (63.3 %) of them had been in contact with other conscripts who were suffering from AGE. Discussion This study revealed that NoV was present on the envi- ronmental surfaces of two Finnish garrisons for several weeks in spring 2013. During the first visit to each gar- rison, one quarter of the surface swabs were NoV-positive, which is in line with other studies that have been conducted during, or shortly after, an identified NoV outbreak (Cheesbrough et al. 2000; Wu et al. 2005; Jones et al. 2007; After our swab sampling period was finished in May 2013, we were informed that the personnel of garrison A had suspected a gastroenteritis outbreak, and collected 11 fecal samples from conscripts suffering from gastroenteritis 123 Food Environ Virol (2017) 9:62–71 69 Table 5 Conscripts’ reports of their recent acute gastroenteritis (AGE) symptoms (diarrhea and either abdominal pain, vomiting, or both) and contacts with other conscripts or non-military persons suffering from AGE. Gar = garrison Category Year P valuesa 2013 2014 Gar A 95 % CI Gar B 95 % CI Gar B 95 % CI 1 2 Conscripts who had AGE symptoms within 6 days before hand swabbing (no./total; %) 0/28 (0.0) 0.0–14.3 15/49 (30.6) 19.4–44.6 3/32 (9.4) 2.5–25.0 \0.001* 0.025* Conscripts who had been in contact with another conscript who had AGE symptoms within 6 days before hand swabbing (no./total; %) 8/28 (28.6) 15.1–47.2 31/49 (63.3) 49.2–75.4 15/32 (46.9) 30.9–63.6 0.004* 0.156 Conscripts who had been in contact with non-military persons who had AGE symptoms within 6 days before hand swabbing (no./total; %) 3/28 (10.7) 2.9–28.0 20/49 (40.8) 28.2–54.8 9/32 (28.1) 15.4–45.5 0.005* 0.257 a P values were calculated with mid-P exact test. P value 1 is calculated between the questionnaire results collected in garrisons A and B in 2013. P value 2 is calculated between the questionnaire results collected in garrison B in 2013 and 2014 * P values \ 0.05 were considered statistically significant Table 5 Conscripts’ reports of their recent acute gastroenteritis (AGE) symptoms (diarrhea and either abdominal pain, vomiting, or both) and contacts with other conscripts or non-military persons suffering from AGE. Gar = garrison Table 5 Conscripts’ reports of their recent acute gastroenteritis (AGE) symptoms (diarrhea and either abdominal pain, vomiting, or both) and contacts with other conscripts or non-military persons suffering from AGE. Gar = garrison a P values were calculated with mid-P exact test. P value 1 is calculated between the questionnaire results collected in garrisons A and B in 2013. Discussion Unfortunately, we were not informed if the health care personnel of garrison B had suspected a gas- troenteritis outbreak in spring 2013, and no fecal samples were obtained. NoV was, however, detected in two of the hand swabs collected in this garrison. According to Box- man et al. (2009) and Liu et al. (2013), infected persons often have detectable NoV on their hands, both in labora- tory and outbreak settings. It has also been demonstrated that NoV remains detectable on finger pads only for a couple of hours (Liu et al. 2009), which implies that NoV contamination on the hands of these conscripts must have happened soon before the hand swabbing. The presence of the same NoV GII.4 variant both on the surfaces and in a hand swab further supported that NoV was at least one of the causative agents of these gastroenteritis cases. In our study, the presence of eAdV did not coincide with that of NoV. Non-eAdVs were, however, frequently pre- sent both on the surfaces and hands. The non-eAdVs on the surfaces were distributed between the lavatories and the other places similar to that found for NoVs; however, because some non-eAdVs are also excreted in feces (Russell et al. 2006; Lynch et al. 2011; Rusin˜ol et al. 2014; Verani et al. 2014), it is not possible to tell whether the source of non-eAdV was contamination from feces or from other bodily excretions. Other studies have also reported of detecting non-eAdVs frequently on lavatory surfaces and air (Russell et al. 2006; Verani et al. 2014). The prevalence of non-eAdV on the hands of conscripts was somewhat lower than that reported by Russell et al. (2006); in their study, 69 % of conscripts with febrile respiratory AdV illness had AdV 4 DNA on their hands. In our study, the 12 Food Environ Virol (2017) 9:62–71 70 hands illustrate route of transmission: A case study. Journal of Food Protection, 72(8), 1753–1755. conscripts were not questioned about recent symptoms of respiratory-or other illnesses, so based on our results we cannot exclude the possibility that an outbreak of non- eAdV was ongoing. Boxman, I. L., Verhoef, L., Dijkman, R., Ha¨gele, G., Te Loeke, N. A., & Koopmans, M. (2011). Year-round prevalence of norovirus in the environment of catering companies without a recently reported outbreak of gastroenteritis. Applied and Environmental Microbiology, 77(9), 2968–2974. Discussion We conclude by stating that NoV cases occurred in both garrisons during the study period in 2013, and the detection of NoV on the surfaces during the same period was frequent. This was in contrast to the 2014 results, when both AGE cases and NoV findings on the surfaces were rare. We were not able to draw any conclusions on whether there was a correlation between the viral findings on hands and AGE symptoms because of the low number of NoV- or eAdV-positive hand swabs. Some swab samples remained negative for the process control viruses, which indicates that viruses are lost during sample pro- cessing. Therefore, it is possible that some of our swabs were false-negative for NoV and AdV. We find that routine surface swabbing, however, provides valuable information on the presence of both of these viruses, and we believe that in our study, the rapidly disseminated information of the virus-positive surfaces to the garrisons’ personnel had a role in preventing larger scale outbreaks caused by NoV. Cheesbrough, J. S., Green, J., Gallimore, C. I., Wright, P. A., & Brown, D. W. (2000). Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiology and Infection, 125(1), 93–98. da Silva, A. 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Acknowledgments This study was funded by the Centre for Military Medicine (Finnish Defence Forces) research project ‘‘Prevalence and transmission routes of norovirus in the Finnish Defence Forces,’’ the European Community’s 7th Framework Programme ‘‘Aquavalens’’ (Grant 311846), and the Walter Ehrstro¨m Foundation. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Conflict of Interest The authors declare that they have no conflict of interest. Jothikumar, N., Cromeans, T. L., Hill, V. R., Lu, X., Sobsey, M. D., & Erdman, D. D. (2005a). Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41. Applied and Environmental Microbiology, 71(6), 3131–3136. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Jothikumar, N., Lowther, J., Henshilwood, K., Lees, D., Hill, V., & Vinje´, J. (2005b). Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Applied and Environmental Microbiology, 71(4), 1870–1875. Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S., Hoshino, F. B., et al. (2003). Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. Journal of Clinical Microbiology, 41(4), 1548–1557. 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The National Infectious Diseases Register. https://www.thl.fi/ ttr/gen/rpt/tilastot.html. Accessed on 18 Aug 2015. van Maarseveen, N. M., Wessels, E., de Brouwer, C. S., Vossen, A. C., & Claas, E. C. (2010). Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus, Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex real-time PCR assays. Journal of Clinical Virology, 49(3), 205–210. Martella, V., Medici, M. C., De Grazia, S., Tummolo, F., Calderaro, A., Bonura, F., et al. (2013). Evidence for recombination between pandemic GII.4 norovirus strains New Orleans 2009 and Sydney 2012. Journal of Clinical Microbiology, 51(11), 3855–3857. Matthews, J. E., Dickey, B. W., Miller, R. D., Felzer, J. R., Dawson, B. P., Lee, A. S., et al. (2012). The epidemiology of published norovirus outbreaks: a review of risk factors associated with attack rate and genogroup. Epidemiology and Infection, 140(7), 1161–1172. 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Microstructure-Property Relationship in Advanced Ni-Based Superalloys
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1. Introduction Ni based superalloys have been developed more or less empirically over the past 60 years from a simple Ni-Cr matrix to the present multi element and phase systems[1], having a fully austenitic face centred cubic (fcc) structure which maintains a superior tensile, fatigue and creep properties at high temperature to a body centred cubic (bcc) alloy[1]. One of the major applications of Ni superalloys is gas turbine engines. They comprise over 50% of the weight of advanced aircraft engines and include wrought and cast turbine blades and powder metallurgy (P/M) route turbine discs [1]. One of the most important goals of engine design is increasing turbine entry temperature (TET): the temperature of the hot gases entering the turbine arrangement [2]. This implies that the resistance against the environmental attack, i.e. high temperature, under a severe mechanical force is the priority challenge and indeed Ni based superalloys are used in the hottest as well as the highest tensile pressure of the gas turbine engine component as shown in the schematic diagram in Fig. 1. Nowadays, for the advanced cast single crystal superalloys in the turbine blades, the alloy capability exceeds 1,000ºC [2]. In this chapter, the polycrystalline Ni superalloys, which have slightly less temperature capability up to 800°C, applied in the turbine discs and the adjoined shafts, will be introduced focusing on their microstructures correlating with the mechanical properties. http://dx.doi.org/10.5772/52011 http://dx.doi.org/10.5772/52011 © 2012 Kitaguchi, licensee InTech. This is an open access chapter 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. Microstructure-Property Relationship in Advanced Ni-Based Superalloys Hiroto Kitaguchi Additional information is available at the end of the chapter 2.1. Hardening precipitates On the other hand, the precipitates arise at lower temperature such as during cooling after heat treatment stay small (Fig. 4 (left hand side of the image)). γ´ has the perfect coherency with the γ matrix, hence their morphologies are mostly sphere, whereas γ´´ has a tall crystal unit tetragonal structure where a axis has the identical lattice parameter with the γ matrix but c axis has nearly double the length of the γ, hence γ´´ always precipitate with the perfect coherency on the basal plane with γ and grow along the longitudinal direction (Fig. 5). 2.1. Hardening precipitates From the point of view of microstructure, Ni superalloys are complex [4]. The fcc matrix, known as γ, mainly consists of nickel, cobalt, iron, chromium and molybdenum. The strength of superalloys are conferred by the hardening precipitates known as γ´ (Ni3Al based L12 Metallurgy – Advances in Materials and Processes 20 Figure 1. (a) Schematic diagram of a turbine engine Ref. [3]. (b) Schematic diagram of the temperature and pressure gradients throughout the engine component correlating with the diagram (a) Ref. [4] Figure 1. (a) Schematic diagram of a turbine engine Ref. [3]. (b) Schematic diagram of the temperature and pressure gradients throughout the engine component correlating with the diagram (a) Ref. [4] structure) (Fig. 2). In some nickel – iron superalloys such as IN718 and IN706, which contain niobium, they are hardened by γ´´ (Ni3Nb based D022 structure) (Fig. 3) [2]. Homogeneously distributed coherent hardening precipitates confer excellent tensile and fatigue life properties at high temperatures. Their volume fraction is controlled by the nominal chemical composition. The size and the morphology are controlled by the process and their crystallographic relations with γ matrix. The precipitates arise close to the solvus temperature grow larger which subsequently restrict the grain growth pinning grain boundaries (Fig. 4). On the other hand, the precipitates arise at lower temperature such as during cooling after heat treatment stay small (Fig. 4 (left hand side of the image)). γ´ has the perfect coherency with the γ matrix, hence their morphologies are mostly sphere, whereas γ´´ has a tall crystal unit tetragonal structure where a axis has the identical lattice parameter with the γ matrix but c axis has nearly double the length of the γ, hence γ´´ always precipitate with the perfect coherency on the basal plane with γ and grow along the longitudinal direction (Fig. 5). structure) (Fig. 2). In some nickel – iron superalloys such as IN718 and IN706, which contain niobium, they are hardened by γ´´ (Ni3Nb based D022 structure) (Fig. 3) [2]. Homogeneously distributed coherent hardening precipitates confer excellent tensile and fatigue life properties at high temperatures. Their volume fraction is controlled by the nominal chemical composition. The size and the morphology are controlled by the process and their crystallographic relations with γ matrix. The precipitates arise close to the solvus temperature grow larger which subsequently restrict the grain growth pinning grain boundaries (Fig. 4). 2.2. Carbides and borides Carbon and boron are added as a grain boundary strengthener by segregating in the grain boundaries and forming carbides and borides. They are believed to be formed during solidification, aging treatment which strengthen grain boundaries at elevated temperatures Microstructure-Property Relationship in Advanced Ni-Based Superalloys 21 but the ones arising during service must be controlled carefully since they can impair properties [4]. p p [ ] Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple Carbides are traditionally classified by their chemical composition, mainly MC, M6C and M23C6, where M stands for metal elements such as Ti, Cr, Nb, Mo, Hf and Ta [4]. MC carbides are usually coarse (Fig. 6), having a fcc densely packed structure [4]. Ti, Nb, Hf and Ta are the main metal elements. They are very strong and are normally considered to be some of the most stable compounds in nature, justified by their high precipitation and Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple Carbides are traditionally classified by their chemical composition, mainly MC, M6C and M23C6, where M stands for metal elements such as Ti, Cr, Nb, Mo, Hf and Ta [4]. MC carbides are usually coarse (Fig. 6), having a fcc densely packed structure [4]. Ti, Nb, Hf and Ta are the main metal elements. They are very strong and are normally considered to be some of the most stable compounds in nature, justified by their high precipitation and melting temperature: they are believed to precipitate during processing shortly after solidification of the superalloy [4]. They usually have little or no orientation relationship with the alloy matrix [4]. 2.2. Carbides and borides M6C carbides have a complex cubic structure and they precipitate when the alloy contains highly refractory elements, for example Mo and W. These carbides are believed to be the product of MC carbide decomposition during service or relatively high heat treatment between 815 and 980ºC [4]. The examples of the micrographs of M6C can be found in Ref. [5, 6]. Metallurgy – Advances in Materials and Processes 22 Figure 4. TEM dark field (DF) image. γ΄ pinning grain boundary, shown by the white dashed line. The small spherically shaped precipitates inside the grain are also γ´. (g = 01 1 B = [111]) Figure 4. TEM dark field (DF) image. γ΄ pinning grain boundary, shown by the white dashed line. The small spherically shaped precipitates inside the grain are also γ´. (g = 01 1 B = [111]) Figure 5. TEM DF image of the γ΄΄ in IN718. The growth direction is c axis parallel to the a axis of γ ( (gγ´´ = 0 0 2) B = [100]). Figure 5. TEM DF image of the γ΄΄ in IN718. The growth direction is c axis parallel to the a axis of γ ( (gγ´´ = 0 0 2) B = [100]). Microstructure-Property Relationship in Advanced Ni-Based Superalloys 23 Figure 6. Coarse Nb and Ti based carbide in IN718 Figure 6. Coarse Nb and Ti based carbide in IN718 Figure 6. Coarse Nb and Ti based carbide in IN718 M23C6 carbides (Fig. 7) form mainly along grain boundaries at a relatively low temperature for carbides: between 760 and 980ºC. The crystal structure is complex cubic structure. The lattice parameter is exactly three times larger than γ matrix, hence they precipitate with cube- cube orientation with the matrix (Fig. 8). They are believed to form either by the decomposition of MC or M6C or they nucleate directly on the grain boundaries. They are known as having a high content of Cr. M23C6 carbides have a significant effect on Ni based superalloy properties [4] since they are profuse in alloys with moderate to high Cr content [4] Figure 7. Fine M23C6 type carbides precipitate along the grain boundary running diagonally. Figure 7. Fine M23C6 type carbides precipitate along the grain boundary running diagonally. Metallurgy – Advances in Materials and Processes 24 and are controversial carbides. 2.2. Carbides and borides Firstly, this is because their different morphologies: the blocky shaped ones at grain boundaries have a beneficial effect on rupture strength; on the contrary the film ones are regarded as promoting early rupture failure [4]. Secondly, this is because that they make a Cr depleted zone (Fig. 9) around the precipitate. In this area, it is difficult to form a protective oxide, namely Cr2O3, due to lack of Cr. Figure 8. M23C6 and γ matrix perfect coherent diffraction pattern (left) and the bright field image from another beam direction to make M23C6 outstanding (right) Figure 8. M23C6 and γ matrix perfect coherent diffraction pattern (left) and the bright field image from another beam direction to make M23C6 outstanding (right) Figure 9. Left: STEM EDX line scan results across M23C6 revealed the Cr depletion as indicated by the arrows (Cr nominal composition is 15 wt%). Right: STEM bright field image of the M23C6 (the thin arrow shows the length and the direction of the EDX line scan) Figure 9. Left: STEM EDX line scan results across M23C6 revealed the Cr depletion as indicated by the arrows (Cr nominal composition is 15 wt%). Right: STEM bright field image of the M23C6 (the thin arrow shows the length and the direction of the EDX line scan) 25 Microstructure-Property Relationship in Advanced Ni-Based Superalloys It is broadly acknowledged that boron segregation along the grain boundary increases the cohesive strength of the grain boundaries. The role of borides is, however, still under open discussion. Those so far identified have a base centred tetragonal (BCT), M3B2 [4] or M5B3 [7] formula, where M is typically a refractory element, namely Mo or Cr. They appear as various shapes such as blocky to half-moon [4]. The examples shown in Fig. 10 were found in an advanced polycrystalline Ni superalloy after a thermal exposure at 980°C for 1 hour. Figure 10. Some examples of M5B3 type boride appeared in TEM bright field (left) and in SEM (right) Figure 10. Some examples of M5B3 type boride appeared in TEM bright field (left) and in SEM (right) 2.3. Other phases Adding excess quantity of refractory elements, such as Mo, W and Re, promotes the precipitation of hard intermetallic phases [2], so called TCP phase, which are believed to deteriorate the alloy ductility [4] and the creep life [8]. In the ternary phase diagrams for superalloy elements, such as Ni-Cr-Mo, there are two phase spaces: one is austenite (γ) fcc and the other is bcc [4]. Between these two major fields, a band of numerous small phase volumes can be identified such as σ, μ, R and so on [4], which are characterized firstly as having a high and uniform packing density of atoms[2] and secondly as having complex crystal structures [2], either hcp, body centred tetragonal or rhombohedral. With the careful control of these refractory elements, TCP phases occur after a long time service or a prolonged heat treatment [9]. Some are believed to be the products of transformation from another beneficial phase: for example η(Ni3X) results from γ΄ [4] and σ has the same crystal structure as that of M23C6, but without the carbon atoms. The example of σ phase shown in Fig. 11 was found to be Cr, Mo and Co based chemistry after a thermal exposure at 720°C for 1,100 hours in a newly developed advanced Ni superalloy. The second phases introduced above and some other important second phases for the Ni superalloy microstructure are summarized in Table 1. Metallurgy – Advances in Materials and Processes 26 Figure 11. Sigma (σ) phase precipitates on the grain boundary running diagonally from top left to bottom right Figure 11. Sigma (σ) phase precipitates on the grain boundary running diagonally from top left to bottom right Figure 11. Table 1. Summary of second phases in the polycrystalline Ni based superalloys [10] The lattice parameter may vary (less than 5%) by changing chemical composition. rameter may vary (less than 5%) by changing chemical composition. 2.3. Other phases Sigma (σ) phase precipitates on the grain boundary running diagonally from top left to bottom right Phase Prototype Pearson symbol Strukturbericht symbol Lattice [nm] Chem Comp Phase Prototype Pearson symbol Strukturbericht symbol Lattice [nm] Chemical Composition (Appx) γ' Cu3Au cP4 L12 a 0.36 (Ni Co)3(Al Ti) γ'' Al3Ti tI8 D022 a 0.36 c 0.74 (Ni Fe)3(Nb Al Ti) MC NaCl cF8 B1 a 0.44 (Ti Ta)C or TiC, TaC, NbC, WC M6C Fe3W3C cF112 E93 a 1.11 (Mo Cr W)6C M7C3 Cr7C3 oP40 D101 a 0.45 b 0.70 c 1.21 Cr7C3 M23C6 Cr23C6 cF116 D84 a 1.07 Cr21Mo2C6 M5B3 Cr5B3 tI32 D8l a 0.55 c 1.06 (Cr Mo)5B3 M3B2 Si2U3 tP10 D5a a 0.60 c 0.32 (Mo Cr)3B2 σ CrNi tP30 D8b a 0.88 c 0.46 Cr Mo Co based δ Cu3Ti (β) oP8 D08 a 0.51 b 0.43 c 0.46 Ni3Nb η Ni3Ti hP16 D024 a 0.51 c 0.83 Ni3(Ti Ta) μ Fe7W6 hR13 D85 a 0.48 c 2.5 Mo Co based Table 1. Summary of second phases in the polycrystalline Ni based superalloys [10] The lattice parameter may vary (less than 5%) by changing chemical composition. Phase Prototype Pearson symbol Strukturbericht symbol L Table 1. Summary of second phases in the polycrystalline Ni based superalloys [10] The lattice parameter may vary (less than 5%) by changing chemical composition. Microstructure-Property Relationship in Advanced Ni-Based Superalloys 27 27 3. Microstructures and mechanical properties It is worth noting the microstructure related mechanical properties in detail. We will discuss briefly how microstructure affects various mechanical properties in polycrystalline Ni superalloys. Altering grain sizes results in various effects with regard to the different mechanical properties. Tensile and fatigue life properties are optimized by a fine grain microstructure, on the other hand, good creep and fatigue crack growth properties at elevated temperature are favoured by a coarse grain microstructure [2]. The former is a result of grain orientation and stress concentration by dislocation movement along the slip plane [2]. The latter is about intergranular crack propagation susceptibility. For example, Bain et al [11] showed the significance of the grain size for the crack growth rate using UDIMET720. Testing at 650°C, the crack growth rate reduced by more than two orders of magnitude by changing the size from 20 to 350 μm in diameter. (Fig. 12). Figure 12. UDIMET 720 fatigue crack growth rate for different grain sizes (ASTM grain size between 0 and 8.5: 360μm and 19μm in diameter) tested at 650ºC [11] 650ºC Figure 12. UDIMET 720 fatigue crack growth rate for different grain sizes (ASTM grain size between 0 and 8.5: 360μm and 19μm in diameter) tested at 650ºC [11] The size of the hardening precipitates significantly affects the yield strength of the material via the interaction between the precipitate and the dislocation. If the precipitates are large, dislocation bowing around the precipitates becomes dominant; for small sized precipitates, dislocation cutting becomes dominant. For bowing For bowing ߬ൌܩ∗ܾ ܮെ2ݎ (1) and for cutting ߬ൌݎ∗ߛ∗ߨ ܾ∗ܮ (2) ߬ൌܩ∗ܾ ܮെ2ݎ (1) (1) and for cutting and for cutting ߬ൌݎ∗ߛ∗ߨ ܾ∗ܮ (2) (2) τ is the strength of the material, G is the shear modulus, b is the magnitude of the Burgers vector, L is the distance between the hardening precipitates, r is the radius of the precipitates and γ is the surface energy. In general in Ni-Al binary system, the optimum size to τ is the strength of the material, G is the shear modulus, b is the magnitude of the Burgers vector, L is the distance between the hardening precipitates, r is the radius of the precipitates and γ is the surface energy. In general in Ni-Al binary system, the optimum size to Metallurgy – Advances in Materials and Processes 28 maximize the strength is found to be around 5 - 30 nm in diameter (Fig. 3. Microstructures and mechanical properties 13). The size of the precipitates also affects the creep strain as shown in Fig. 14. In their study [12], the size of the precipitate was changed by changing the heat treatment temperature and time and found that the smaller the precipitate the slower the creep strain rate is, which is achieved via the smaller γ´ - γ΄ channel width [12, 13]. Figure 13. γ´ particle diameter against the critical shear stress in Ni-Al system [28] Figure 13. γ´ particle diameter against the critical shear stress in Ni-Al system [28] Figure 14. Creep strain tested at 700ºC for different heat treatments (HT1, HT2 and HT3) The size of γ´: HT2>HT1>HT3 [12]. It is well known fact that in general both the yield strength and the creep rupture strength increases by increasing the hardening precipitate volume fraction [2] Historically low cycle Figure 13. γ´ particle diameter against the critical shear stress in Ni-Al system [28] Figure 14. Creep strain tested at 700ºC for different heat treatments (HT1, HT2 and HT3) The size of γ´: HT2>HT1>HT3 [12]. Figure 14. Creep strain tested at 700ºC for different heat treatments (HT1, HT2 and HT3) The size of γ´: HT2>HT1>HT3 [12]. It is well known fact that in general both the yield strength and the creep rupture strength increases by increasing the hardening precipitate volume fraction [2]. Historically, low cycle fatigue life was the main concern for turbine disc alloys, but fatigue crack growth rate and damage tolerant design have attracted more attention over the last two decades [11, 14]. 29 Microstructure-Property Relationship in Advanced Ni-Based Superalloys They can be strongly influenced not only by the size of the grains as introduced above, but also by the size of the precipitates; the striking results were shown in Ref. [15, 16]. The results show that the larger the hardening precipitates the better the crack growth property. However, this conflicts with the creep life property as mentioned above. Research on damage tolerant design originally started to investigate the grain boundary chemistry since fast crack growth (FCG) is always observed with intergranular cracks and tends to disappear at low temperature. Additionally, transgranular ductile cracking replaces intergranular crack when the tests carried out in the reduced oxygen partial pressure [17, 18] (Fig. 15) Thus, FCG embrittlement has been attributed to oxidation [11, 19]. Grain boundary engineering has been explored by changing the morphology of the grain boundary. 3. Microstructures and mechanical properties Optical microscopy image of serrated grain boundaries. The arrows indicate the serrated boundaries [15] 3. Microstructures and mechanical properties For example, Ref. [15, 20] reported a complex grain boundary geometry, so called ‘serrated’ (Fig. 16), by slow cooling after solution treatment. The result showed slower intergranular crack growth rate than with a normal grain boundary [15]. However, the improvement above did not account for the property change by the different size of the hardening precipitate mentioned above. The fast intergranular crack growth at high temperature in superalloys added a new dimension after intensive studies with regard to the correlation between the hardening precipitate distribution and the crack growth rate. Ref. [15, 16, 21] claimed that the prevention of stress relaxation of the crack tip by the hardening precipitates can increase the crack growth rate. Some experimental work support the idea, for example Andieu et al [22] carried out a unique dwell fatigue crack propagation test where oxygen was introduced in different phases of the low cycle fatigue crack growth test and found that it is potent for the fast crack growth when oxygen is introduced at the beginning of the loading rather than introducing in the later part of the loading. This may imply that the oxidation at the crack tip happens during the stress concentrated at the crack tip. Molins et al [23, 24] concluded that the local microstructure at the crack tip, which can be controlled by an appropriate heat treatment against the stress accumulation, can significantly affect the crack propagation behaviour in Ni superalloys. This conclusion recalls an arguable grain boundary microstructure feature, namely the precipitate free zone (PFZ). One suggested that the PFZ would promote plastic deformation and fracture [25, 26]. Another suggested that the PFZ in some nickel alloys is beneficial for crack tip stress relaxation [27]. Figure 15. Typical intergranular (left) and transgranular (right) fracture surfaces. Alloy 718 tested at 650ºC in air (left) and vacuum (right) [18] Figure 15. Typical intergranular (left) and transgranular (right) fracture surfaces. Alloy 718 tested at 650ºC in air (left) and vacuum (right) [18] Metallurgy – Advances in Materials and Processes 30 These findings above suggest that not only the macroscopic structure such as the grain size and the distribution of the hardening precipitates, but also the microscopic structure, such as the grain boundary shape and the relationship with the hardening precipitates, can significantly affect the mechanical properties. Figure 16. Optical microscopy image of serrated grain boundaries. The arrows indicate the serrated boundaries [15] Figure 16. 4. Polycrystalline superalloy grain boundary structure The details of the Ni superalloy grain boundary microstructure will be demonstrated in this section. Particular attention will be paid to the relationship between the hardening precipitates and the high grain boundaries. Fig. 17 shows the STEM bright field image of the grain boundary and the hardening precipitate morphology in an advanced polycrystalline superalloy. The grain boundary running top left to bottom right cuts through γ´. This was confirmed by the conventional TEM image analysis combining with the crystallographic analysis that the either side of the γ´ keeps the coherency with the matrix (Fig. 18). With respect to the morphologies of γ´ on the grain boundaries, it is the same as those inside the grains. It has, however, two different crystallographic orientations keeping the coherency with the either side of the matrix. This morphology is believed to form during the process with the high boundary mobility [29]. There are at least four different possibilities of interactions between the migrating grain boundaries and the precipitates, which are illustrated in Fig. 19. Following Fig. 19, a. the boundary migrates with no effect on the precipitates; the precipitates thus become incoherent after the migrating grain boundary passes through them. a. the boundary migrates with no effect on the precipitates; the precipitates thus become incoherent after the migrating grain boundary passes through them. Microstructure-Property Relationship in Advanced Ni-Based Superalloys 31 31 b. the precipitates dissolve in contact with migrating boundary and reprecipitate coherently within the new grain. b. the precipitates dissolve in contact with migrating boundary and reprecipitate coherently within the new grain. c. the grain boundary is held by the coherent precipitates which then coarsen, leading to complete halting of the boundary movement. c. the grain boundary is held by the coherent precipitates which then coarsen, leading to complete halting of the boundary movement. d. the grain boundary can pass through the coherent precipitate which undergoes the same orientation change as the grain surrounding it and thereby retains the coherent low-energy interface between the precipitate and the matrix. d. the grain boundary can pass through the coherent precipitate which undergoes the same orientation change as the grain surrounding it and thereby retains the coherent low-energy interface between the precipitate and the matrix. Figure 17. General aspect of the high angle grain boundary and γ´ (dark spheres). The grain boundary is running diagonally from top left to bottom right 100nm Figure 17. 4. Polycrystalline superalloy grain boundary structure General aspect of the high angle grain boundary and γ´ (dark spheres). The grain boundary is running diagonally from top left to bottom right Figure 18. A crystallographic analysis of the cutting γ´. The dark field images of the lower and the upper grain are in (I) and (II), respectively. The white arrows indicate the grain boundary Figure 18. A crystallographic analysis of the cutting γ´. The dark field images of the lower and the upper grain are in (I) and (II), respectively. The white arrows indicate the grain boundary Metallurgy – Advances in Materials and Processes 32 With regard to theory a), this is often observed with high solvus temperature precipitates, such as carbides and oxides. b) is not applicable in this study. This can be, however, the case for less γ΄ volume fraction superalloys with small amount of nominal Al content such as Nimonic PE16 or the case in high temperature very close to the γ´ solvus. With regard to c), it can be applicable in the case of the larger γ´ such as the one in Fig. 4. d) is relevant to explain the results of Fig. 17 and Fig. 18. As the grain boundary impinges on the γ΄, the grain boundary apparently cuts off γ΄. Firstly, the interface free energy between γ´ and the grain boundary increases. This results in dissolving the γ´ at the interface but due to the supersaturation of γ´ formers, such as Al and Ti, γ´ immediately re - nucleates in the next grain coherent with the next grain discontinuously [30, 31]. Thus, this phenomenon can be concluded the result of γ΄ dissolution and subsequent (discontinuous) precipitation. Figure 19. The possible interactions between the migrating grain boundary and the precipitate [29] gure 19. The possible interactions between the migrating grain boundary and the precipitate [29] Another example is forming a precipitate free zone (PFZ) as shown in Fig. 20 in IN718. γ´ and γ´´ coexist in IN718, however, γ´´ denude along the grain boundary and form a γ´´ PFZ. On the contrary, the minor hardening precipitate in IN718: γ´ exist along the grain boundary. Vacancy deficiency is one of the causes of the PFZ along grain boundaries due to lack of the nucleation sites as grain boundary acts as a good vacancy sink [32]. 4. Polycrystalline superalloy grain boundary structure One of the important factors to create the PFZ in the superalloys can be the difference of the interfacial free energy, i.e. the free energy between γ´-matrix and γ´´-matrix. The γ´´-γ´´ nucleus channel distance along the grain boundary can be larger than the critical distance to aggregate two γ´´ nuclei. On the other hand, the critical distance for the γ´ is smaller than that of γ´´ or γ´ can nucleate their precipitate independently as γ´ has smaller interfacial energy. Thus, the γ´ nuclei can grow and form precipitates along the grain boundary but not for γ´´ and the γ´´ PFZ arises. Microstructure-Property Relationship in Advanced Ni-Based Superalloys 33 Figure 20. γ´´ PFZ appeared in IN718, although γ´ still exist in the γ´´PFZ adjacent to the grain boundary Figure 20. γ´´ PFZ appeared in IN718, although γ´ still exist in the γ´´PFZ adjacent to the grain b Figure 20. γ´´ PFZ appeared in IN718, although γ´ still exist in the γ´´PFZ adjacent to the grain boundary 5. High temperature oxidation along grain boundaries The context of ‘High temperature’ in this section is the temperature range of 600-700°C which is the high temperature regime of the disc in the turbine engine application. It is more than a half century ago, the investigation of the oxidation assisted fast crack growth started. Cr is believed to be an important element for the oxidation assisted crack growth. For example, as shown in Fig. 21, crack growth tests were conducted under various oxygen partial pressure on Ni-Cr binary alloys with 5, 20 and 30 wt% of Cr. It was found that the higher the Cr content the higher the transition oxygen partial pressure from transgranular to intergranular cracking is. The highest Cr content alloy: Ni-30wt%Cr did not show a transition pressure. Oxidation process on a freshly exposed alloy surface had been characterised intensively and well understood. As illustrated in Fig. 22, both Ni and Cr oxide formation takes place at the beginning of the oxidation [33]. This is particularly important for alloys on the borderline between protective and non-protective behaviour [34]. But in the early stage, the fast kinetics Ni oxide grows quickly and dominates the oxide. In general, there are two types of oxidation: the cation diffusion type and the anion diffusion type [34] . The difference between the two is the movement of the ions; the former involves cation (metal ion) transport, the latter anion (oxygen ion) transport. For the cation diffusion type the oxides form between the oxide and the free surface, but, for the anion diffusion, the oxides form between the oxide and the metal Metallurgy – Advances in Materials and Processes 34 interface. During the transient stage which corresponds to the middle of the illustrations in Fig. 22, Cr2O3 particles are embedded inside the NiO layer. As NiO grows and the oxidation rate becomes slower, Cr2O3 soon establishes its own layer underneath the NiO layer, which implies that Cr2O3 can be the anion diffusion. Eventually the Cr2O3 layer is completed, where the layer prevents further diffusion of oxygen into the alloy, called passivation [34]. Figure 21. Transition oxygen partial pressure from the transgranular to intergranular cracking against the Cr concentration in Ni alloys after crack propagation tests at 650°C [24] Figure 21. Transition oxygen partial pressure from the transgranular to intergranular cracking against the Cr concentration in Ni alloys after crack propagation tests at 650°C [24] Figure 22. 5. High temperature oxidation along grain boundaries Schematic diagram of the oxidation process (from top to bottom) of the freshly exposed Ni alloy [33] e 22. Schematic diagram of the oxidation process (from top to bottom) of the freshly exposed Ni [33] Figure 22. Schematic diagram of the oxidation process (from top to bottom) of the freshly exposed Ni alloy [33] 35 Microstructure-Property Relationship in Advanced Ni-Based Superalloys 35 Nevertheless, the oxidation process at (ahead) of the crack tip has remained under debate. This is due to the technical difficulties of studying the microstructure of such small regions, which is predictable taking consideration of the size of the crack tip: less than a micron and even smaller for the oxides adjacent. To overcome the problem, so called ‘site specific specimen preparation’ has been developed since the late 90’s using focused ion beam (FIB) technique [35, 36]. The use of gallium ions in a focused ion beam accelerated in a FIB apparatus to energies up to 30 keV enables us to mill specimens selectively to reveal structural features and to deposit films at selected locations. An example carried out in the University of Birmingham UK (2009) is shown in Fig. 23. The specimen is an advanced Ni based superalloy after an interrupted crack propagation test at 650°C in air. A plan-view crack tip TEM specimen was prepared [37]. Using the two different modes: the tungsten deposition to protect the region of interest (Fig. 23 a)) and the milling (trenching) (Fig. 23 b)), the crack tip was transported to a TEM copper grid (c) and d)). The size of the focused Ga ion beam can be achieved as small as a few nanometres in radius. It is possible to prepare the site specific TEM specimen foil as thin as 50 nm in thickness. Fig. 24 shows TEM bright field images; they are from the same material: polycrystalline advanced superalloy, but they are after different testing conditions. Fig. 24 (a) is from a specimen after interrupting a crack propagation test at 650°C; the intergranular crack propagation was identified. Fig. 24 (b) is from a specimen after interrupting the same crack propagation test mentioned above except for the crack growth rate: 0 μm/s (~0.9 Kth: just below the crack growth threshold) held for 5 hours. The oxides ahead of the crack tip are also along grain boundaries. 5. High temperature oxidation along grain boundaries The dashed line boxes indicate the area analysed by EDX shown later in this section. Comparing the two bright field images in Fig. 24, it is apparent that the oxides penetration in the metal ahead of the crack tip is approximately 5 times longer for the static crack specimen (b). The EDX mapping and the EDX line scan of the oxides close to the crack tip for the moving crack specimen from the region I in Fig. 24 are shown in Fig. 25. It is apparent that the grain boundary is completely filled with oxides (oxygen map). There is a Co and Ni rich oxide in the middle. There are Cr rich oxide areas on both sides of the oxide. Cr thus forms a thin layer between the Ni (Co) oxides and the alloy. EDX line scans across the oxide revealed that Ti, Al are also segregating in this region. Crystallographic analysis using selected aperture diffraction confirmed that the middle oxide is cubic (Nix Co1-x)O and the rim oxide is hcp (Cr Al Ti)2O3. The higher oxygen partial pressure region in the middle of the oxide is consisted by the Ni and Co oxide. The rim of the oxide between the Ni (Co) oxide and the matrix are consisted by the passive Cr, Al and Ti layer. This is correlating with the freshly exposed Ni alloy oxidation process described above. Fig. 26 shows the oxide chemistry at the tip of the oxide corresponding to the region II in Fig. 24. According to the EDX mapping, the oxides formation manner looks similar to that of the region I; Ni(Co) oxide in the middle and the Cr, Al and Ti oxide in the rim. It is questionable to argue the stoichiometry of the oxide from the results of the EDX cross section line scan due to the x-ray emission from the matrix, however it revealed from the line scan in Fig. 26 that the oxide is Cr based; Ni, Co, Al and Ti deplete in the very tip of the oxide. Fig. 27 shows the chemistry of the oxides in the middle of the oxide ahead of the crack tip for the static crack corresponding to the region x in Fig. 24. EDX mapping revealed that the Ni and Co are depleted even in the middle of the oxide and the Metallurgy – Advances in Materials and Processes 36 Figure 23. 5. High temperature oxidation along grain boundaries SEM secondary electron images of the process of the site specific TEM sample preparation e 23. SEM secondary electron images of the process of the site specific TEM sample preparation Microstructure-Property Relationship in Advanced Ni-Based Superalloys 37 Figure 24. TEM bright field images of the two specimens. The crack tips locate just next to the specimen and the crack propagate from the left to right. Figure 24. TEM bright field images of the two specimens. The crack tips locate just next to the specimen and the crack propagate from the left to right. Figure 25. STEM dark field image from the region I in Fig. 24 (top left); the thick white arrow indicates the crack growth direction and the thin black arrow indicates the area and the direction of the EDX line scan. The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 25. STEM dark field image from the region I in Fig. 24 (top left); the thick white arrow indicates the crack growth direction and the thin black arrow indicates the area and the direction of the EDX line scan. The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Metallurgy – Advances in Materials and Processes 38 Figure 26. STEM dark field image from the region II in Fig. 24 (top left). The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 26. STEM dark field image from the region II in Fig. 24 (top left). The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 27. STEM dark field image from the region x in Fig. 24 (top left). The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 27. STEM dark field image from the region x in Fig. 24 (top left). The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Microstructure-Property Relationship in Advanced Ni-Based Superalloys 39 line scan revealed that the oxide is consisted mainly by Cr, Al. Fig. 28 shows the tip of the oxide for the static crack corresponding to the region z in Fig. 24. 6. Summary One of the challenges of the advanced Ni based superalloys is in the damage tolerance properties without reducing their superior strength at high temperature. The microstructure, particularly the grain boundaries, was found to be controlled by the two factors in this study. Firstly, it is the nominal chemical composition, especially the hardening precipitate participants. Secondly, heat treatment has a profound influence of microstructure. The damage tolerance properties are also concerned with the environmentally assisted crack propagation along grain boundaries, which is essentially the oxidation assisted crack propagation in this study. In general, chromium oxide (Cr2O3) has been regarded as a healing agent of the oxidation process in Ni alloys. The state-of-the-art technique enabled us to observe the crack tip oxidation. In this study, it was successfully presented that the oxidation sequence is following the free energies for the oxides to form. Thus, for example, Cr2O3 is one of the earliest oxides to form at the crack tip. Understanding the environmentally assisted crack propagation is one of the crucial fields of research to increase the turbine entry temperature (TET), which is indeed one of the most significant attainments in the propulsion industries. Author details Hiroto Kitaguchi Department of Materials, University of Oxford, OX1 3PH, Oxford, UK 5. High temperature oxidation along grain boundaries Cr still exists in the oxide in the middle, but particularly at the very tip of the oxide approximately 100 nm or so, Cr is depleted and only Al and Ti enriched at the tip of the oxide (see also the cross section line scan). It is also difficult in this case due to the thickness effect to discuss the stoichiometry of the oxide, however, in this region, Al and Ti based oxide formation takes place. Figure 28. STEM dark field image from the region z in Fig. 24 (top left). The EDX line scan across the oxide(top right) and the EDX mapping results of the oxide (bottom). Table 2 shows the oxygen dissociation pressure to form the oxide and the free energy for the elements forming oxides introduced above. It is clear that the formation of the oxide at the Figure 28. STEM dark field image from the region z in Fig. 24 (top left). The EDX line scan across the oxide(top right) and the EDX mapping results of the oxide (bottom). Table 2 shows the oxygen dissociation pressure to form the oxide and the free energy for the elements forming oxides introduced above. It is clear that the formation of the oxide at the crack tip follows the thermodynamics suggesting the oxygen partial pressure gradient along the oxide tip ahead of the crack tip. Metallurgy – Advances in Materials and Processes 40 Oxide formation Free energy Oxygen dissociation pressure in log10 [kJ/mole] [bar] Al Al2O3 -1425 -48 Ti Ti2O3 -1295 -44 TiO2 -798 -40 Cr Cr2O3 -877 -31 Co CoO -166 -18 Ni NiO -149 -16 Table 2. Oxide formation free energy and the dissociation pressure at 727°C for the elements introduced in this section [38] Table 2. Oxide formation free energy and the dissociation pressure at 727°C for the elements introduced in this section [38] Author details Hiroto Kitaguchi Department of Materials, University of Oxford, OX1 3PH, Oxford, UK Acknowledgement The author would thank to Profs. I.P. Jones and P. Bowen at The University of Birmingham and Dr. M.C. Hardy at Rolls-Royce plc for their advice and the guidance. The joint support of the UK Engineering and Physical Sciences Research Council (EPSRC) and Rolls-Royce plc is also gratefully acknowledged. Many thanks are also due to Dr H.Y. Li, Research Fellow at the School of Metallurgy and Materials, The University of Birmingham and Dr Y.Y. 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Andrieu, Acta Materialia, 45 (1997) 663- 674. Metallurgy – Advances in Materials and Processes 42 [25] R.G. Baker, J. Nutting, Iron and Steel, 32 (1959) 606-612. [25] R.G. Baker, J. Nutting, Iron and Steel, 32 (1959) 606-612. [26] G. Thomas, J. Nutting, Institute of Metals -- Journal, 86 (1957) 7-14. [27] The Journal of the institute of metals; Discussion, 91 (1963-64) 153. [28] H. Gleiter, E. Hornbogen, Materials science and engineering, 2 (1968) 285-302. [29] E. Grant, A. Porter, B. Ralph, Journal of Materials Science, 19 (1984) 3554-3573. [30] A. Porter, B. Ralph, Journal of Materials Science, 16 (1981) 707-713. [31] R.D. Doherty, Metal Science, 16 (1982) 1-13. [32] G.W. Lorimer, R.B. Hicholson, Acta Metallurgica, 14 (1966) 1636. [33] G.C. Wood, F.H. Stott, Materials Science and Technology, 3 (1986) 519-530. [34] L.L. Shreir and R.A. Jarman and G.T. Burstein, Corrosion, Butterworth-Heinemann Ltd, Oxford, 1963. [35] S. Lozano-Perez, Y. Huang, R. Langford, J.M. Titchmarsh, Electron Microscopy and Analysis 2001. Proceedings, 5-7 Sept. 2001, IOP Publishing, Bristol, UK, 2001, pp. 191- 194. [36] D.M. Longo, J.M. Howe, W.C. Johnson, Ultramicroscopy, 80 (1999) 69-84. [37] F.A. Stevie, R.B. Irwin, T.L. Shofner, S.R. Brown, J.L. Drown, L.A. Giannuzzi, Characterization and Metrology for ULSI Technology. 1998 International Conference, 23-27 March 1998, AIP, USA, 1998, pp. 868-872. [38] G.V. Samsonov, The oxide handbook, Plenim Publishing Company, Ltd, London, 1973, pp. 23.
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Dynamic and transient processes in warm dense matter
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Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Queen s University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal y Link to publication record in Queen's University Publisher rights Copyright 2023 the authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. g Copyright 2023 the authors. py g This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Dynamic and transient processes in warm dense matter White, T. G., Dai, J., & Riley, D. (2023). Dynamic and transient processes in warm dense matter. Philosophical Transactions of the Royal Society A, 381(2253), Article 20220223. https://doi.org/10.1098/rsta.2022.0223 General rights g Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Published in: Philosophical Transactions of the Royal Society A Document Version: Publisher's PDF, also known as Version of record Document Version: Publisher's PDF, also known as Version of record Dynamic and transient processes in warm dense matter royalsocietypublishing.org/journal/rsta royalsocietypublishing.org/journal/rsta Open Access Thi h Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love t this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 royalsocietypublishing.org/journal/rsta Introduction Cite this article: White TG, Dai J, Riley D. 2023 Dynamic and transient processes in warm dense matter. Phil.Trans.R.Soc.A 381: 20220223. https://doi.org/10.1098/rsta.2022.0223 Received: 5 May 2023 Accepted: 22 May 2023 One contribution of 11 to a theme issue ‘Dynamic and transient processes in warm dense matter’. Subject Areas: astrophysics, high-pressure physics, plasma physics Keywords: warm dense matter, review, plasmas, experimental techniques, simulation Author for correspondence: David Riley e-mail: d.riley@qub.ac.uk Subject Areas: This article is part of the theme issue ‘Dynamic and transient processes in warm dense matter’. astrophysics, high-pressure physics, plasma physics Introduction 1Department of Physics, University of Nevada, Reno, NV 89557, USA 2College of Science, National University of Defense Technology, Changsha 410073, People’s Republic of China 3School of Mathematics and Physics, Queen’s University Belfast, Belfast BT7 1NN, UK 1Department of Physics, University of Nevada, Reno, NV 89557, USA 2College of Science, National University of Defense Technology, Changsha 410073, People’s Republic of China 3School of Mathematics and Physics, Queen’s University Belfast, Belfast BT7 1NN, UK Cite this article: White TG, Dai J, Riley D. 2023 Dynamic and transient processes in warm dense matter. Phil.Trans.R.Soc.A 381: 20220223. TGW, 0000-0002-3865-4240; DR, 0000-0002-6212-3212 https://doi.org/10.1098/rsta.2022.0223 In this paper, we discuss some of the key challenges in the study of time-dependent processes and non- equilibrium behaviour in warm dense matter. We outline some of the basic physics concepts that have underpinned the definition of warm dense matter as a subject area in its own right and then cover, in a selective, non-comprehensive manner, some of the current challenges, pointing along the way to topics covered by the papers presented in this volume. One contribution of 11 to a theme issue ‘Dynamic and transient processes in warm dense matter’. 1. Introduction warm dense matter, review, plasmas, experimental techniques, simulation The term warm dense matter (WDM) was coined over 20 years ago, e.g. [1–3] to describe matter that is at high pressures, generally over 100 GPa, but at the same time, at high temperatures of order 104–106 K. This range of conditions leads to partially degenerate and partially ionized matter, with strong correlations between charged particles and both populated excited and bound states playing a role in the microscopic structure and particle dynamics [4]. It is a transitional state between solid and plasma and exhibits properties of both. Its inherent complexities make perturbative techniques unreliable, leading to significant differences in theoretical and computational predictions of important quantities [5]. Moreover, due to the difficulty of creating and 2. Background Much of the physics relevant to WDM was studied in the fields of dense plasma physics [7,8] and liquid metal physics [9] long before the term was coined. The remit of these two fields includes a wider range of conditions than is usually included within the definition of WDM but they do share some issues, such as the effect of degeneracy and particle correlation on basic properties such as thermal and electrical conductivity. A principal and oft-cited motivation for the study of WDM is that the relevant conditions are applicable to many planetary interiors, especially for large planets such as Jupiter and Saturn. For the former, the expected conditions at the core, depending on the equation of states used, include a pressure of order 4 TPa and temperature approximately 104 K. A key point emerging from work such as that by Nettelmann and colleagues [10] is that, by using different equation of state models for the hydrogen and helium within the planetary core, various models of the interior layers and conditions can be constructed that still agree with observations of surface temperature and gravitational moments observed by missions such as Voyager and Cassini, e.g. [11–13]. The significantly improved gravity data from the Juno mission (see [14–16] and references therein) means that more detailed models of the interior of Jupiter can be considered and compared. For example, the more accurate measurements of the J4 and J6 moments have suggested a more diluted core with heavy and light element mixing. This means that more exact information on the equation of state for hydrogen and helium has more impact than ever and efforts to improve measurements in the laboratory are more important than ever. These considerations highlight the necessity of laboratory experiments to determine equations of states for the WDM regime. The discovery over recent decades of thousands of exoplanets [17,18] only gives increased motivation in this respect. Interest is not limited to planetary interiors. For any matter taken on a path from a cold solid to hot plasma, such as components of an inertial confinement fusion (ICF) capsule, the sample must pass through the WDM regime, and thus the properties of WDM may affect the trajectory. The design of ICF implosions depends on the thermal conductivity of both the ablator materials and the fuel mixture [19–21]. 2 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . diagnosing WDM in the laboratory [6], there is a shortage of experimental results with which to benchmark predictions. diagnosing WDM in the laboratory [6], there is a shortage of experimental results with which to benchmark predictions. 2 Author for correspondence: David Riley 2023 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/ by/4.0/, which permits unrestricted use, provided the original author and source are credited. 2. Background [27,28] can only probe a peripheral part of the WDM regime. (Adapted from [29].) y yp g g j . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 1000 DAC Fe (Dubrovinsky) Ramp compression Fe (Wang) Uranus core 'Super' Earth explosive compression Saturn core Jupiter Brown Dwarf Fe Hugoniot Earth core DAC Fe (Yoo) Pressure (GPa) 0.1 1 temperature (eV) 10 10 000 Γ = 50 for Fe H Hugoniot EF/KT = 1 Fe 3 temperature (eV) 1 Figure1. Mappingofthewarmdensematterregion.Theboundariesareabroadguide.Wecanseethewayinwhichconditions in Jupiter as a function of depth (dashed blue line) pass through the WDM regime, as do conditions in a Brown Dwarf (dashed orange line). We note that static methods of compression and heating, such as diamond anvil cells (DAC), e.g. [27,28] can only probe a peripheral part of the WDM regime. (Adapted from [29].) can see that the regime extends across a wide range of pressures and temperatures from a few eV to several 10s of eV. The significance of planetary interiors as a reason to study WDM means that the conditions we are motivated to study are often in a steady or quasi-steady state with a very long timescale of evolution. For static methods of producing WDM states, Anzellini & Boccato [30] have recently reviewed the accessible range of DACs for both laser and resistive heating. Temperatures of up to 6000 K are reachable for pressures in the region of more than 100 GPa. Tateno et al. [31] reported measurements of iron up to 5700 K and 377 GPa, while Dubrovinsky et al. [32] report pressures above 750 GPa with a double-stage DAC. Although these extreme conditions are relevant, for example, to the structure of the Earth’s core, as we can see from figure 1, the temperatures reached are at the lower border of the WDM regime and the interiors of larger planets may be out of reach for DAC and other static methods. 2. Background At the same time, the efficiency of the process can be reduced by fuel degradation through mechanisms like diffusion [22–24]. The Rayleigh–Taylor instability, which can affect the interface between the ablator and fuel within the fusion capsule, is particularly sensitive to the diffusion coefficient [25]. The recent successes at the National Ignition Facility, e.g. [26] can only serve to further increase interest in WDM research within the ICF community. In figure 1, we see the WDM regime mapped out in pressure–temperature space. We can see how the shock Hugoniots for two elements of astrophysical interest, hydrogen and iron, pass through the WDM regime. The label, Γ , against the grey dashed curve refers to the ion–ion coupling parameter, which measures the ratio of Coulombic potential energy to thermal energy: Γ = Z2e2 akBT (2.1) (2.1) where Z is the average charge on the ions, and a is a characteristic distance between ions (Wigner– Seitz radius) given by a =  3 4πni 1/3 (2.2) (2.2) where ni is the average density of the ions. For strong shocks, we typically see values of Γ ≲50, and the thermal energy stops being a small perturbation to the Coulomb interaction. The yellow line denotes where the Fermi energy (EF) for Fe is equal to the temperature, in eV, indicated in the figure. In this regime, we cannot treat the free electrons in the sample as fully degenerate and we where ni is the average density of the ions. For strong shocks, we typically see values of Γ ≲50, and the thermal energy stops being a small perturbation to the Coulomb interaction. The yellow line denotes where the Fermi energy (EF) for Fe is equal to the temperature, in eV, indicated in the figure. In this regime, we cannot treat the free electrons in the sample as fully degenerate and we 100 1000 DAC Fe (Dubrovinsky) Ramp compression Fe (Wang) Uranus core 'Super' Earth explosive compression Saturn core Jupiter Brown Dwarf Fe Hugoniot Earth core DAC Fe (Yoo) Pressure (GPa) 0.1 1 temperature (eV) 10 10 000 Γ = 50 for Fe H Hugoniot EF/KT = 1 Fe Figure1. Mappingofthewarmdensematterregion.Theboundariesareabroadguide.Wecanseethewayinwhichconditions in Jupiter as a function of depth (dashed blue line) pass through the WDM regime, as do conditions in a Brown Dwarf (dashed orange line). We note that static methods of compression and heating, such as diamond anvil cells (DAC), e.g. 2. Background This presents a challenge for experimental study, and it is for this reason we often look to dynamic methods of production, such as shocks and other rapid heating mechanisms, as discussed in the next section. 3. Timescale considerations for dynamic production of warm dense matter However, answering this question is not a simple matter, as samples expand upon heating, and the hydrodynamic timescale is dependent on both the pressure and sample size. For example, if we take a sound speed given by cs ≈  P ρ , (3.1) (3.1) for a sample at pressure 100 GPa and density of 103 kgm−3, we can see that the sound speed is of order 104 m s−1. For a millimetre-sized sample, the estimated timescale for decompression is t ∼d/cs ∼100 ns, where d is the size of the sample. In this sense, we can say that experiments carried out with nanosecond creation and probing can be applicable to steady-state conditions. p g pp y However, it is important to note that hydrodynamic expansion timescales are not the only timescale of interest. Not all species in a sample (e.g. ions and electrons) are initially heated in the same way when creating WDM. For example, shock compression heats ions, e.g. [43,44], which transfer energy to electrons through collisions. By contrast, X-ray heating, e.g. [45] transfers energy to electrons via photo-ionization and these transfer energy to ions via collisional processes and electron–phonon coupling. The rate at which energy is transferred between ions and electrons, and indeed the rate at which processes, such as melting, occur, is an important factor to consider when studying the formation of WDM and forms an important topic in this collection addressed by several papers. Not only will this rate determine if we have conditions relevant to equilibrium situations, but macroscopic quantities such as electrical and thermal conduction depend on this rate. These latter quantities are of vital importance in planetary interiors. Interest in the issue of electron–ion equilibration is further motivated by the fact that short- pulse laser systems have also been used to extend WDM experiments to the other end of the temporal scale, for example, Lecherbourg et al. [46] will present results on the evolution of solid copper into a warm dense matter state, in this case, after excitation with a femtosecond laser pulse. More recently, and of particular significance to the work in this issue, the advent of X-ray free-electron lasers has made it possible to achieve volumetric heating on sub-picosecond timescales [45,47]. Using this property, Ren et al. 3. Timescale considerations for dynamic production of warm dense matter As noted in the previous section, when attempting to access conditions relevant to either planetary interiors or inertial fusion efforts, the appropriate conditions can be well outside the range accessible to static methods. Therefore, to reach conditions within the range of interest, we must turn to dynamic heating and compression methods, which can take several forms. For example, strong shocks (more than 100 GPa) can generate matter at several times solid density and temperatures well in excess of 104 K [33,34]. Shocks strong enough to heat a solid beyond this temperature can be generated in a variety of ways; for example, intense lasers of nanosecond duration have commonly been used to generate pressures ranging from 0.1 to 1 TPa [34]. Intense lasers [35,36] or Z-pinch machines [37] can also be used to generate in the range of 100s kJ of X-ray radiation in the sub-keV to keV photon range that can, on timescales of several nanoseconds, volumetrically heat a sample into the WDM region. In a similar vein, intense short-pulse lasers can be used to generate high-intensity electron [38] or proton beams [39] to heat solid targets volumetrically on sub-nanosecond timescales. There are also longer timescale experiments; for example, Gardelle et al. used beams of intense electrons from pulsed power generators to generate WDM on 100 ns timescales [40]. Finally, the longest-lived experiments utilize flyer plates driven by explosives in gas-gun experiments that create shocks on microsecond timescales that can reach pressures in excess of 600 GPa [41]. Z-pinches can, in addition to being used to drive X-rays, also be used to accelerate flyer plates to over 40 km s−1 using the enormous JXB forces possible, see [42] for a review of Z-pinch HED science. 4 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Considering the timescale of experiments raises concerns regarding the validity of extrapolating laboratory experiments to conditions of interest, such as those encountered in a planetary core. 4. Experimental approaches It is essential to validate our theoretical knowledge of warm dense matter through experiments, and significant effort has been invested in producing benchmark-quality experimental results. A significant portion of the experimental effort has been dedicated to measuring transport coefficients, including attempts to measure electrical conductivity via the reflectivity of incident electromagnetic radiation from a solid target [50–53]. An early example was the use of a nanosecond optical probe as a diagnostic looking at the time resolved reflectivity history of the rear surface of a shock-compressed foil, e.g. [54] with approximately 10 ps resolution. With the development of chirped pulse amplification (CPA) laser technology in the late 1980s, Strickland & Mourou [55] allowed for much shorter, sub-picosecond pulses. This made it possible to conduct experiments where the reflectivity of the target is measured before any substantial decompression from the well-defined solid density is possible. This can be done for a target heated by the probe itself, e.g. [56] or with a pump-probe arrangement in a relatively simple reflectivity measurement, e.g. [57]. For a more complex arrangement, such as Fourier domain interferometry (FDI), e.g. in [58–60], the motion of the heated surface can be followed and factored into calculations and experimental conclusions. An important issue closely related to conductivity measurements is the experimental investigation into electron–ion equilibration in the WDM regime. However, hindered by the opacity of WDM, there are limited experimental measurements of the electron–ion equilibration rate in this regime, with only a few model-dependent techniques in use. Historically, the response of the electron subsystem was measured in pump–probe experiments that used optical [57] reflectivity, surface optical emission [44,61] or X-ray absorption spectroscopy [62,63]. Likewise, the bulk ion temperature has only been inferred from the atomic structure, measured through ultrafast electron [64] and X-ray diffraction [38,39,65,66]. For the case of X-ray diagnostics of WDM, the opacity is an important quantity, and Hansen [67] presents a method of calculating non-equilibrium opacity using a DFT-based average atom approach that can be applied across a wide range of conditions including rapidly heated, non-equilibrium samples. g g p y q p The experiments of Chen et al. [57] on warm dense gold revealed an electron–ion coupling parameter that was notably lower than predicted by classical Spitzer theory or the Fermi Golden Rule approach, which allows for electron–ion energy exchange through normal modes of the electron fluid interacting with normal modes of the ions. 5 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3. Timescale considerations for dynamic production of warm dense matter [48] discuss the application of inner shell photoionization to creation of a solid density X-ray laser using volumetric pumping with a free electron laser, with particular attention paid to collisional effects of photo-ejected electrons, an issue of relevance to volumetric X-ray heating of WDM. In another paper, Hu et al. [49] discuss electron-lattice equilibration within the context of ultra-fast X-ray diagnostics, but again, with physics encompassing processes relevant to WDM production. The development of the field of WDM physics means experimental and modelling techniques progress together, often hand-in-hand. However, for convenience, in the next two sections we present their discussion separately. 6 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . terahertz (THz) radiation, derived from intense ultra-short pulse optical interaction, e.g. [71]. The advantage is that, for a Drude-type model of the conductivity, we can relate the ac conductivity to the dc value via terahertz (THz) radiation, derived from intense ultra-short pulse optical interaction, e.g. [71]. The advantage is that, for a Drude-type model of the conductivity, we can relate the ac conductivity to the dc value via 6 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . σ(ω) = σDC 1 −iωτ . (4.1) (4.1) In reality, of course, there may be complicating factors such as band structure at higher density and lower temperatures. Indeed, non-Drude like behaviour has been reported with DFT calculations of the dynamic conductivity of warm dense Al for energy transfers large enough to cause excitations, e.g. [72]. The significantly lower frequencies in the THz regime mean the initial measurements are closer to the dc values pursued. Despite being complex and difficult to implement, one key advantage of terahertz radiation is that it can penetrate into solid-density samples and, thus, is capable of probing bulk properties of highly excited solid foil samples, e.g. [73]. A more common approach to probing bulk properties of a WDM sample has been to use laser-produced X-rays. Some important early experiments used X-ray Thomson scattering to extract thermodynamic quantities such as temperature and density [74]. However, careful measurement of the plasmon spectrum encodes dynamic information on particle collisions as well as detailed balance [75]. The use of X-ray Thomson scattering to extract thermodynamic properties from experimental data is complex and depends on making approximations in the various theoretical models used. Dornheim et al. [76] address this by discussing a new approach based in use of imaginary-time correlation functions. More recently, high-resolution X-ray radiography experiments are being developed with the aim of directly observing the effects of transport properties, such as the thermal conductivity and particle diffusion, across interfaces in WDM [77–80]. 4. Experimental approaches Instead, it was concluded that coupled modes of the electrons and ions likely play an important role. The observed lower coupling parameter is in line with earlier results from Ng et al. [61] and Celliers et al. [44], who used temporally resolved measurement of the optical emission from the rear surface of shock-compressed WDM to infer electron-equilibration rates. These studies also indicated that the timescale for electron–ion coupling in strongly coupled plasma was over an order of magnitude longer than expected from equilibrium models such as Spitzer [68] and Lee & More [69], including degeneracy corrections [70], suggesting that equilibration could take hundreds of picoseconds. This has significant implications for shock measurements [44], which commonly use the Rankine– Hugoniot equations to connect shock and particle speeds to density, pressure, and internal energy density in order to assess the equation of state for WDM. These equations rely on accurate temperature measurements to connect to internal energy, which can be affected by equilibration effects and are often obtained using techniques such as streaked optical pyrometry as used by Ng et al. [61] and Celliers et al. [44]. Many experiments, such as those cited above, are naturally carried out in the optical region where ultra-short pulse pump-probe experiments down to 10 s of femtoseconds duration are available. However, the reflectivity measured then gives access to the ac conductivity. There is much interest in dc conductivity as this is more relevant to understanding WDM in many situations of interest. To this end, there has been a strong development of probing capability using 5. Modelling approaches This volume includes papers that discuss various numerical simulation techniques used in the field of WDM, and their recent advancements. As noted in the Introduction, sometimes approaches from liquid metal theory have been used. An example used to investigate the ion–ion structure is the use of the Ornstein–Zernicke equation and hypernetted chain closure relations (e.g. [90]), where a given interionic potential is used to develop a self-consistent solution to the pair-distribution function, g(r). This given potential might, for example, take the form of a screened Coulombic potential introduced by Wünsch et al. [91]. This potential included the addition of a stiff repulsive term to account for the overlap of bound shells as ions approach closely, V(r) = A r4 + Z2 e4 r e(−r/λ), (5.1) (5.1) where the strength of the first term is determined by the parameter A, and there is a screening length, λ, which is typically set somewhere between the Debye and Thomas–Fermi screening lengths, depending on the degeneracy [92]. The HNC approach is practical and fast for an assumed steady state with a fixed, equilibrated temperature, a known interaction potential and moderate coupling [93]. However, this is not the case for many of the cases of interest here. For example, solids that are rapidly heated, such as those produced by FEL sources, may experience rapid changes in temperature as the energy exchange between electrons and ions takes place. This can result in both thermal melting, in which the ions gain enough energy to reach the melting point at the given pressure, and non-thermal melting, in which the ejection of electrons through photo-ionization and subsequent electron–electron collisions leads to a potential surface where the ions are no longer bound to each other. For more strongly coupled systems, it may be necessary to use other techniques, such as ab initio molecular dynamics, to understand the material behaviour and evolution. Molecular dynamics is an important class of simulation techniques that can model a system of many particles with differing levels of physics assumptions made. A classical approach can be taken, with a predetermined pairwise potential to govern the forces between ions, in a similar way to the HNC approach above. Historically, a Coulomb [94] or screened Coulomb potential [95] was used. However, recent more advanced potentials have been developed, including short- range repulsion effects [91,96] and thermally damped Friedel oscillations [97]. 6 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [89] will discuss the related issue of diffusion of ions in warm dense matter. This is important in the field of fusion pellets, where fuel mixing is a key factor in capsule performance. 7 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The development of new facilities such as free-electron lasers (FELs) in the extreme ultraviolet radiation (XUV) to hard X-ray range has revitalized research on WDM, allowing for the heating and probing of samples on ultrafast timescales of 1–100 fs, which is shorter than the inverse phonon frequency of solids. This opens up the possibility of conducting experiments that, rather than trying to achieve an equilibrated system, intentionally create a non-equilibrium state with hot electrons and cold ions in order to study non-equilibrium effects [81,82]. However, the novel sources provided by FELs not only provide an ability to isochorically heat systems to well-defined conditions, but are also an extremely bright source for scattering, imaging, and spectroscopic probing [83]. A particular improvement in diagnostic capability provided by FELs is in X-ray Thomson scattering, with some key advantages over the use of X-rays from laser plasmas. The enhanced collimation and monochromatic nature of the probe are extremely well suited to this diagnostic and this has allowed many advances. For example, XUV Thomson scattering experiments have been used to test electron–ion equilibration models on ultra-short timescales [84]. At the same time, X-ray scattering experiments have been able to probe the structure and dynamics of warm dense matter with unparalleled resolution [85], with measurements of the damped electron plasmon wave by Sperling et al. being used to show that the dynamic conductivity exhibits non-Drude-like behaviour that must be considered when determining the optical properties of WDM, rather than relying on the widely used random phase approximation [86]. In addition to transport and equilibration, another important aspect of the electronic properties of warm dense matter is the stopping power of charged particles, which is relevant to fields such as planetary science, geophysics and fusion research, where the stopping of alpha particles can have a significant impact on capsule performance. Experiments on stopping power in warm dense matter, such as those conducted by Malko et al. [87], have found deviations from classical models for protons in warm dense carbon but have shown better agreement with first-principles calculations using time-dependent DFT, as demonstrated by Ding et al. when applied to warm dense Be experiments performed by Zylstra et al. [88]. This time-dependent DFT approach showed a significant deviation from classical models when applied to alpha-particle stopping in warm dense DT plasmas relevant to fusion capsules. Angermeier et al. 8 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . matter at high temperatures due to the need to include a large number of excited state orbitals in the simulation, which scales cubically with the number of orbitals. Using DFT, Mazevet et al. [106] have studied the time evolution of a sample of gold assuming an initial, essentially instantaneous, input of energy to the electrons. Using a combination of classical molecular dynamics with ab initio methods, where the forces acting are derived in a time-dependent way, governed by the configuration of the ions at a particular point in the simulation, they were able to follow the melting of the lattice and equilibration of the ion and electron temperatures. 8 g q p These ab initio methods are well suited to the study of time-dependent problems [107], in which the electronic states and orbitals are calculated from the atomic structure, which then determines the forces that move the particles—essentially, using DFT to calculate the potential for MD on-the-fly. With increased computing power, such simulations have become possible over the last decade, with DFT-MD being used to simulate the dynamic properties of WDM with increasing levels of complexity [108–110]. Of course, there have also been significant developments in the method. White & Collins [111] developed algorithms that allow temperatures in the 50 eV range to be handled with acceptable computing times while considerable effort is now being spent on the development of high-temperature exchange and correlation functionals [112]. With the increased interest in machine learning, the accurate interatomic potentials from DFT data can be produced for WDM, so that we can extend the study of WDM to millions of atoms within ab initio accuracy [113,114]. Incorporating electronic quantum effects into first-principles simulations is inherently computationally demanding, limiting their applicability to larger spatiotemporal scales and non- equilibrium systems; one way around this is to use reduced models. Quantum hydrodynamics, which has recently gained renewed interest [115], is able to include the important role of quantum effects in cases where the quantum correlations are not too prominent [116] and consider the role of quantum non-locality through the inclusion of the Bohm potential, which is neglected in classical hydrodynamics. It is noteworthy that in certain scenarios, the Bohm force arising from this approach can be comparable in magnitude to the forces generated by exchange and correlation pressures, highlighting the relevance of quantum hydrodynamics in capturing essential quantum phenomena [117]. 5. Modelling approaches These classical simulations have been used to investigate many WDM properties such as density fluctuations [96,98], transport coefficients [99,100] and electron–ion equilibration [101]. Molecular dynamics is able to capture the strongly coupled nature of warm dense matter, but the quantum effects are hidden in the details of the interatomic potential that ultimately defines the accuracy of these simulations. In many cases, the parameters and even the form of the potential are unknown. In such cases, we must use more advanced techniques, such as density functional theory (DFT), to calculate the potential. As an example of the power of the molecular dynamics approach, Ling et al. [102] used molecular dynamics to simulate the formation of warm dense Cu using the shock compression method, and find the complicated evolution of the thermodynamic paths during the compressing processes. p g p DFT is a popular simulation technique in the WDM field, with DFT molecular dynamics (DFT-MD) a popular method of choice. The theory is based on the concept that the total energy is a function of the electron density distribution, and by minimizing this energy, we can determine the actual electron distribution. The Thomas–Fermi model is often seen as a precursor to DFT, but it was Hohenberg & Kohn’s [103] and Kohn & Sham’s [104] work that truly advanced the field. For more information about DFT, see Kohanoff’s work [105]. One challenge for DFT is properly formulating expressions for exchange and correlation energies in non-uniform systems. While DFT is commonly used in solid-state physics, it can be computationally intensive for warm dense 9 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . heat, momentum and mass, respectively, are a necessary input for continuum models such as magnetohydrodynamics, and all three coefficients play a vital role in our understanding of planetary interiors and the design and analysis of inertial confinement fusion (ICF) implosions. As highlighted at a recent workshop [126], there still exist large order-of-magnitude differences between theoretical predictions. 9 Several authors have carried out extensive theoretical discussions of electron-ion relaxation times for dense plasmas and warm dense matter (e.g. [127]). Dharma-Wardana & Perrot [128] used a Fermi Golden Rule (FGR) approach to calculate the interaction between the normal modes of independent ion and electron subsystems. Depending on whether they assumed the subsystems to have independent spectra or a spectrum of modes generated by the interaction between the subsystems, they found for solid density Al at temperatures in the range 3–40 eV, the coupling rate was 4 or 5 orders of magnitude lower than calculated with the classical Spitzer–Brysk method. For systems where the ion acoustic or phonon modes interact strongly with the electrons, coupled modes may form, further reducing the equilibration rate and forming a relaxation bottleneck [39,128]. Gericke et al. [129] investigated the validity of ad-hoc approximations to the Coulomb logarithm that are designed to extend a classical Landau–Spitzer approach to strongly coupled systems where linear approximations to the electron trajectory are not valid and both close and weak collisions are included. In highly non-equilibrium systems undergoing rapid changes, such as in laser-excited solids, using FGR is thought to overestimate the transition rates on short time scales [130,131]. Models based on the extension of FGR, such as those based on the Eliashberg spectral function formalism [132], are thought to overestimate the coupling strength compared to experimental data. The most successful descriptions take into account the varying coupling between electrons and different phonon modes [133], with a hybrid approach based on tight-binding molecular dynamics code showing good agreement with the limited experimental data [134]. In this volume, the issue of electron–ion equilibration is addressed further in the paper of Ziaja et al. [135]. 8 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A further important development has been wave packet molecular dynamics (WPMD). This time-dependent quantum mechanical method simultaneously simulates the classical point particle motion of ions and the quantum-mechanical behaviour of electrons [118]. It has become popular in recent years as a computationally fast way to study dynamic processes in warm dense matter that go beyond the Born–Oppenheimer approximation [119,120]. Such non-adiabatic techniques are necessary for describing states that are not in local thermal equilibrium (Te ̸= Ti), in which energy can be exchanged between electrons and ions through collisions [121]. However, recent research has indicated that these techniques may also be important for studying systems in equilibrium [122,123]. Angermeier et al. [89] investigate the role non-adiabatic effects have in particle diffusion in warm dense hydrogen, while Svensson et al. [124] present an extension of the wave packet description of quantum plasmas, in which non-isotropic wave packets can be elongated in arbitrary directions. As we have noted, molecular dynamics methods, particularly those based on DFT, have been de-rigeur in warm dense matter modelling. However, we should not ignore the potential contributions of other methods. For example, Filinov et al. [125] present ab initio calculations of helium based on path integral Monte-Carlo methods that could be applied across a range of conditions, including WDM. The evolution of the microscopic structure of warm dense matter in rapidly evolving systems is clearly a key interest for this volume. However, the calculation of transport coefficients, which describe the rate at which key properties are transferred throughout the plasmas, is another important category of dynamic processes. Transport properties such as thermal conductivity, viscosity and diffusion are some most fundamental dynamical parameters that reflect the nature of the interatomic potential and characterize the thermodynamics of the system. The corresponding transport coefficients, which encapsulate the rate of transfer of 6. Conclusion It is clear that warm dense matter is a field that is of wide interest for applications in fusion sciences, geophysics and planetary sciences. It is also a vibrant field with many advances, both in experimental techniques, computational simulation and underlying theory. These advances are exemplified by the papers presented and the works that they build upon and reference. The literature on warm dense matter is wide enough and varied enough that even within the scope of this special edition, concentrating on issues of time dependence and dynamic processes, we can see a huge variety of techniques and topics addressed. Data accessibility. This article has no additional data. Data accessibility. This article has no additional data. Authors’ contributions. D.R.: conceptualization, project administration, writing—original draft, writing—review and editing; D.J.: writing—original draft, writing—review and editing; T.G.W.: writing—original draft, writing—review and editing. All authors gave final approval for publication and agreed to be held accountable for the work performed therein.l Conflict of interest declaration. This theme issue was put together by the Guest Editor team under supervision from the journal’s Editorial staff, following the Royal Society’s ethical codes and best-practice guidelines. The Guest Editor team invited contributions and handled the review process. Individual Guest Editors were not involved in assessing papers where they had a personal, professional or financial conflict of interest with the authors or the research described. Independent reviewers assessed all papers. Invitation to contribute did not guarantee inclusion. Funding. This work was supported by the National Science Foundation (grant no. PHY-2045718). This work was funded in part by the US Department of Energy, National Nuclear Security Administration (NNSA) (Award no. DE-NA0004039). D.J.Y. thanks the support of the Science and Technology Innovation Program of Hunan Province China (grant no. 2021RC4026). D.R. thanks the UK Engineering and Physical Sciences Research Council for funding (grant no. EP/P010059/1). Acknowledgements. The authors thank Prof. Ronald Redmer for useful discussions. References 10 1. Lee R et al. 2002 Plasma-based studies with intense X-ray and particle beam sources. Laser Part. Beams 20, 527–536. (doi:10.1017/S0263034602202293) 2. Ng A. 2002 Shock waves and plasma physics. In Proceedings of the Conference of the American Physical Society, Topical Group on Shock Compression of Condensed Matter, vol. 620, Atlanta, GA, 24–29 June 2001, pp. 53–58. 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Implications of providing social support to close network members on the social well-being of older people in Kitui County, Kenya
Frontiers in aging
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TYPE Original Research PUBLISHED 16 November 2022 DOI 10.3389/fragi.2022.897508 TYPE Original Research PUBLISHED 16 November 2022 DOI 10.3389/fragi.2022.897508 TYPE Original Research PUBLISHED 16 November 2022 DOI 10.3389/fragi.2022.897508 KEYWORDS social well-being, social support, instrumental support, emotional support, informational support, close network members Implications of providing social support to close network members on the social well-being of older people in Kitui County, Kenya OPEN ACCESS EDITED BY Joyce S. Pang, Nanyang Technological University, Singapore REVIEWED BY Pauline Thuku, Karatina University, Kenya Jan Oyebode, University of Bradford, United Kingdom *CORRESPONDENCE Kezia Mbuthia, mbuthiakezia@gmail.com SPECIALTY SECTION This article was submitted to Interventions in Aging, a section of the journal Frontiers in Aging RECEIVED 16 March 2022 ACCEPTED 24 October 2022 PUBLISHED 16 November 2022 CITATION Mbuthia K, Mwangi S and Owino G (2022), Implications of providing social support to close network members on the social well-being of older people in Kitui County, Kenya. Front. Aging 3:897508. doi: 10 3389/fragi 2022 897508 OPEN ACCESS EDITED BY Joyce S. Pang, Nanyang Technological University, Singapore REVIEWED BY Pauline Thuku, Karatina University, Kenya Jan Oyebode, University of Bradford, United Kingdom Kezia Mbuthia*, Samuel Mwangi and George Owino Department of Sociology, Gender and Development Studies, Kenyatta University, Nairobi, Kenya Social support is a strong predictor of social well-being. Older people are key providers of social support to others, but an overemphasis on received social support in gerontological literature portrays them as mere recipients. We examined the association between social support provided by older people and its influence on their social well-being. Data were collected from 369 respondents residing in rural dwellings in Kitui County using mixed methods and were analyzed for association using chi-square statistics. Instrumental, emotional, and information support provision was determined by asking four questions in each category about whether the respondents provided social support to close network members. The subjective experience of support provision using a satisfaction question for each domain of social support was used to determine the influence of providing social support on the social well-being of older people. Provision of social support across the three domains was found to be significantly associated with social well-being. The level of statistical significance was highest for emotional and information support compared to instrumental support. Older people are important providers of social support. The majority of those who provided social support reported being satisfied. Therefore, offering social support, especially emotional and information support, is an important contributor to satisfaction with these aspects of social well-being. COPYRIGHT © 2022 Mbuthia, Mwangi and Owino. 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. social well-being, social support, instrumental support, emotional support, informational support, close network members (Elliott, 2017). In addition, studies on social support infrequently examine whether helping others has any benefits. According to Brown et al. (2003), the effects are frequently attributed to receiving support or occasionally to reciprocal support. Even if some social support measures actually appear to examine giving, they do so possibly inadvertently. Therefore, the study aims to investigate the effects of providing instrumental, emotional, and informational social support to others on the social well-being of the providers (i.e., older people). According to Dunaeva (2018), social support is a significant element of social well-being. It relates to social exchanges that are regarded as beneficial (Dykstra, 2015), such as advice, encouragement, and love (Thoits, 2011). These supportive qualities sustain social relationships (Umberson & Montez, 2010). From this definition, similar to Schwarzer, Knoll, and Rieckmann (2004), social support is investigated in several types such as emotional (i.e., love), informational (i.e., giving advice), and instrumental (i.e., assistance with a problem). It serves as a potential resource to individuals and is associated with well-being benefits, especially among older adults (Mohd et al., 2019). 2 Materials and methods Social support is described as either perceived or received support. Received social support refers to the actual reported exchanges, while perceived social support refers to potential access to support by an individual (Haber et al., 2007; Uchino, 2009). The multidimensionality of social support has led to some of its aspects being given more attention than others, which may be attributed to the lack of a standardized method of measuring and reporting the multiple dimensions (Mohd et al., 2019). Specifically, perceived social support has attracted wider scholarly attention than received social support (Mohd et al., 2019). 1 Introduction Social well-being can be defined as a person’s evaluation of their situation, social relations, and how well they can function in a community (Dunaeva, 2018). This construct has been classified into two dimensions: subjective experiences and objective circumstances (Boreham, Povey, & Tomaszewski, 2013). Objective circumstances relate to support that is apparent, tangible, and direct (Yu et al., 2020). Frontiers in Aging 01 frontiersin.org Mbuthia et al. 10.3389/fragi.2022.897508 Mbuthia et al. Subjective experiences reflect a person’s internal thoughts and feelings that are not visible to outside observers (Lucas, 2018). A singular objective life situation or circumstance can be evaluated differently by different people. The subjective experiences are evaluations of life in terms of satisfaction (Keyes, Shmotkin, & Ryff, 2002) based on objective circumstances. According to Sen, Prybutok, and Prybutok (2021) social well-being involves both external and internal factors, such as the presence of exchanges or connections and satisfaction with the quality of those exchanges or connections. Therefore, in the context of this study, social well-being refers to satisfaction with the quality of social support provided. However, although the social well-being of older people is a topic that has drawn scholarly consideration in high-income nations, it has received little attention in low- and middle-income countries (Elliott, 2017). people who did not. Other studies however, found that providing social support elicits feelings of burden and frustration (Thomas, 2010; Morelli et al., 2015) and is overwhelming (Krause & Shaw, 2002) for older people. The mixed and contradictory findings call for more research to understand the influence of providing social support on well-being. Numerous crucial aspects of social support provision and its effects on social well-being have not been systematically documented (Thomas, 2010). This is because prior works focus on two categories of social support, instrumental, and emotional, with each category affecting support providers differently (Morelli et al., 2015). Therefore, this study focused on three categories of social support, instrumental, emotional, and information, to assess their effect on support providers because the findings in prior studies are mixed and contradictory. people who did not. Other studies however, found that providing social support elicits feelings of burden and frustration (Thomas, 2010; Morelli et al., 2015) and is overwhelming (Krause & Shaw, 2002) for older people. The mixed and contradictory findings call for more research to understand the influence of providing social support on well-being. 1 Introduction Numerous crucial aspects of social support provision and its effects on social well-being have not been systematically documented (Thomas, 2010). This is because prior works focus on two categories of social support, instrumental, and emotional, with each category affecting support providers differently (Morelli et al., 2015). Therefore, this study focused on three categories of social support, instrumental, emotional, and information, to assess their effect on support providers because the findings in prior studies are mixed and contradictory. 2.1 Participants A cross-sectional survey design was employed to collect primary data in June and July 2021. The total study population of older people in four sampled sub-counties of Kitui County was 32,839, according to the Kenya National Bureau of Statistics (KNBS, 2019). Using Yamane’s (1967) formula at a 95% confidence level and p = 0.05, a sample of 396 older people was determined. Multistage cluster sampling was employed, and participants within each of the clusters were selected using a simple random sampling technique. Received social support is said to be provided and received in social relationships. Received social support has attracted wide theoretical and empirical attention, unlike provided social support (Song, Son, and Lin, 2011). This is especially the case when investigating social support among older people. Received social support has been found useful in providing protection against threats to well-being and in helping people to deal with setbacks that are detrimental to social well-being (Dykstra, 2015). The overemphasis in gerontological literature of social support received by older people portrays them as mere recipients and disregards their role as its providers (Albertini, Kohli, & Vogel, 2007; Dykstra, 2015). Some studies found that provided social support was beneficial to providers. For instance, Bai et al. (2020) observed that providing social support enhanced well-being in older people who took care of their grandchildren compared to In the first stage, administrative locations in the four sub- counties were identified. In each sub-county, three locations were selected using the fish bowl draw method (Kumar, 2011). In the second stage, two sub-locations were purposively identified from the selected locations, and a sampling frame (i.e., list of older people living within the sub-locations) was obtained from the area administrative chiefs and village elders. These steps were necessary because the population was geographically heterogeneous (Sedgwick, 2015), and the sampling units for the different stages were different (Acharya et al., 2013). The names of older people provided by the area chiefs and village elders were entered into Microsoft Excel 2016. Older people in the sampled sub-locations were randomly selected using the Rand function. Frontiers in Aging frontiersin.org 02 10.3389/fragi.2022.897508 10.3389/fragi.2022.897508 Mbuthia et al. Mbuthia et al. 2.4 Data analysis Statistical Package for the Social Sciences (SPSS) Version 21.0 was used to analyze the data. The chi-square test of association was used to establish the association between the independent and the dependent variables. For each item in the provision of social support questionnaire, the relationship to overall satisfaction in that domain of social support was assessed to establish whether it was significant. Because the sample sizes were small, Fisher’s exact test was preferred because it does not depend on any large-sample asymptotic distribution assumptions (Field & Miles, 2010). The results were reported with a 95% confidence interval (CI) and a p- value of 0.05. Descriptive statistics (frequencies, percentages) were also calculated and are presented in tables. Thematic analysis was used to analyze qualitative data. Thematic analysis is a technique for assessing qualitative data that involves looking through data to find, examine, and report recurring themes (Viji & Benedict, 2014). Kiger and Varpio (2020) contend that rather than choosing an easy-to-use method of analysis, deciding to utilize thematic analysis should be based on the objectives of the study itself. In 2.2.2 Dependent variable: Social well-being The study measured social well-being by assessing how the respondents appraised the social support that they provided using a satisfaction question for each domain. The satisfaction question was indexed by responses (from 1 = extremely dissatisfied, 2 = dissatisfied, 3 = satisfied, and 4 = extremely satisfied) to the following statement: In the last 12 months, how satisfied are you with the social support that you provided to CNMs? The responses were highly skewed because very few respondents selected extremely dissatisfied and extremely satisfied in all the three domains of social support. At the analysis stage, responses for 1 and 2 were collapsed from the original scale to 1 = dissatisfied and those for 3 and 4 were collapsed to 2 = satisfied, creating a dichotomized scale. The research instrument was pre-tested with 30 respondents from a subsample of older people, and a post-test was conducted 3 weeks later. The test–retest reliability was reasonably measured by the correlation coefficient between the two scores, which was 0.718. 2.2.1 Independent variable: Social support 2.2.1 Independent variable: Social support Participants were asked to report whether they provided social support to close network members (CNMs), including spouse, children, neighbors, relatives, and friends, in the 12 months prior to the study. The study measured three domains of social support with four items in each: (a) instrumental social support—lend or give money, help with chores/errands, provided care in sickness, lent items or tools; (b) emotional support—cheered up or helped CNM feel better, showed interest in their personal life, did or said things that were kind or considerate, trusted CNM to solve their problems; (c) informational support—gave helpful advice when needed to make important decision, agreed with CNM’s actions or thoughts, gave information to understand an issue, and gave CNM feedback on an action they wanted to take. These items in the three domains of social support were drawn from the previous studies (Morelli, Lee, Arnn & Zaki, 2015; Schwarzer et al., 2004; Thoits, 2011; Newsom et al., 2005). Participant’s response categories were: Yes [1] or No [2]. 2.2 Measures With the assistance of local leaders, a distinct group of older people from those who filled out the questionnaire were purposively sampled. This was done in order to include individuals who could stimulate one another to explore their opinions and experiences in light of the discussion topics (Moser & Korstjens, 2018). The focus group discussion themes were developed using the quantitative data from the questionnaire. For instance, the questionnaire was divided into five sections: demographic characteristics of the respondents, instrumental, emotional, and informational social support, and each domain’s satisfaction questions. These social support domain questions served as the basis for the focus group template and were the starting point for addressing the crucial concerns of “why” and “how” providing social support influenced satisfaction with doing so. This was done in order to understand the reasoning underlying participants’ feelings and actions. The discussants were welcomed to the FGD, and the purpose of the study was explained to them. The researcher moderated the focus group discussions as the research assistants allocated each participant’s initials and transcribed the discussion of each participant verbatim. The socio-demographic questions for the focus group participants included gender, marital status, level of education attainment, and their source of livelihood (multi-response question). Because demographic characteristics can influence sharing within the group discussion, homogeneity with sufficient variation among members to support opposing viewpoints was established. Frontiers in Aging 2.3 Procedure A questionnaire was developed for this study that covered the three domains of social support and the social well-being question, as well as socio-demographic questions, including gender, participant’s marital status, level of education attainment, source of livelihood, and average monthly income. These were administered by the researcher and the research assistants at the respondent’s homes. Four focus group discussions comprising older men and women were also conducted, and the data were used to complement the quantitative data from the questionnaire. Frontiers in Aging frontiersin.org 03 Mbuthia et al. 10.3389/fragi.2022.897508 TABLE 1 Demographic profiles of FGD participants. Variable Categories n = 369 (%) n = 32 (%) Gender Male 162 40.9 14 47.3 Female 234 59.1 18 56.3 Age 60–69 200 50.5 18 56.3 70–79 110 27.8 12 37.5 80–89 63 15.9 1 3.1 90–99 17 4.3 1 3.1 100+ 6 1.5 0 0 Marital Status Single 13 3.3 2 6.3 Married 228 57.6 15 46.9 Divorced 3 0.8 1 3.1 Separated 9 2.3 1 3.1 Widowed 143 36.1 13 40.6 Level of education No formal education 142 35.9 6 18.7 Primary 165 41.7 14 43.8 Secondary 74 18.7 7 21.9 Tertiary 15 3.8 5 15.6 Source of livelihood Farming 284 57.8% 25 43.9 Older persons cash transfer (OPCT) 94 19.1% 6 10.5 Pension 31 6.3% 0 0 Business 31 6.3% 0 0 Casual labor 25 5.1% 2 8.8 Support by children and other kin 18 3.7% 22 38.6 Employment 8 1.6% 2 8.8 Average monthly Ksh Below 1,000 99 25.0 1,001–5,000 227 57.3 5,001–10,000 25 6.3 Over 10,000 45 11.4 qualitative studies and, in this case, demonstrated that the emerging themes were trustworthy and plausible. this case, the focus group template of the key themes that were developed from the questionnaire study was checked against the focus group transcripts. Verbatim quotations from each of the domains were chosen to complement the quantitative data that represented the main themes that emerged from the discussions. To ascertain the trustworthiness of FGDs, the researcher and two research assistants engaged in transcribing and categorizing the themes. Final agreement on the themes for each domain of social support was reached by comparing the three researchers’ independently drawn theme categories. The data were triangulated across the four sub-counties by conducting one FGD in each. 2.3 Procedure Verbatim quotations that were selected also reflected the main themes that emerged from the discussions, and there was prolonged engagement with the transcriptions as well as persistent observation of the emerging themes. According to Guion et al. (2011) triangulation establishes the validity of 3.1 Socio-demographic characteristics Most respondents were women (234, 59.1%) aged between 60 and 69 years. Of which, 200 (50.5%) were married, 228 (57.6%) had attained a primary level of education, 165 (41.7%) earned their livelihood from farming, and 224 (57.8) reported on average a monthly income of KES 1,001–5,000 (equivalent to USD 10–50). Results are shown in Table 1. I left my watchman job to attend to my sick brother because he is my brother and needed me (AG, 67-year-old male FGD participant). I help in cleaning utensils because I can’t help in the farms and am happy am useful (JK, 84-year-old female). I feel good when I help the people close to me with chairs and sufurias (cooking pot) because I know that they have sought my assistance since they don’t have the items (FK, 75-year-old female participant). 3 Results My son was having a very hard time paying rent in Nairobi because of COVID-19 lockdown, I gave my son money to rent a cheaper house and felt good helping him (JM, 68-year-old male FGD participant). My son was having a very hard time paying rent in Nairobi because of COVID-19 lockdown, I gave my son money to rent a cheaper house and felt good helping him (JM, 68-year-old male FGD participant). I left my watchman job to attend to my sick brother because he is my brother and needed me (AG, 67-year-old male FGD participant). 3.2.1 Instrumental social support The results show that most respondents provided social support within each item of instrumental social support and were satisfied with the social support they provided to CNMs. The calculated chi-square statistic using Fisher’s exact test demonstrated a highly statistically significant association between the instrumental social support variables and satisfaction with provided social support [satisfaction with lending money (χ2 = 6.05, p < 0.014)], satisfaction with providing care to CNM in sickness (χ2 = 13.26, p < 0.000), satisfaction with providing help with chores/errands (χ2 = 4.48, p < 0.034), satisfaction with lending household tools/items (χ2 = 8.90 p < 0.017)]. Results are shown in Table 2. 2.5 Ethical approval Study participants were informed about the study by the researcher and research assistants. They were notified about the procedure to be followed and that participation was voluntary. They were made aware that they could withdraw from the study at any time and that the information they provided would be used for academic purposes only. The approval to conduct the study was granted by the Kenyatta University Graduate School and permitted by the Kenyatta University Ethical Review Committee (KU-ERC), approval number PKU/2235/11379, and the National Commission for Science, Technology, and Innovation (NACOSTI) [Permit number NACOSTI/P/21/11012. Frontiers in Aging frontiersin.org 04 10.3389/fragi.2022.897508 Mbuthia et al. TABLE 2 Cross-tabulation of social support provided and satisfaction with provided instrumental support. Support provided Responses Satisfaction with provided instrumental support χ2 df p-value Satisfied % Dissatisfied % Lend money Yes 281 (97.2%) 74.1% 8 (2.8%) 47.1% 6.05 1 0.014 e* No 98 (91.6%) 25.9% 9 (8.4%) 52.9% Care in sickness Yes 325 (97.3%) 85.8% 9 (2.7%) 52.9% 13.26 1 0.000 e* No 54 (87.1%) 14.7% 8 (12.9%) 47.1% Help with household chores Yes 287 (97.0%) 74.7% 9 (3.0%) 52.9% 4.475 1 0.034 e* No 92 (92.0%) 25.3% 8 (8.0%) 47.1% Lending household items/tools Yes 358 (96.5%) 94.5% 13 (3.5%) 76.5% 8.901 1 0.017 e* No 21 (84.0%) 5.5% 4 (16.0%) 23.5% *Fisher’s exact test. 3.2 Social support and social well-being These narratives demonstrate that older people felt useful when they were able to meet the needs of their CNM. Therefore, providing instrumental support was satisfying for older people because it elicited a feeling of being useful to others when they met the needs of CNMs. 3.2.2 Emotional social support The results show that most respondents provided social support within each item of emotional social support and were satisfied with the social support that they provided to CNMs. The smallest expected frequency in the cross- tabulation Table 3 did not exceed 5, and the assumption of chi-square was not met. Fisher’s exact test (Fisher, 1922) was used to compute the exact probability of the chi-square statistic. The association between emotional support provided and satisfaction with providing emotional support was highly significant [satisfied cheering up or helping CNM feel better (χ2 = 32.54, p < 0.000), satisfaction with showing interest in their personal life (χ2 = 40.44, p < 0.000), satisfied doing or saying things that were kind or considerate (χ2 = 67.41, p < 0.000), satisfied helping CNM to solve their problems (χ2 = 41.83, p < 0.000)]. The results are shown in Table 3. Participants in the focus group discussions agreed with the study findings that providing instrumental social support is pervasive among older people. They further alluded that providing for others is a good thing to do as long as it is within a person’s ability to provide. The narrative below demonstrates that providing instrumental social support elicited positive effects on older peoples’ social well-being: Frontiers in Aging frontiersin.org 05 10.3389/fragi.2022.897508 Mbuthia et al. TABLE 3 Cross-tabulation of social support provided and satisfaction with provided emotional support. Support provided Responses Satisfaction with provided emotional support χ2 df p-value Satisfied % Dissatisfied % Cheered CNM up Yes 351 (97.8%) 92.4 8 (2.2%) 50.0 32.538 1 0.000* No 29 (78.4%) 76 8 (21.6%) 50.0 Showed interest in the personal life of CNM Yes 350 (98.0%) 92.1 7 (2.0%) 43.8 40.435 1 0.000* No 30 (76.9%) 7.9 9 (23.1%) 56.3 Did or said things that were kind or considerate Yes 374 (97.4%) 98.4 10 (2.6%) 62.5 67.419 1 0.000* No 6 (50.0%) 1.6 6 (50.0%) 37.5 Trusted CNM to solve your problems Yes 351 (98.0%) 92.4 7 (2.0%) 43.8 41.834 1 0.000* No 29 (96.0%) 7.6 9 (4.0%) 56.3 *Fisher’s exact test. These findings were congruent with the voices of the FGD participants, who reported that they provide emotional support to CNMs and found fulfillment in this activity. This support is meant to help CNMs when they face difficulties, as explained in the excerpts below: they succeed at it, their own social well-being is enhanced. 3.2.2 Emotional social support Providing the emotional support was satisfying to older people by increasing their social connection and engagement with CNMs. 3.2.3 Informational social support Anytime they are unhappy, it affects me too (MK, a 70-year-old male participant). The voices of FGD participants echo the findings from the quantitative analysis. I am always happy when my children are happy. I celebrate when they celebrate and thank God for them. Anytime they are unhappy, it affects me too (MK, a 70-year-old male participant). My neighbor has been quarreling with his neighbor about a land boundary and wanted to take the case to court. I suggested to him that the case could be solved at the clan level instead of going to court. He heard my advice, and the issue has been resolved (AW, 72-year-old male participant). I am the treasurer of the community self-help group. My son needed some money and borrowed it from me. I took some from the kitty and trusted my son to refund on time before our next community meeting. He refunded the money on time, and I was very proud of him for being worthy of my trust (AN, a 68-year-old male participant). y My neighbor had kept a man (co-habit), and the neighbors thought her actions were not right and the man was not good for her, but I saw him as a good man and agreed with her action to stay with him. I told her that she made a good decision to think about herself for once since her husband died ten years ago (AM, 72-year-old female participant). My grandchildren have been raised in Nairobi and do not understand much about our Kamba culture. I explained to them when they visited about our clan, which is called Anzauni, about how hardworking we are. It felt good My neighbor had kept a man (co-habit), and the neighbors thought her actions were not right and the man was not good for her, but I saw him as a good man and agreed with her action to stay with him. I told her that she made a good decision to think about herself for once since her husband died ten years ago (AM, 72-year-old female participant). These voices from the FGD participants imply that older people go to great lengths to provide help to their CNMs. This is because when CNMs are facing challenges, the older people are also affected and thus put effort in helping the CNM return to a state that can potentially elicit positive emotions. 3.2.3 Informational social support Out of my four children, three of them lost their jobs due to the COVID-19 pandemic. I cheered them up by reminding them of where we have come from and also sent them maize and beans from the village and encouraged them not to give up despite the hard times they were going through (DK, a 73-year-old FGD male participant). The association between information support and satisfaction with providing information support was computed. The expected frequencies in the distribution were not greater than 5, and so the chi- square assumption was not met. Fisher’s exact test, a precise test that is suitable when samples sizes are small (Kim, 2017), was used, and it demonstrated significance between the variables giving helpful advice when needed to make important decision and satisfaction providing information support (χ2 = 73.02, p < 0.000), satisfaction agreeing with CNM’s actions or thoughts (χ2 = 41.55, p < 0.000), gave information to understand an issue (χ2 = 49.62, p < 0.000), gave CNM feedback on an action they wanted to take (χ2 = 39.01, p < 0.000). Results are shown in Table 4. My son had a fight with the wife and chased her away. She went to town and got a house for herself and the kids. I knew my son was in the wrong, and he was not listening to my scolding. I left my son in the village and joined his estranged wife in town and stayed with her until my son accepted her back. She is a good wife and exactly the person my son needs. I feel good they reconciled even if it compelled me to intrude in their personal lives (SK, a 69-year-old female participant). My son had a fight with the wife and chased her away. She went to town and got a house for herself and the kids. I knew my son was in the wrong, and he was not listening to my scolding. I left my son in the village and joined his estranged wife in town and stayed with her until my son accepted her back. She is a good wife and exactly the person my son needs. I feel good they reconciled even if it compelled me to intrude in their personal lives (SK, a 69-year-old female participant). I am always happy when my children are happy. I celebrate when they celebrate and thank God for them. Frontiers in Aging 3.2.3 Informational social support My brother came to me because he had problems paying school fees for his son who was to join university. He wanted to sell a portion of land that our parents left me to oversee. I did not have a response at the time and told him I would think about it. A few days later, I went to him and gave him reasons why we cannot sell that portion of land. I suggested ways we could raise the money without having to dispose our only prime land. My brother’s patience and willingness to look for alternative ways showed how much he respects me (SM, a 68- year-old male participant). My brother came to me because he had problems paying school fees for his son who was to join university. He wanted to sell a portion of land that our parents left me to oversee. I did not have a response at the time and told him I would think about it. A few days later, I went to him and gave him reasons why we cannot sell that portion of land. I suggested ways we could raise the money without having to dispose our only prime land. My brother’s patience and willingness to look for alternative ways showed how much he respects me (SM, a 68- year-old male participant). Generally, the significant finding that providing instrumental social support was associated with social well-being has been observed in the literature. Providing instrumental social support was associated with higher levels of life satisfaction (Brown et al., 2003) and greater life satisfaction among rural Taiwanese older adults (Ku et al., 2013). However, in a study on contributory behaviors and life satisfaction among older Chinese adults, Liu et al. (2019) found that providing instrumental social support and satisfaction with life were unrelated. From these voices, it is evident that an effect is generated that has the potential to influence social well-being. Older people weigh in on whether the information support they provide will benefit the CNM. In addition, they feel knowledgeable and needed by the CNM, which benefits their social well-being. Actively offering information support to others was satisfying to older people by making them part and parcel of the decision-making process. Additionally, it increased communication between them and their CNM and gave them a sense of resourcefulness. 3.2.3 Informational social support In the first instrumental item [lending money and satisfaction with lending money (χ2 = 6.05, p < 0.014)], satisfaction was attributed to the feelings of being useful to CNMs and the assurance of support during their own time of need. Older people have support expectations from children and grandchildren that they assist, in the present or past, in their time of need (Schatz & Ogunmefun, 2007; Chitaka, 2017). These findings resonate with a longitudinal study in Ireland that found that older people who provide financial support to CNMs have the highest quality of life compared to those who only receive support (Ward & McGarrigle, 2017). Similarly, spending money on others was found to produce positive effect in a study of 136 higher- and lower-income countries around the world (Aknin et al., 2013). The results are reinforced In summary, the study shows that each dimension of social support provided by older people was significantly associated with social well-being, as measured by participants’ reports of their satisfaction with that dimension. However, emotional and informational support had a stronger association than instrumental support. 3.2.3 Informational social support When My grandchildren have been raised in Nairobi and do not understand much about our Kamba culture. I explained to them when they visited about our clan, which is called Anzauni, about how hardworking we are. It felt good Frontiers in Aging frontiersin.org 06 10.3389/fragi.2022.897508 Mbuthia et al. TABLE 4 Cross-tabulation of social support provided and satisfaction with provided informational support. Support provided Responses Satisfaction with provided informational support χ2 df p-value Satisfied % Dissatisfied % Offered helpful advice Yes 358 (98.9%) 93.7 4 (1.1%) 28.6 73.025 1 0.000* No 24 (70.6%) 6.3 10 (29.4) 71.4 Agreed with CNM’s thoughts/actions Yes 347 (98.6%) 90.8 5 (1.4%) 35.7 41.549 1 0.000* No 35 (79.5%) 9.2 9 (20.5%) 64.3 Gave CNM information to understand an issue Yes 337 (99.1%) 88.2 3 (0.9%) 21.4 49.620 1 0.000* No 45 (80.4%) 11.8 11 (19.6%) 78.6 Gave CNM feedback on an action they wanted to take Yes 326 (99.1%) 85.6 3 (0.9%) 21.4 39.013 1 0.000* No 56 (83.6%) 14.4 11 (16.4%) 78.6 *Fisher’s exact test. sharing about our clan with the children (AP 78-year-old male participant). sharing about our clan with the children (AP 78-year-old male participant). instrumental, informational, and emotional. Instrumental support includes doing things for others (DeHoff et al., 2016) and offering goods and services (Shakespeare-Finch & Obst, 2011; Southwick et al., 2016) that help solve practical problems. Generally, a statistically significant (p < 0.05) association was reported between providing instrumental social support and social well-being. The high significance recorded between the two variables could be attributed to the expectation that such support will be availed to older people at a later date. According to Chitaka (2017), the expectations of older people that the support they offered can be reciprocated when they need it is satisfying. instrumental, informational, and emotional. Instrumental support includes doing things for others (DeHoff et al., 2016) and offering goods and services (Shakespeare-Finch & Obst, 2011; Southwick et al., 2016) that help solve practical problems. Generally, a statistically significant (p < 0.05) association was reported between providing instrumental social support and social well-being. The high significance recorded between the two variables could be attributed to the expectation that such support will be availed to older people at a later date. According to Chitaka (2017), the expectations of older people that the support they offered can be reciprocated when they need it is satisfying. 4.1 Instrumental social support Social support is a multi-dimensional construct that Schaefer, Coyne, and Lazarus (1981) classified into three types: tangible/ Frontiers in Aging frontiersin.org 07 10.3389/fragi.2022.897508 Mbuthia et al. emotional support (χ2 = 32.54, p < 0.000)] was statistically significant. This was associated to the bond shared between the older person and a CNM as well as the opportunity to share knowledge and experience gained over the years. This finding agrees with Carstensen, Freedman, and Larson (2016), who noted that older people are in a unique position to serve as supporters and guides to CNMs, which allows the older people to experience fulfillment and purpose in their own lives. Similarly, in Kenya, Kimamo and Kariuki (2018) noted that older people often cheer their CNM, especially during major public holidays, by slaughtering chicken, sheep, or goats and joyfully sharing a meal with CNMs, while at the same time enquiring how each is doing in their personal lives. It is satisfying for older people when their CNMs are all cheerful in their home. emotional support (χ2 = 32.54, p < 0.000)] was statistically significant. This was associated to the bond shared between the older person and a CNM as well as the opportunity to share knowledge and experience gained over the years. This finding agrees with Carstensen, Freedman, and Larson (2016), who noted that older people are in a unique position to serve as supporters and guides to CNMs, which allows the older people to experience fulfillment and purpose in their own lives. Similarly, in Kenya, Kimamo and Kariuki (2018) noted that older people often cheer their CNM, especially during major public holidays, by slaughtering chicken, sheep, or goats and joyfully sharing a meal with CNMs, while at the same time enquiring how each is doing in their personal lives. It is satisfying for older people when their CNMs are all cheerful in their home. by the Blau (1968) tenet, which stresses that social actors engage in activities that entail some costs in order to obtain desired goals. The second instrumental item [providing care in sickness and satisfaction with providing instrumental support (χ2 = 13.26, p < 0.000)] was also significantly associated with satisfaction with providing instrumental support. This significant finding was attributed to the unintentionality of being/getting sick as well as how providing care perpetuates life and survival. 4.1 Instrumental social support According to Inagaki and Orehek (2017), caring for others is not only a good thing to do but also necessary for the survival of our species. However, these findings are not in agreement with Milne et al. (2014), who found that the likelihood of older people providing care to sick CNMs is high and has the potential to affect their own health problems, which is not satisfying. The significant results reported on the third item of the instrumental social support investigated [helping with household chores and satisfaction with providing instrumental support (χ2 = 4.48, p < 0.034)] could be attributed to this activity’s effect of keeping older people busy and active. According to Koblinsky et al. (2021), household chores may include activities such as cleaning, tidying, dusting, and home maintenance work like yard work and home repairs. These household chores keep older people active and serve as a low-risk form of beneficial exercise (Ward & McGarrigle, 2017; Koblinsky et al., 2021). The findings of this study corroborate those of Adjei and Brand (2018), which showed that older people were satisfied with their household chores because those chores were beneficial to their health and well-being. Similarly, the study by Crisp and Robinson (2010) also revealed that household chores enhanced the social well-being of older people and their relationships with CNMs. Showing interest in the personal life of a CNM was also significantly associated with satisfaction with providing emotional social support (χ2 = 40.44, p < 0.000). This significant association was attributed to CNMs and their personal concerns being important to older people. According to Vaillant (2012), older people are motivated to get involved in the lives of others. This interest in others is not limited to some aspects of life but to their great desire to lavish time, affection, information, concern, and resources on others (Erikson, 1994). These results resonate with Amati et al.’s (2018) findings that older people provide CNMs with emotional support by showing interest in their personal or family matters, serving as a resource pool to help the CNMs solve their problems. The older people find it satisfying to be part of the solution. 4.1 Instrumental social support The findings of this study, however, do not concur with those of Juma, Okeyo, and Kidenda (2004), which showed that showing interest in the life of CNMs, especially those under their care like orphans, cause older people to worry about who will continue providing care when they die. The significant (p < 0.05) association between lending household tools/items and satisfaction with providing instrumental support (χ2 = 8.90 p < 0.017) was attributed to the feelings of being helpful to others and meeting a need. Lending household tools/items is a form of assistance in social relationships that serves as a resource to meet needs (Amurwon et al., 2017). This study’s results conform with Wang et al.’s (2019) findings that social reciprocity takes place in close relationships like those of CNMs and older people, which eases access to what one has and the other does not in a time of need and is thus beneficial to social well-being. The third emotional item was doing or saying things that were kind or considerate and was significantly associated with satisfaction with providing emotional social support (χ2 = 67.41, p < 0.000). This significance was attributed to the effect generated when something kind was said to a CNM. When the CNM experienced pleasant emotions because of something the older person said or did, it resultantly produced a positive effect in that older person. Oerlemans, Bakker, and Veenhoven (2011) stated that people experience happiness as a pleasant and somewhat stimulated emotional state in their daily lives. The kind and considerate words or deeds from older people to CNMs stimulated pleasant emotions, which elicited satisfaction. These results are in agreement with a South African study by Makiwane (2010)thatshowed that older people are great encouragers who are happy about the success of the CNM and also offer support when things are not working well. Because the older people have amassed much experience, they can offer hope and wisdom to calm or cheer CNMs. 4.2 Emotional social support Emotional social support includes being there for someone (DeHoff et al., 2016) and behavior that fosters and expresses feelings of being loved, respected, and cared for (Shakespeare-Finch & Obst, 2011; Southwick et al., 2016). The first item of emotional social support [cheering up or helping a CNM feel better and satisfaction with providing Frontiers in Aging frontiersin.org 08 Mbuthia et al. Mbuthia et al. 10.3389/fragi.2022.897508 10.3389/fragi.2022.897508 Lastly, trusting CNMs to solve their problems was significantly associated with satisfaction with providing social support (χ2 = 41.83, p < 0.000). This was attributed to the presence of a trustworthy person and the relief obtained when a viable solution to an issue was provided by a CNM. The presence of trustworthy CNMs whom older people can confide in provides access to social support that addresses older people’s problems (Storchi, 2017). These findings contradict those of the Clough et al. (2007) study, which reported that older people expressed concerns about not knowing whom to trust to solve their problems. He argued that older people fear for their safety and lives when they do not know or have someone they can trust to solve their problems. They face challenges and lack trustworthy people, which creates worry that affects their social well-being. desire to give back. These findings are in tandem with the Michel et al. (2019) study on the roles of a grandmother in African societies. They concluded that older people provide invaluable and unbiased advice to every family member and are satisfied taking the role of an adviser. Similarly, Crisp and Robinson (2010) found that older people offer useful advice to CNMs, especially on navigating life’s challenges, which makes them feel good about themselves. desire to give back. These findings are in tandem with the Michel et al. (2019) study on the roles of a grandmother in African societies. They concluded that older people provide invaluable and unbiased advice to every family member and are satisfied taking the role of an adviser. Similarly, Crisp and Robinson (2010) found that older people offer useful advice to CNMs, especially on navigating life’s challenges, which makes them feel good about themselves. In the second item, agreeing with a CNM’s actions or thoughts was significantly associated with satisfaction with providing emotional support (χ2 = 41.55, p < 0.000). The significant result was associated with the CNM’s decision to act according to the older person’s expectations. 4.2 Emotional social support Otherwise, the older people could not accept the CNM’s decision to act contrary to what they had strongly suggested and advised. According to Noftle and Fleeson (2010), older adults are significantly more agreeable than younger adults. This is because they possess higher emotional control (Charles & Carstensen, 2007) to weigh the costs and benefits of the thoughts or actions presented to them, which corroborates this study. Generally, the highly significant association observed in the four items of emotional support and social well-being was attributed to the presence of CNMs who have a close relationship with the older person. According to Thomas, Liu, and Umberson (2017), closeness in family ties, particularly to adult children who give social support to their elderly parents, can have a major impact on well-being. Providing emotional support has been reported in prior studies as significantly associated with reduced stress (Shakespeare-Finch, & Obst, 2011), beneficial for the life satisfaction of aging parents (Silverstein, Cong, and Li, 2006) and has the potential to enhance satisfaction with life for older people regardless of gender (Liu et al., 2019). Similarly, Morelli et al. (2015) also observed that emotionally supportive individuals also report higher levels of satisfaction. Older people gave their CNM information to understand an issue, which was significantly associated with satisfaction with providing informational support (χ2 = 49.62, p < 0.000). This was attributed to the positive effects elicited when older people gave a CNM information to understand an issue, which was satisfying to them. Annear et al. (2017) indicated that older people have wisdom, values, and skills that can benefit the younger generation. They possess a wide array of information, knowledge, and expertise that they pass along to CNMs (Michel et al., 2019). These findings are in agreement with those of Chadha (1999), who found that CNMs consult older people on a majority of life’s major issues, which makes older people feel useful and valued. Amurwon, J., Hajdu, F., Yiga, D B., and Seeley, J. (2017). ‘Helping my neighbour is like giving a loan’–the role of social relations in chronic illness in rural Uganda. BMC Health Serv. Res. 17 (1), 1–12. 4.3 Informational social support inally, giving feedback to a CNM on an action that they wanted to take was significantly related to satisfaction with providing information support (χ2 = 39.913, p < 0.05). This was attributed to the feelings of being valued arising from being consulted on a matter and being given time to think things through and offer feedback. According to Storchi (2017), when contacted about a subject, older people contribute feedback to a CNM because they feel valued and significant. Therefore, they take time to find the best possible response in a situation because they are strategic communicators who want to make a difference in the lives of others (Carstensen et al., 2016). The findings of this study are in agreement with Carstensen et al. (2016), who reported that before giving feedback on any issue, older people tend to rely on their past experiences, which are directly linked to their emotions, short- and long-term goals, and desire to give back. Being in a position to provide feedback when requested contributes to their satisfaction with life. Informational social support is sharing knowledge and resources (DeHoff et al., 2016), provision of advice and guidance (Lu et al., 2016; Southwick et al., 2016), or advice regarding the environment (Shakespeare-Finch & Obst, 2011), which is intended to help individuals accomplish a task or cope with a difficult life situation. The results observed in the four items studied are significantly associated with satisfaction with providing information support. In the first item, providing helpful advice when needed to make an important decision was significantly associated with the older person’s satisfaction with providing information support (χ2 = 73.02, p < 0.000). This was attributed to the feeling of being knowledgeable on a matter and contributing to a CNM’s ability to make sound decisions. According to Carstensen et al. (2016), older people possess an almost mystical ability to assist CNMs to develop capabilities to deal with daily concerns. The authors argued that older people tend to rely on past experiences, which are directly linked to their emotions, temporary and permanent goals, and Overall, providing information support, as evidenced in the four items, significantly influenced satisfaction with providing information support. Having gone through different challenges Frontiers in Aging frontiersin.org 09 10.3389/fragi.2022.897508 Mbuthia et al. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability statement 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. The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author. Ethics statement and experiences, older people are able to understand the informational needs of a CNM and offer useful information (Heaney & Israel, 2008). This finding concurs with other studies that found that providing information support to others is linked to better health outcomes and well-being (Brown et al., 2003). Generally, providing social support in the three dimensions requires both cognitive judgments of satisfaction with social support and affective appraisals of moods and emotions about that support (Tov & Diener, 2013). The older people had to consider the help they offered to CNMs and how doing so affected their moods and emotions. The studies involving human participants were reviewed and approved by the Kenyatta University Institutional Ethical Review Committee, Kenya, approval number PKU/2235/11379, and the National Commission for Science, Technology and Innovation (NACOSTI) Kenya, approval number NACOSTI/P/21/11012. The patients/participants provided their written informed consent to participate in this study. Author contributions The findings of this study contribute significantly to the limited research evidence on the relationship between social support in the three categories and social well-being among older individuals in Kenya. The strengths of this study lie in its use of a randomly selected sample of older people, a high response rate, and detailed information on provided social support. The use of mixed methods—quantitative data from the questionnaire and qualitative data from focus group discussions—allowed the study to examine the association between social support and social well-being and obtain a deeper understanding through narratives, revealing subjective appraisals of providing support. The potential in the data lies in its scope and detail of the social support variables provided across the three domains. KM conceived the idea for the study, collected data, analyzed, and developed the manuscript. SM and GO revised the draft manuscript critically for important intellectual content. All authors approved the final manuscript to be published. Acknowledgments The study acknowledges the study participants who provided important data for this study. The main shortcoming of this study was that the data are cross- sectional, and thus, the causal ordering of providing social support and social well-being is precluded. 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Joint genome-wide association study of progressive supranuclear palsy identifies novel susceptibility loci and genetic correlation to neurodegenerative diseases
Molecular neurodegeneration
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Permalink https://escholarship.org/uc/item/79n4d1xn ISSN 1750-1326 Authors Chen, Jason A Chen, Zhongbo Won, Hyejung et al. Publication Date 2018-08-01 DOI 10.1186/s13024-018-0270-8 Peer reviewed UCLA UCLA Previously Published Works UCLA UCLA Previously Published Works UCLA Previously Published Works Title Joint genome-wide association study of progressive supranuclear palsy identifies novel susceptibility loci and genetic correlation to neurodegenerative diseases. Joint genome-wide association study of progressive supranuclear palsy identifies novel susceptibility loci and genetic correlation to neurodegenerative diseases. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Progressive supranuclear palsy (PSP) is a rare neurodegenerative disease for which the genetic contribution is incompletely understood. Methods: We conducted a joint analysis of 5,523,934 imputed SNPs in two newly-genotyped progressive supranuclear palsy cohorts, primarily derived from two clinical trials (Allon davunetide and NNIPPS riluzole trials in PSP) and a previously published genome-wide association study (GWAS), in total comprising 1646 cases and 10,662 controls of European ancestry. Results: We identified 5 associated loci at a genome-wide significance threshold P < 5 × 10−8, including replication of 3 loci from previous studies and 2 novel loci at 6p21.1 and 12p12.1 (near RUNX2 and SLCO1A2, respectively). At the 17q21.31 locus, stepwise regression analysis confirmed the presence of multiple independent loci (localized near MAPT and KANSL1). An additional 4 loci were highly suggestive of association (P < 1 × 10−6). We analyzed the genetic correlation with multiple neurodegenerative diseases, and found that PSP had shared polygenic heritability with Parkinson’s disease and amyotrophic lateral sclerosis. Conclusions: In total, we identified 6 additional significant or suggestive SNP associations with PSP, and discovered genetic overlap with other neurodegenerative diseases. These findings clarify the pathogenesis and genetic architecture of PSP. Keywords: Genome-wide association study, Progressive supranuclear palsy, Neurodegeneration dementia, characterized by pathological tau aggregation and a clinical syndrome of postural instability, falls, and supra- nuclear ophthalmoplegia [1]. It shares symptomatic and neuropathologic overlap with a large group of diseases, that are collectivity known as “tauopathies” due to characteristic tau deposits; however, compared to these diseases, PSP appears to be more clinically, neuropathologically, and genet- ically homogenous [2–4]. Notably, the clinical syndrome has high correlation with the neuropathology [5]. These charac- teristics have thrust PSP into a central role for studying Powered by the California Digital Library University of California eScholarship.org Chen et al. Molecular Neurodegeneration (2018) 13:41 https://doi.org/10.1186/s13024-018-0270-8 RESEARCH ARTICLE Open Access Joint genome-wide association study of progressive supranuclear palsy identifies novel susceptibility loci and genetic correlation to neurodegenerative diseases Jason A. Chen1, Zhongbo Chen2, Hyejung Won3, Alden Y. Huang1, Jennifer K. Lowe3, Kevin Wojta4, Jennifer S. Yokoyama5, Gilbert Bensimon6,7,8, P. Nigel Leigh9, Christine Payan6,7, Aleksey Shatunov2, Ashley R. Jones2, Cathryn M. Lewis10, Panagiotis Deloukas11, Philippe Amouyel12, Christophe Tzourio13, Jean-Francois Dartigues13, Albert Ludolph14, Adam L. Boxer5, Jeff M. Bronstein3, Ammar Al-Chalabi2, Daniel H. Geschwind1,3 and Giovanni Coppola1,3,4,15* Open Access Joint genome-wide association study of progressive supranuclear palsy identifies novel susceptibility loci and genetic correlation to neurodegenerative diseases Jason A. Chen1, Zhongbo Chen2, Hyejung Won3, Alden Y. Huang1, Jennifer K. Lowe3, Kevin Wojta4, Jennifer S. Yokoyama5, Gilbert Bensimon6,7,8, P. Nigel Leigh9, Christine Payan6,7, Aleksey Shatunov2, Ashley R. Jones2, Cathryn M. Lewis10, Panagiotis Deloukas11, Philippe Amouyel12, Christophe Tzourio13, Jean-Francois Dartigues13, Albert Ludolph14, Adam L. Boxer5, Jeff M. Bronstein3, Ammar Al-Chalabi2, Daniel H. Geschwind1,3 and Giovanni Coppola1,3,4,15* * Correspondence: gcoppola@ucla.edu 1Interdepartmental Program in Bioinformatics, University of California, Los Angeles, CA 90095, USA 3Program in Neurogenetics, Department of Neurology and Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA Full list of author information is available at the end of the article Background Tau pathology is a prominent hallmark of neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotem- poral dementia (FTD). Progressive supranuclear palsy (PSP) is a relatively pure tauopathy associated with parkinsonism - * Correspondence: gcoppola@ucla.edu 1Interdepartmental Program in Bioinformatics, University of California, Los Angeles, CA 90095, USA 3Program in Neurogenetics, Department of Neurology and Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA Full list of author information is available at the end of the article Page 2 of 11 Chen et al. Molecular Neurodegeneration (2018) 13:41 subjects. For the Illumina HumanQuad 660 W Array (Hoglinger et al. study), we used phs000103.v1.p1 “Genome-- Wide Association Studies of Prematurity and Its Complica- tions”, phs000289.v1.p1 “National Human Genome Research Institute (NHGRI) GENEVA Genome-Wide Association Study of Venous Thrombosis”, phs000188.v1.p1 “Vanderbilt Genome-Electronic Records (VGER) Project: QRS Dur- ation”, phs000203.v1.p1 “A Genome-Wide Association Study of Peripheral Arterial Disease”, phs000237.v1.p1 “Northwest- ern NUgene Project: Type 2 Diabetes”, phs000234.v1.p1 “Group Health/UW Aging and Dementia eMERGE study”, and phs000170.v1.p1 “A Genome-Wide Association Study on Cataract and HDL in the Personalized Medicine Research Project Cohort”. For the Illumina HumanOmni 2.5 Array (UCLA – this study, and NNIPPS study), we used phs000371.v1.p1 “Genetic Modifiers of Huntington’s Dis- ease”, phs000429.v1.p1 “NEI Age-Related Eye Disease Study (AREDS) - Genetic Variation in Refractive Error Substudy”, and phs000421.v1.p1 “A Genome-Wide Association Study of Fuchs’ Endothelial Corneal Dystrophy (FECD)”. For the Illumina HumanCore Array (UCLA – this study), we used the WTCCC2 cohort, which was typed on the related Illu- mina OmniExpress Array. Subjects with an ascertained phenotype (e.g., disease) were removed. More detailed in- formation regarding these datasets is available in Add- itional file 1: Table S1. neurodegeneration, enabling clinical trials of a relatively homogenous patient population with potentially more uni- form response to treatment. Therefore, PSP has become a target of intense clinical research [6, 7]. While the disease shares neuropathological overlap with other tauopathies, the polygenic genetic correlation with other neurodegenerative diseases remains to be clarified. The major known genetic risk factor is an extended H1 haplotype on chromosome 17q21.31, which includes MAPT (the gene encoding the tau protein), and is homozygous in almost all PSP patients [2]. Other risk factors identified include genome wide significant associ- ations at loci near MAPT, MOBP, STX6, and EIF2AK3, suggesting a strong contribution of common variation in its genetic architecture [8]. Data preprocessing p p g Genotypes for all datasets were converted to the forward strand, and converted into coordinates based on the hg19 reference sequence using UCSC liftOver [10]. The genotypes were then merged and pre-processed according to platform. Determination of cryptic relatedness (pairwise proportion IBD, PI-HAT > 0.2), sample missingness (> 0.05), genotype missingness (> 0.05), Hardy-Weinberg equilibrium p-value (< 10−5), and sex-matching was performed in PLINK v1.90b3.28 [11] and used to quality-control (QC) samples using standard parameters [12]. Ancestry was predicted by multidimensional scaling based on raw Hamming distances, implemented in PLINK. Only samples of presumed European ancestry that clustered with known Europeans from the HapMap3 cohort [13] were included. Preprocessing steps are further elaborated in Additional file 2: Figure S1. Imputation Imputation was performed separately for each geno- typing platform using the IMPUTE v2.3.2 algorithm [14]. Prephasing of chromosomes using the Segmented HAPlotype Estimation & Imputation Tool (SHAPEIT) v2.r837 was performed as previously described [15, 16]. IMPUTE2 was run on the prephased haplotypes using the 1000 Genomes Project Phase 3 reference in non-overlapping 5 megabase chunks with a 250 kilobase buffer and an effect- ive population size of 20,000. Imputed variants with an Genotyping Genotyping in the UCLA study cohort was performed as a prelude to whole-genome sequencing, and was per- formed by Illumina (using the Illumina HumanOmni 2.5 Array) and the New York Genome Center (using the Illumina HumanCore Array). Genotyping calls were made using the Illumina GenomeStudio software. Methods Cohort Three cohorts of primarily European ancestry were in- cluded in the study – “UCLA”, a combination of 349 PSP patients and 130 controls from the UCSF Memory and Aging Center [2, 9] and the Allon Therapeutics Davunetide trial [6]; “NNIPPS”, a group of 341 PSP pa- tients from the Neuroprotection and Natural History in Parkinson Plus Syndromes (NNIPPS) trial [7] and the Blood Brain Barrier in Parkinson Plus syndromes (BBBIPPS) study; and “Hoglinger”, 1112 PSP patients from a previously published GWAS [8]. The UCLA co- hort was divided into two, because of differences in genotyping platform: “UCLA Omni 2.5” and “UCLA HumanCore”. Further details are available in the Supple- mentary Methods. Background We reasoned that the inclu- sion of additional cases and controls could increase the statistical power for genome-wide association, potentially yielding novel loci that could provide insight into the molecular mechanisms of PSP and other more common tauopathies. Credible set of causal variants at PSP GWAS loci Credible set of causal variants at PSP GWAS loci A credible set (potential causal variants) was identified at each of total of seven genome-wide significant loci identified in this study using the CAusal Variants Identi- fication in Associated Regions (CAVIAR) software pack- age [28]. Because of the extended linkage disequilibrium patterns in the chromosome 17q21.31 haplotype region, causal variants were not identified at this associated locus. Within each of the selected loci, the SNP with the minimum joint association p-value was chosen as the index SNP, and variants with p-value < 10−5 and in LD (r2 > 0.6) with the index SNP were input into CAVIAR. The CAVIAR-identified credible set contains potential causal variants (with a confidence level of 95% under the statistical model) that could explain the association at each locus. Prediction of gene expression differences associated with PSP-associated SNPs Genetic associations with PSP may be due to genetic control of gene expression. We used TWAS to pre- dict differential gene expression in PSP from the joint analysis summary statistics, integrating paired geno- typing and gene expression data from the GTEx Consortium [27]. Correcting for approximately 5000 effective independent tests per brain region (taking into account 5483 genes with significantly heritable weights and the interdependence of gene expression, particularly across tissues), the significance threshold was set at P < 1 × 10−5. Public datasets Genotypes from the Hoglinger et al. GWAS [8] (cases only – no controls) were obtained from the NIAGADS database. Out-of-sample controls were obtained from dbGAP Authorized Access to match each genotyping platform. In total, for the HumanOmni 2.5 M platform, we obtained 2364 subjects; for the OmniExpress platform, 870 subjects; and for the HumanQuad 660 W platform, 8756 Chen et al. Molecular Neurodegeneration (2018) 13:41 Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 3 of 11 calculated and used as covariates for restricted max- imum likelihood (REML) analysis. imputation genotype probability < 0.9, missingness > 0.05, or minor allele frequency < 0.01 were removed, and genotypes across platforms were merged. Cryptic relatedness across co- horts was assessed, and related/duplicated samples were removed. Association Association was performed using a linear mixed model to correct for population structure, using BOLT-LMM [17]. The genotyping platform was used as a categorical covariate. The standard infinitesimal model p-values were chosen for downstream anaylsis. Odds ratios were calculated as exp. (beta). Because some of the individual cohort sizes violate the large sample size assumptions of BOLT-LMM, odds ra- tios for association (for individual cohorts) were computed using a logistic regression model in PLINK, using the first 5 eigenvectors, derived from Principal Component Analysis (PCA), as covariates. Power calculations were performed using the Genetic Power Calculator [18], assuming a variant with risk allele frequency of 0.5 and relative risk of 1.3 in an additive genetic model, a disease with a prevalence of 10 in 100,000, and a p-value threshold of 5 × 10−8, using a geno- typic, 2 df case-control test. QQ and Manhattan plots were constructed using the R package “qqman” [19]. Forest plots were constructed using the R package “metafor” [20]. The genomic inflation factor λ was computed with PLINK. Correction of the genomic inflation factor to an equivalent sample size of 1000 cases and 1000 controls was performed as previously described [21]. To control for the extended haplotype on chr17q21 and to identify independent associ- ation signals, we performed association as before, but includ- ing the haplotype (tagged by the SNP rs1560310) [22] as a covariate. Proportion variance in liability explained p y p The explained variance in liability at each of the genome-wide significant loci was calculated according to the method of So et al., which requires the frequency of the risk allele, the relative risk of the heterozygous geno- type, the relative risk of the homozygous risk genotype, and the prevalence of the disease in the population [23]. The allele frequencies were calculated from the control population of the joint genotyping cohort. Relative risks were approximated with the corresponding odds ratios, which converges to relative risk when the prevalence of disease is rare. Genotypic odds ratios were estimated by assuming an additive model. The prevalence of PSP was estimated at 6.5 per 100,000 in accordance with prior epidemiological studies [24, 25]. The genome-wide poly- genic variance in liability explained was calculated using GCTA v1.24.7 [26]. The genetic relationship matrix was calculated chromosome-by-chromosome and then re-combined. The first 5 principal components were Genetic variation can result in changes to the coding se- quence of a gene (e.g., nonsense and missense variants) or can regulate the gene’s expression (e.g., by affecting transcription factor binding in promoter or enhancer regions). We first identified credible SNPs as “func- tional” (stopgain variant, frameshift variant, splice donor variant, nonsense-mediated decay transcript variant, or missense variant). Of the remaining credible SNPs, we identified those in the promoter region of a gene, de- fined as the range 2 kb upstream to 1 kb downstream relative to the transcription start site (TSS). Finally, the remaining credible SNPs were considered possible regu- latory variants and tested for short- or long- range inter- action with other regions of chromatin to identify potential downstream target genes. The interactions were determined by Hi-C experiments in IMR90 and embryonic stem cells from public data [29, 30], and fetal brain germinal zone (ventricular and subventricular Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 4 of 11 Chen et al. Molecular Neurodegeneration (2018) 13:41 zone) and cortical plate (intermediate zone and marginal zone) from our group [31]. zone) and cortical plate (intermediate zone and marginal zone) from our group [31]. To increase statistical power, we combined all three cohorts in a joint analysis framework. We estimated that this combined cohort had 90% power to detect associ- ation of a variant with allele frequency of 0.5 and relative risk of 1.3. Results We analyzed subjects from three GWAS cohorts, including 1) a multi-center cohort [2, 6] in whom we performed genotyping using the Illumina HumanOmni2.5 BeadChip and the Illumina HumanCore BeadChip (“UCLA”); 2) pa- tients from centers in France, Germany, and the United Kingdom as part of the Neuroprotection and Natural History in Parkinson Plus Syndromes (NNIPPS) study, a double-blind randomized placebo-controlled clinical trial of riluzole [7], genotyped with the Illumina HumanOmni2.5 BeadChip (“NNIPPS”), and 3) a cohort of autopsy-proven cases from a previously published [8] GWAS (“Hoglinger”). A more detailed description of the cohorts is provided in Additional file 1: Table S1. We combined each cohort with platform-matched, out-of-sample controls from dbGAP (Additional file 1: Table S1). Stringent QC – excluding SNPs that had low genotype call rates (< 0.95) or did not follow Hardy-Weinberg equilibrium, and excluding subjects with low sample call rate (< 0.95), non-European ancestry, incompatible sex, cryptic relatedness, or duplication across cohorts (Additional file 2: Figure S1) – was applied to each cohort (including platform-matched controls). We then im- puted variants implementing IMPUTE2 [14] using the 1000 Genomes Phase 3 Reference Panel to estimate geno- types at more than 77,000,000 SNPs. Imputed variants with imputation quality scores (r2 < 0.9) or low minor allele frequency (< 0.01) were filtered, and genotypes across all cohorts were combined in a joint analysis. In total, we ex- amined 6,419,662 SNPs in 1646 PSP cases and 10,662 controls. The results of the joint analysis genome-wide association are shown in Fig. 1 and Additional file 2: Figure S4. SNPs at 5 loci, in cytobands 17q21.31 (in an extended haplotype con- taining MAPT, lead SNP rs71920662, odds ratio OR = 0.19, p = 3.9 × 10−113), 3p22.1 (within MOBP, rs10675541, OR = 0.71, p = 7.2 × 10−19), 1q25.3 (within STX6, rs57113693, OR = 1.3, p = 8.7 × 10−16), 6p21.1 (within RUNX2, rs35740963, OR = 0.77, p = 1.8 × 10−8), and 12p12.1 (within SLCO1A2, rs7966334, OR = 1.5, p = 3.2 × 10−8), reached genome-wide significance (p < 5 × 10−8) (Additional file 1: Table S2, Additional file 2: Figure S5). An additional SNP reported in a previous GWAS [8], rs7571971, was also analyzed. Although this SNP did not reach genome-wide significance in the joint analysis (OR = 1.18, p = 2.7 × 10−5), the direction of the asso- ciation was consistent with the previous association in each cohort. Proportion variance in liability explained For a cohort of the sample size of that in a previous PSP GWAS from Hoglinger et al., the power to detect association was only 33%. In the primary analysis, we assessed the genome-wide association between the genotype at each SNP and case-control status using a linear mixed model to correct for population stratifica- tion. The genomic inflation factor λ for the joint analysis was 1.05; for the UCLA-Omni2.5, UCLA-HumanCore, NNIPPS, and Hoglinger cohorts, λ was 1.03, 1.02, 1.11, and 1.11, respectively (Fig. 1, Additional file 2: Figure S2 and S3). We considered the joint inflation factor to be acceptable in the setting of a relatively large joint ana- lysis sample size [33]. Scaled for sample size, the ad- justed genomic inflation factor λ1000 was 1.02. Data availability Full and imputed genotyping results from the UCLA and NNIPPS cohorts will be made available on the NIAGADS database. Genetic correlation with neurodegenerative diseases g Genetic correlation was assessed from GWAS summary sta- tistics using the Linkage Disequilibrium Score Regression method (LDSC) [32]. Summary statistics were filtered by only considering SNPs that overlap with the HapMap3 refer- ence panel. Refer to the Supplementary Methods for further details. Results 1 Genome-wide SNP association in the joint analysis. a Manhattan plot indicating the SNP association P values. The vertical axis displays the strength of association (−log10 P value) as a function of genomic position, with alternating colors for sequential chromosomes. Genome-wide significant and suggestive loci are labeled with the nearest gene symbol. The thresholds for significant (P < 5 × 10−8, red horizontal line) and suggestive (P < 1 × 10−6, blue horizontal line) associations are shown. b-d Quantile-quantile plots for: b all SNPs, including the strongly associated extended haplotype on chromosome 17; c SNPs excluding chromosome 17; and d SNPs excluding genome-wide significant and suggestive loci. The 95% confidence interval for the expected distribution of p-values is shaded To further understand how variation at each of the loci contributes to disease risk, we assessed the functional con- sequences of significant SNPs. We first identified a set of potential causal SNPs using the CAVIAR method, which identifies a “credible set” of SNPs that encompasses those likely to be causal [28]. The 17q21.31 locus was excluded from the analysis because of its unusual, long-range link- age disequilibrium pattern. In some loci, potentially causal coding variants were identified (in genome-wide signifi- cant loci, at 6p21.1, in RUNX2, and at 12p12.1, in SLCO1A2; and in suggestive loci, at 8q24.21, in ASAP1, and at 12q13.13, in AMHR2; Additional file 1: Table S5). Other SNPs in the credible set fell within regulatory re- gions; we identified the gene associated with each SNP using data from Hi-C experiments, mapping chromosome conformation patterns on a genome-wide scale from four human cell types (IMR-90 fetal lung fibroblasts, embry- onic stem cells, fetal brain, and fetal brain germinal zone). Each potential regulatory SNP in the credible set was then associated with genes in close proximity by chromosomal conformation, yielding potential downstream causal genes (Additional file 1: Table S5). entire set of genotyped SNPs explains 9.4 ± 0.8% (estimate ±standard error) of the variance on the liability scale, sug- gesting that many loci are yet to be found. The association between PSP and the chr17q21 haplo- type (H1/H2) has been widely characterized, but inde- pendent SNPs in the chr17q21 region may also contribute to disease susceptibility. To test this, we per- formed linear regression, taking haplotype as a covariate. Results In order to decrease the likelihood that the results were influenced by population stratification, we assessed association at the loci in each of the study cohorts (Fig. 2). Associations at the lead SNPs in each of the regions were consistent across the three most well-powered study cohorts (Hoglinger, NNIPPS, and UCLA Omni2.5) while in general, the HumanCore subset of the UCLA cohort was underpowered to detect association. An add- itional 4 loci demonstrated suggestive association (1 × 10−6 < P < 5 × 10−8), in 1q41 (intergenic, near DUSP10, rs12125383, OR = 1.28, p = 5.3 × 10−8), 12q13.13 (within SP1, rs147124286, OR = 0.74, p = 4.1 × 10−7), 8q24.21 (within ASAP1, rs2045091, OR = 1.25, p = 4.7 × 10−7), and 1p22.3 (near WDR63 and MIR4423, rs114573015, OR = 2.1, p = 5.9 × 10−7) (Additional file 1: Table S3). Overall, the genome-wide significant loci explained a combined 5.9% of the variance in heritable liability of PSP (Additional file 1: Table S4). The locus tagging the chr17q21 haplotype sur- rounding MAPT contributed the majority (5.0%), while new loci contributed an additional 0.2% of the total liability. Using a polygenic model implemented in GCTA [26], the Association was initially performed using the 616 cases represented from the UCLA and NNIPPS cohorts. Genome-wide significant association was detected at loci near MAPT and largely corresponded to the haplotype region (lead SNP rs79730878, p = 5.4 × 10−45; Additional file 1: Table S2). Other top associations were found at loci near MOBP, STX6, SEMA4D, DDX27, and SP1, though these did not reach genome-wide significance. Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 5 of 11 Fig. 1 Genome-wide SNP association in the joint analysis. a Manhattan plot indicating the SNP association P values. The vertical axis displays the strength of association (−log10 P value) as a function of genomic position, with alternating colors for sequential chromosomes. Genome-wide significant and suggestive loci are labeled with the nearest gene symbol. The thresholds for significant (P < 5 × 10−8, red horizontal line) and suggestive (P < 1 × 10−6, blue horizontal line) associations are shown. b-d Quantile-quantile plots for: b all SNPs, including the strongly associated extended haplotype on chromosome 17; c SNPs excluding chromosome 17; and d SNPs excluding genome-wide significant and suggestive loci. The 95% confidence interval for the expected distribution of p-values is shaded Fig. Results At a threshold of P < 1 × 10−5, we identified a number of genes that were called as differentially expressed (Additional file 1: Table S6). As expected due to the length and lack of recombination in the region, most of these genes (17) clustered around the chromo- some 17 haplotype. Notably, MAPT (within the associ- ated 17q21.31 locus) was among the genes predicted to be differentially expressed, as well as STX6 (within the associated 1q24 locus), SP1 (within the suggestive 12q13.13 locus), SKIV2L (within 6p21.33, nearby the as- sociated 6p21.1 locus), and RPSA (within the associated 3p22.1 locus). Other genes that were pinpointed outside of association regions were CEP57 (in 11q21) and RPS6KL1 (in 14q24.3). many common neurodegenerative diseases. Altogether, from a joint analysis of three disease cohorts, we have found 2 novel genome-wide significant susceptibility loci in PSP and replicated 3 previously reported loci. Of the loci identified in this study, three (within MAPT, MOBP, and STX6) were reported significant in a previous GWAS [8]. In the MAPT region, a third independent association was identified, also in MAPT intron 1, speculatively sug- gesting important regulatory functions in this region; however, no effects in differential expression have been uncovered. Overall, the mechanisms of the MAPT asso- ciations have been unclear. The larger H1/H2 haplotype appears to affect splicing at MAPT exon 3 but not over- all tau expression [34]; while other MAPT variants, such as rs242557, may affect tau expression in some tissues [35], the effect is not robust in brain tissue. The additional association identified here may provide an or- thogonal point of investigation into this curious region. An additional locus near the EIF2AK3 gene (encoding PERK, a key component of the unfolded protein re- sponse) was also previously identified; however, the re- ported SNP did not reach genome-wide significance in this joint analysis or in the new “Hoglinger” cohort (using different controls). The strong neuropathological overlap of PSP with other tauopathies suggests that genetic overlap may exist. Using the LDSC software [32], we assessed genetic overlap of PSP with other neurodegenerative diseases, including AD, behavioral variant FTD (bvFTD), Parkin- son’s disease (PD), and amyotrophic lateral sclerosis (ALS), by using GWAS summary statistics. Results Additionally, we identified subjects that were homozy- gous for the risk allele (H1/H1), and performed associ- ation in this subset of patients. Both approaches identified similar independent associations from the H1 haplotype in the 17q21.31 region, with the most signifi- cant SNPs at rs8078967 (P = 1.9 × 10−14) and rs9904290 (P = 8.9 × 10−12) in the haplotype-regressed and H1/H1 only datasets, respectively (Additional file 2: Figure S5). These SNPs did not appear to be in strong linkage dis- equilibrium with a previously reported SNP association, rs242557 [8] (r2 = 0.008 and 0.007 in the 1000 Genomes Project data – EUR super-population, respectively) that was filtered from this dataset in variant QC; however, they were highly correlated with each other (r2 = 0.996). Additionally, both variants and rs242557 are within the first intron of the MAPT gene. To supplement the mapping information from Hi-C, we also identified the functional consequences of GWAS Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 6 of 11 Fig. 2 (See legend on next page.) Fig. 2 (See legend on next page.) Chen et al. Molecular Neurodegeneration (2018) 13:41 Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 7 of 11 (See figure on previous page.) Fig. 2 Forest plots showing association across each individual cohort for selected SNPs. A total of six genome-wide significant loci were identified, with representative SNPs: a rs71920662 in 17q21.31, near MAPT; b rs57113693 in 1q25.3, near STX6; c rs10675541 in 3p22.1, near MOBP; d rs35740963 in 6p21.1, near RUNX2; and e rs7966334 in 12p12.1, near SLCO1A2. An additional four suggestive loci were also identified: f rs12125383 in 1q41, near DUSP10 in an intergenic region; g rs147124286 in 12q13.13, near SP1; h rs2045091 in 8q24.21, near ASAP1; and i rs114573015 in 1p22.3, near WDR63. j Additionally, a previously reported GWAS SNP rs7571971 in 2p11.2, near EIF2AK3, was not identified as genome-wide significant in the joint analysis hits using the TWAS method to predict genes that may be affected by risk alleles [27]. TWAS estimates gene ex- pression values using paired reference transcriptome/ genotyping datasets (e.g., for expression quantitative trait loci - eQTL studies) and genotype information from summary statistics, and predicts differential expression between cases and controls. Using reference data from the GTEx Consortium, TWAS predicted the effect of gene expression from the risk haplotypes in multiple tissues. Results As controls, we included summary statistics from GWAS for heritable, non-neurodegenerative diseases of brain (schizophrenia and bipolar disorder), a quantitative trait (height), and a non-brain disease (type 2 diabetes) (for further details, refer to the Supplementary Methods). Each of these traits was shown to be heritable. Statistically significant genetic correlations were identified for PD (P = 9.7 × 10−5) and ALS (P = 1.8 × 10−3), but not for non-neurodegenerative disease control GWAS (Fig. 3). We also identified 2 novel genome-wide significant susceptibility loci at 6p21.1 and 12p12.1 (near RUNX2 and SLCO1A2, respectively). At 6p21.1, we identified a lead SNP as well as several coding SNPs in the credible set within RUNX2, a gene thought to be a transcriptional factor involved in regulation of osteoblastic differentiation [36]. While seemingly unrelated to PSP, a curious number of neurodegeneration-related genes are also involved in bone diseases (e.g. TREM2, which has been linked to AD and Nasu-Hakola disease [37, 38], and VCP, linked to amyotrophic lateral sclerosis and Paget’s disease of bone [39, 40]). At 12p12.1, we identified a lead SNP and credible set coding SNPs within SLCO1A2, a transporter present (among other places) at the blood-brain barrier, where it regulates solute trafficking [41]. An additional four loci (near the genes DUSP10, SP1, ASAP1, and WDR63/ MIR4423) were suggestive of association, but did not reach genome-wide significance. While this study raises the possi- bility of involvement of these genes in PSP pathogenesis, further fine-mapping and functional studies are needed to confirm their possible roles. Discussion Phenotypes that share a common polygenic background are positively correlated 1 0.5 0 0.5 1 PSP_Chen ALS_ProjectMinE AD_IGAP bvFTD_Ferrari PD_23&Me_meta SCZ_PGC BIP_PGC Height_GIANT T2D_DIAGRAM PSP_Chen ALS_ProjectMinE AD_IGAP bvFTD_Ferrari PD_23&Me_meta SCZ_PGC BIP_PGC Height_GIANT T2D_DIAGRAM 1 (0) 0.52 (0.002) 0.12 (0.4) 0.6 (0.2) 0.4 (1e 04) 0.18 (0.05) 0.06 (0.6) 0.02 (0.9) 0.053 (0.6) 0.52 (0.002) 1 (0) 0.15 (0.5) 0.45 (0.5) 0.11 (0.5) 0.32 (0.007) 0.083 (0.5) 0.29 (0.2) 0.003 (1) 0.12 (0.4) 0.15 (0.5) 1 (0) 0.29 (0.5) 0.077 (0.5) 0.039 (0.6) 0.023 (0.8) 0.055 (0.8) 0.016 (0.9) 0.6 (0.2) 0.45 (0.5) 0.29 (0.5) 1 (0) 0.31 (0.4) 0.054 (0.8) 0.16 (0.6) 0.11 (0.8) 0.034 (0.9) 0.4 (1e 04) 0.11 (0.5) 0.077 (0.5) 0.31 (0.4) 1 (0) 0.079 (0.3) 0.12 (0.1) 0.16 (0.3) 0.034 (0.6) 0.18 (0.05) 0.32 (0.007) 0.039 (0.6) 0.054 (0.8) 0.079 (0.3) 1 (0) 0.66 (2e 27) 0.062 (0.6) 0.089 (0.2) 0.06 (0.6) 0.083 (0.5) 0.023 (0.8) 0.16 (0.6) 0.12 (0.1) 0.66 (2e 27) 1 (0) 0.009 (1) 0.12 (0.1) 0.02 (0.9) 0.29 (0.2) 0.055 (0.8) 0.11 (0.8) 0.16 (0.3) 0.062 (0.6) 0.009 (1) 1 (0) 0.1 (0.4) 0.053 (0.6) 0.003 (1) 0.016 (0.9) 0.034 (0.9) 0.034 (0.6) 0.089 (0.2) 0.12 (0.1) 0.1 (0.4) 1 (0) Fig. 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy, ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease), calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Phenotypes that share a common polygenic background are positively correlated Fig. 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy, ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease), calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Phenotypes that share a common polygenic background are positively correlated Fig. Discussion As a prototypical tauopathy, insight into PSP susceptibility alleles can help to illuminate the downstream molecular ef- fects of tau pathology, which is a major component of Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 8 of 11 1 0.5 0 0.5 1 PSP_Chen ALS_ProjectMinE AD_IGAP bvFTD_Ferrari PD_23&Me_meta SCZ_PGC BIP_PGC Height_GIANT T2D_DIAGRAM PSP_Chen ALS_ProjectMinE AD_IGAP bvFTD_Ferrari PD_23&Me_meta SCZ_PGC BIP_PGC Height_GIANT T2D_DIAGRAM 1 (0) 0.52 (0.002) 0.12 (0.4) 0.6 (0.2) 0.4 (1e 04) 0.18 (0.05) 0.06 (0.6) 0.02 (0.9) 0.053 (0.6) 0.52 (0.002) 1 (0) 0.15 (0.5) 0.45 (0.5) 0.11 (0.5) 0.32 (0.007) 0.083 (0.5) 0.29 (0.2) 0.003 (1) 0.12 (0.4) 0.15 (0.5) 1 (0) 0.29 (0.5) 0.077 (0.5) 0.039 (0.6) 0.023 (0.8) 0.055 (0.8) 0.016 (0.9) 0.6 (0.2) 0.45 (0.5) 0.29 (0.5) 1 (0) 0.31 (0.4) 0.054 (0.8) 0.16 (0.6) 0.11 (0.8) 0.034 (0.9) 0.4 (1e 04) 0.11 (0.5) 0.077 (0.5) 0.31 (0.4) 1 (0) 0.079 (0.3) 0.12 (0.1) 0.16 (0.3) 0.034 (0.6) 0.18 (0.05) 0.32 (0.007) 0.039 (0.6) 0.054 (0.8) 0.079 (0.3) 1 (0) 0.66 (2e 27) 0.062 (0.6) 0.089 (0.2) 0.06 (0.6) 0.083 (0.5) 0.023 (0.8) 0.16 (0.6) 0.12 (0.1) 0.66 (2e 27) 1 (0) 0.009 (1) 0.12 (0.1) 0.02 (0.9) 0.29 (0.2) 0.055 (0.8) 0.11 (0.8) 0.16 (0.3) 0.062 (0.6) 0.009 (1) 1 (0) 0.1 (0.4) 0.053 (0.6) 0.003 (1) 0.016 (0.9) 0.034 (0.9) 0.034 (0.6) 0.089 (0.2) 0.12 (0.1) 0.1 (0.4) 1 (0) Fig. 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy, ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease), calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Discussion 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy, ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease), calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Phenotypes that share a common polygenic background are positively correlated Aside from identifying additional associated loci and highlighting potential PSP susceptibility genes, we ana- lyzed the polygenic overlap between neurodegenerative diseases, identifying shared heritability with PD and ALS. Curiously, these diseases do not have predominant tau neuropathology, as PSP and other tauopathies do. Typic- ally, PD is associated with aggregation of α-synuclein, and ALS with aggregation of TDP43 and other proteins, while tau pathology is prominent in AD. However, there are known shared genetic risk factors among these diseases. The 17q21.31 haplotype is highly associated with PD, in the same direction as in PSP [44], and SNPs near the MOBP gene have been recently associated with ALS [45]. Our results indicate the existence of common neurode- generative disease pathways even across traditional protein aggregate-based subdivisions, and could potentially lead to effective treatment strategies. Our results also implicate possible alternative causal genes in previously reported genome-wide significant loci. At 3p22.1, the gene closest to the GWAS lead SNP was reported as MOBP. This locus has previously been implicated in differential expression of the SLC25A38/ appoptosin gene, which may regulate tau cleavage [42]. Using Hi-C, we have identified chromatin interactions with MYRIP and EIF1B that could also explain this asso- ciation. Similarly, at 1q25.3, the gene closest to the GWAS lead SNP was STX6; by Hi-C, we have also iden- tified XPR1 as a possible candidate gene. Interestingly, our group has previously demonstrated XPR1 mutations in primary familial brain calcification [43], though any mechanistic overlap with PSP is unclear. Analysis of eQTL datasets (in GTEx) suggests that RPSA at 3p22.1 and SKIV2L near 6p21.1 may also be the causal genes but the tissue-relevant datasets were relatively underpowered. Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 9 of 11 Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 9 of 11 A limitation of the study includes the case-control matching design. URLs CAVIAR, http://genetics.cs.ucla.edu/caviar/index.html; dbGAP, http://www.ncbi.nlm.nih.gov/gap; GCTA, http://cnsgenomics.com/software/gcta/index.html; Genetic Power Calculator, http://zzz.bwh.harvard.edu/gpc/; HapMap, ftp://ftp.ncbi.nlm.nih.gov/hapmap//; IMPUTE2, https://mathgen.stats.ox.ac.uk/impute/impute_v2.html; LocusZoom, http://locuszoom.sph.umich.edu/locuszoom/; MetaXcan, https://github.com/hakyimlab/MetaXcan; NIAGADS, https://www.niagads.org/; PLINK2, https://www.cog-genomics.org/plink2; SHAPEIT, https://mathgen.stats.ox.ac.uk/genetics_software/shapeit/ shapeit.html; UCSC Genome Browser https //genome ucsc edu/cgi bin/hgGateway PLINK2, https://www.cog-genomics.org/plink2; SHAPEIT, https://mathgen.stats.ox.ac.uk/genetics_software/shapeit/ shapeit.html; SHAPEIT, https://mathgen.stats.ox.ac.uk/genetics_software/shapeit/ shapeit.html; UCSC Genome Browser, https://genome.ucsc.edu/cgi-bin/hgGateway; Acknowledgements We acknowledge the contributions of Alice Zhang, who has helped with acquiring controls genotyping; Margaret Chu, for administrative support; and all of the patients and their families, to whom this work is dedicated. Abbreviations AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; bvFTD: Behavioral variant frontotemporal dementia; eQTL: Expression quantitative trait loci; FTD: Frontotemporal dementia; GWAS: Genome-wide association study; PCA: Principal component analysis; PD: Parkinson’s disease; PSP: Progressive supranuclear palsy; QC: Quality control; SNP: Single nucleotide polymorphism Conclusion h Summary measures will be made available in the NIAGADS database (https:// www.niagads.org/). Additional data obtained from the NIAGADS and dbGAP databases are available with access restrictions from the corresponding databases. Here, we have increased the number of significant genetic risk locus for PSP, an important advance for understanding its pathophysiology. The power of this study to identify novel loci at genome wide signifi- cance and a large unexplained heritability suggests that PSP may be highly amenable to genetic associ- ation studies in larger sample cohorts using next generation sequencing. Overall, by establishing the genetic correlations of PSP with PD and ALS and identifying novel genome-wide significant and sug- gestive associations, we shed insight into the mecha- nisms of neurodegenerative disease. Discussion The NNIPPS genotyping and analysis was supported under the aegis of JPND – (www.jpnd.eu, United Kingdom), Med- ical Research Council (MR/L501529/1; MR/R024804/1) and Economic and So- cial Research Council (ES/L008238/1). A.A.C. receives salary support from the National Institute for Health Research (NIHR) Dementia Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s Col- lege London. The work leading up to this publication was funded by the European Community’s Health Seventh Framework Programme (FP7/2007– 2013; grant agreement number 259867) and Horizon 2020 Programme (H2020-PHC-2014-two-stage; grant agreement number 633413). We thank the UCLA Neuroscience Genomics Core (www.semel.ucla.edu/ungc) for as- sistance with genotyping data generation. Samples from the National Cell Repository for Alzheimer’s Disease (NCRAD), which receives government sup- port under a cooperative agreement grant (U24 AG021886) awarded by the National Institute on Aging (NIA), were used in this study. We thank contribu- tors who collected samples used in this study, as well as patients and their families, whose help and participation made this work possible. Authors’ contributions Additional file 1: Supplementary Tables S1-S5. (XLSX 636 kb) Additional file 2: Supplementary Figures S1-S5, Supplementary Methods. (DOCX 8021 kb) Additional file 1: Supplementary Tables S1-S5. (XLSX 636 kb) Additional file 2: Supplementary Figures S1-S5, Supplementary Methods. (DOCX 8021 kb) JAC contributed to study design, data collection, experimental design, statistical analysis, and drafting and revision of the manuscript. ZC contributed to data collection, statistical analysis, and drafting of the manuscript. HW contributed to data collection and statistical analysis. AYH contributed to study design and statistical analysis. JKL, KW, GB, PNL, CP, AS, ARJ, CML, WL, PD, PA, CT, J-FD, AL, ALB, and JMB contributed to subject recruitment, data collection, experimental design, and revision of the manuscript. JSY contributed to data collection and statistical analysis. AA-C and DHG contributed to supervision of the project, subject recruitment, data collection, statistical analysis, experimental design, and revision of the manu- script. GC contributed to supervision of the project, subject recruitment, data collection, statistical analysis, experimental design, and drafting and revision of the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Written informed consent was obtained for all patients participating in the Allon Therapeutics davunetide trial and the NNIPPS trial, and IRB approval was obtained from the corresponding Institutional Review Boards. For the NNIPPS study, The protocol and amendments were reviewed and approved by the Comité de Protection des Personnes of Pitié-Salpêtrière Hospital (France), the UK Multicentre Research Ethics Committee (MREC) (UK), Ethikkommission of the University of Ulm (Germany) and by local Institutional Review Boards (Ethics Committees) where appropriate (UK, Germany). For the BBBIPPS study, the protocol and amendments were reviewed and approved by the Comité de Protection des Personnes of Pitié- Salpêtrière Hospital (France). Additional data was obtained via NIAGADS and dbGAP in accordance with data access policies. The Institutional Review Discussion While this design allows for matching by array platforms and avoids stratification due to technical artifacts, stratification based on the ancestral differences may be present. The potential for stratification was reduced by strict filtering based on multidimensional scaling to limit the sample to subjects of European ances- try, and linear mixed model methods to further reduce confounding. Combining multiple cohorts as we have done may also reduce the degree of population stratifica- tion in the joint sample. Overall, the genomic inflation fac- tor (λ = 1.05) suggested an acceptable level of population stratification. The validity of the results and replication of the original GWAS are further reinforced by the consistency of the identified associations across the mul- tiple platform-matched sub-cohorts. study was supported by the French Health Ministry, Programme Hospitalier de Recherche Clinique (AOM04035). The Assistance Publique - Hôpitaux de Paris (France) was the sponsor of the study. The protocol and amendments were reviewed and approved by the Comité de Protection des Personnes of Pitié-Salpêtrière Hospital (France). The NNIPPS genotyping and analysis was supported under the aegis of JPND – (www.jpnd.eu, United Kingdom), Med- ical Research Council (MR/L501529/1; MR/R024804/1) and Economic and So- cial Research Council (ES/L008238/1). A.A.C. receives salary support from the National Institute for Health Research (NIHR) Dementia Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s Col- lege London. The work leading up to this publication was funded by the European Community’s Health Seventh Framework Programme (FP7/2007– 2013; grant agreement number 259867) and Horizon 2020 Programme (H2020-PHC-2014-two-stage; grant agreement number 633413). We thank the UCLA Neuroscience Genomics Core (www.semel.ucla.edu/ungc) for as- sistance with genotyping data generation. Samples from the National Cell Repository for Alzheimer’s Disease (NCRAD), which receives government sup- port under a cooperative agreement grant (U24 AG021886) awarded by the National Institute on Aging (NIA), were used in this study. We thank contribu- tors who collected samples used in this study, as well as patients and their families, whose help and participation made this work possible. study was supported by the French Health Ministry, Programme Hospitalier de Recherche Clinique (AOM04035). The Assistance Publique - Hôpitaux de Paris (France) was the sponsor of the study. The protocol and amendments were reviewed and approved by the Comité de Protection des Personnes of Pitié-Salpêtrière Hospital (France). Author details 1 13. The International HapMap 3 Consortium. Integrating common and rare genetic variation in diverse human populations. 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Funding This work was funded by grants from the Tau Consortium (D.H.G. and G.C.); the National Institutes of Health, F31 NS084556 (J.A.C.) UG3 NS104095 and P30 NS062691 from the National Institute of Neurological Disorders and Stroke (Informatics Center for Neurogenetics and Neurogenomics to G.C.); and the Fu-Hsing and Jyu-Yuan Chen family. Z.C. was supported by funding from the NIHR Academic Clinical Fellowship. Three academic institutions (In- stitute of Psychiatry, Psychology and Neuroscience, King’s College London; Assistance Publique-Hôpitaux de Paris and University of Ulm) were sponsors of the NNIPPS study in each country, and jointly own the data. The BBBIPPS Page 10 of 11 Page 10 of 11 Chen et al. Molecular Neurodegeneration (2018) 13:41 Chen et al. Molecular Neurodegeneration (2018) 13:41 Board at the University of California, Los Angeles, approved the joint study design, including review of outside consent forms. 7. 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Inherently Non-Pulsating Input Current DC-DC Converter for Battery Storage Systems
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Received July 10, 2020, accepted July 27, 2020, date of publication July 29, 2020, date of current version August 11, 2020. Received July 10, 2020, accepted July 27, 2020, date of publication July 29, 2020, date of current version August 11, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3012709 GUIDONG ZHANG 1, (Member, IEEE), NA JIN1, LILI QU 2, AND SAMSON SHENGLONG YU 3, (Member, IEEE) 1School of Automation, Guangdong University of Technology, Guangzhou 510006, China 2School of Automation, Foshan University, Foshan 528225, China 3School of Engineering, Deakin University, Melbourne, VIC 3216, Australia C di h Lili Q ( lili@f d ) GUIDONG ZHANG 1, (Member, IEEE), NA JIN1, LILI QU 2, AND SAMSON SHENGLONG YU 3, (Member, IEEE) 1School of Automation, Guangdong University of Technology, Guangzhou 510006, China 2School of Automation, Foshan University, Foshan 528225, China 3School of Engineering, Deakin University, Melbourne, VIC 3216, Australia Corresponding author: Lili Qu (qulili@fosu edu cn) This work was supported in part by the National Natural Science Foundation of China under Grant 51907032 and Grant 61733015; in part by the Natural Science Foundation of Guangdong Province under Grant 2018A030313365; and in part by the Science and Technology Planning Project of Guangzhou under Grant 201804010310. ABSTRACT With an ever-growing number of batteries being integrated into the electric grid, bidirectional converters with non-pulsating dc input current are required to replace the existing bidirectional converters so as to extend the lifetime of such dc power suppliers. Bidirectional DC-DC converters are increasingly used in a variety of applications including uninterruptable power supplies, electric vehicles and renewable energy systems. In this study, we propose a new topology of bidirectional dc-dc converter with inherently non-pulsating input current (NPIC), which is intended to be used for battery energy storage systems. With simple modifications from the conventional converters, the proposed NPIC converter has no inherent pulsating input current in the step-down (buck) mode under both continuous and boundary conduction modes (CCM and BCM), whereas in the step-up (boost) mode, the proposed NPIC converter retains the desired voltage gain. Underpinning theories, operating principles and steady-state performances are analyzed and presented in detail, which are then corroborated by simulation and experimentation. The proposed converter topology, with simple design principle and ease for implementation, is likely to have wide-ranging applicability in interfacing electrochemically functioned dc sources to modern power systems. INDEX TERMS Bidirectional dc-dc converter, non-pulsating input current, step-up mode, step-down mode, continuous conduction mode, boundary conduction mode. INDEX TERMS Bidirectional dc-dc converter, non-pulsating input current, step-up mode, step-down mode, continuous conduction mode, boundary conduction mode. The associate editor coordinating the review of this manuscript and approving it for publication was Zhilei Yao . This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 I. INTRODUCTION ated by the converter cannot be recovered, causing a low efficiency. Compared to isolated bidirectional dc-dc con- verter, non-isolated converters have smaller volume and higher efficiency due to the absence of transformer [8]–[10]. Conventional buck/boost converters are widely used due to their simple topologies and required control strategies, but these converters cannot operate in extensively wide voltage-conversion range [11], [12]. For zeta/sepic and cuk/cuk type converters, energy transfer efficiency is low because of their two-stage structures [13]–[15]. In order to improve the efficiency and increase the voltage-conversion range, many new non-isolated bidirectional dc-dc con- verters have been proposed. Authors in [16] proposed a non-isolated bidirectional dc-dc converter with high volt- age gain comprised of two boost converters; authors in [17] and [18] proposed two non-isolated bidirectional dc–dc converter topologies, with a simple circuit structure. Their In renewable energy integrated hybrid power systems, hybrid vehicle energy systems and uninterruptible power sup- ply systems, multiple dc power sources need to trans- fer energy to other dc energy storage systems, which requires bi-directional dc-dc converters [1]–[4]. The topol- ogy of bidirectional dc-dc converter can be divided into isolated (transformer-integrated) and non-isolated (without transformer) types. Flyback converters, forward-flyback con- verters, half-bridge converters and full-bridge converters are typical bidirectional isolated dc-dc converters [5]–[7]. By adjusting the turns ratio of the transformer, this con- verter family can obtain large voltage gains in both step-up and step-down modes. However, the power leakage gener- The associate editor coordinating the review of this manuscript and approving it for publication was Zhilei Yao . is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 140293 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 1. (a) Proposed converter. (b) Equivalent circuit in the step-down mode. (c) Equivalent circuit in the step-up mode. experimentally proven superior voltage step-up performances have extended their applicability to wide-ranging voltage levels than conventional converters. However, the above-mentioned converters only consider voltage gain and device stress, without taking into account the impact of dc pulsating input current on the input power supply. II. OPERATING PRINCIPLE OF THE PROPOSED NPIC CONVERTER II. OPERATING PRINCIPLE OF THE PROPOSED NPIC CONVERTER I. INTRODUCTION For example, the buck/boost bidirectional converter operating in step-down mode is equivalent to a buck con- verter, and input current of the converter is equal to the inductor current when the switch is turned on and becomes zero when the switch is turned off, causing the converter to have a pulsating input current [19]. The pulsating input current of these bidirectional converters may degrade the performance and lifespan of the electrochemically functioned power sources or energy storage systems, because the pul- sating current can lead to an accelerated aging rate of the electrodes [20]–[22], which may also cause problems like electromagnetic interference, slow transient response, and reduction of energy efficiency. It is well known that large electrolytic capacitors con- nected to the input port can cope with the inherent pulsating input current issue, however, this solution is executed at the expense of deterioration of the electrolytic capacitor, which can shorten the lifespan of the overall converter system [22], [23]. Another feasible option may be replacing the bulky electrolytic capacitor with an LC filter, which, however, may degrade energy efficiency and affect the dynamic perfor- mance of the converter [24], [25]. Interleaved converter could be another reasonable solution to alleviate the pulsating input current issue, but this method has been proven to be not suitable for bidirectional dc–dc converters [26], [27]. Knowing the need of non-pulsating input current convert- ers and the shortage of a proper design, in this study, we pro- pose a novel bidirectional dc-dc converter, which, compared to its conventional counterparts, is able to draw NPIC from the dc source while operating in the buck mode. At the same time, the proposed NPIC bidirectional converter is also able to retain its high voltage gain when working in the boost mode. Simulation and experimentation are conducted in this study, which verify the functionality and effectiveness and the proposed converter structure. The proposed NPIC converter has a simple topological design, and is easy to implement and apply to industrial uses. FIGURE 1. (a) Proposed converter. (b) Equivalent circuit in the step-down mode. (c) Equivalent circuit in the step-up mode. connected in series with the input source, with contributes to the NPIC feature of the proposed converter. I. INTRODUCTION In order to simplify the analysis, some reasonable assumptions are made as: a) all semi-conductor devices and passive components are considered ideal; b) capacities of the capacitors are large enough to keep the capacitor voltages nearly constant. The remainder of the paper is organized as follows. The structure and operating principle in buck and boost modes are detailed in Section II. Simulations and experiments are con- ducted with their results shown in Section III and Section IV respectively. Finally, a conclusion is drawn in Section V. A. STEP-DOWN MODE OF THE PROPOSED NPIC CONVERTER Fig. 1 shows the configuration of the proposed converter, which mainly consists of two inductors L1 and L2, one capac- itor Cb, two switches S1 and S2. Steady-state analysis of the proposed converter in step-up and step-down modes is discussed as follows. It is worth-noting that inductor L1 is The proposed converter in step-down mode is shown in Fig. 1(b). Characteristic waveforms of the proposed converter in CCM are depicted in Fig. 3, and the current flow path in one switching period is illustrated in Fig. 2(a). 140294 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 2. Key waveforms of the proposed converter in the step-down mode: (a) CCM; (b) BCM. FIGURE 2. Key waveforms of the proposed converter in the step-down mode: (a) CCM; (b) BCM. source delivers energy to inductor L1 and the load. Therefore, inductor L1 starts to store energy, and accordingly the current flowing through inductor L1 (iL1) increases. At the same time, energy saved in capacitor Cb is released to inductor L2 and the load. Correspondingly, the current of inductor L2 (iL2) starts increasing. In addition, current and voltage stresses of switches S1 and S2 in this mode can be found in Fig. 2 (a). Accordingly, the following equations among the components during this time interval can be obtained, FIGURE 3. Equivalent circuits in step-down mode: (a) mode 1; (b) mode 2.    iL1(t) = IL1V + vH −vL L1 t, iL2(t) = IL2V + vCb −vL L2 t, iCb(t) = −IL2V −vCb −vL L2 t, iCL(t) = IL1V + IL2V + (vH −vL L1 + vCb −vL L2 )t −io, (1) (1) where vH is the input voltage, vCb is the voltage of the capacitor Cb, vL is the output voltage and IL1V and IL2V are the minimum values of the currents flowing through inductors L1 and L2, respectively. FIGURE 3. Equivalent circuits in step-down mode: (a) mode 1; (b) mode 2. Mode 2 [t1 < t < t2]: In this mode, S1 is turned off and S2 is turned on at t = t1 as shown in Fig. 3 (b). During this mode, energy stored in L1 and the input power source is delivered to the load and Cb. Hence, the current flowing through L1 decreases. Meanwhile, inductor L2 releases energy to the load and the current flowing through it keeps decreasing as 2) INDUCTOR CURRENTS (19) Applying the ampere-second balance theorem for the capac- itor current, we can have Similarly, the critical inductance of inductor L2 can be derived as, Z Ts 0 iC(t)dt = 0, (6) (6) L2c = VLRL 2VHfs . (20) (20) where Ts is the switching period. where Ts is the switching period. where Ts is the switching period. Considering (1), (2), (4) and (6), the maximum and mini- mum values of the inductor currents can be calculated as, When inductances L1 and L2 are equal to critical values, the converter operates in the boundary-conduction-mode (BCM) mode. Fig. 2 (b) shows some typical waveforms in BCM operation. IL1P = DIo + VH −VL 2L1 DTs, (7) IL1V = DIo −VH −VL 2L1 DTs, (8) IL2P = (1 −D)Io + VH −VL 2L2 DTs, (9) IL2V = (1 −D)Io −VH −VL 2L2 DTs, (10) (7) (7) (8) Then, current ripples of the inductors can be deduced as, Then, current ripples of the inductors can be deduced as, depicted in Fig. 2 (a). Main equations among the components during this time interval can be deduced as, depicted in Fig. 2 (a). Main equations among the components during this time interval can be deduced as, 1iL1 = VH −VL L1 DTs, (13) 1iL2 = VH −VL L2 DTs. (14) (13)    iL1(t) = IL1P −vH −vCb −vL L1 t, iL2(t) = IL2P −vL L2 t, iCb(t) = IL1P −vH −vCb −vL L1 t, iCL(t) = IL1P + IL2P −(vH −vCb −vL L1 + vL L2 )t −io, (2) (14) (2) 4) CRITICAL INDUCTANCE L1c AND L2c ( VCb = VH, VL = DVH. (4) (4) When IL1V = 0, the critical inductance of the inductor L1 can be derived as, According to (4), the voltage conversion ratio of the pro- posed converter can be expressed as, L1c = RL(1 −D)Ts 2D , (18) (18) Gstep−down = VL VH = D. (5) where RL = VL/Io. Incorporating (4), (18) can be rewritten as, where RL = VL/Io. Incorporating (4), (18) can be rewritten as, (5) as, L1c = RL(VH −VL) 2VLfs . (19) 3) VOLTAGE AND CURRENT STRESSES OF POWER DEVICES According to the above analysis, it can be obtained that the voltage stresses of switches S1 and S2 in the proposed converter, namely, VS1 and VS2, are equal to the voltage of the capacitor Cb, i.e., VCb, as shown in (4). where IL1P and IL2P are the maximum values of the currents flowing through the inductors L1 and L2, respectively. b According to Fig. 2(a), peak currents flowing through switches S1 and S2 can be derived as, Assuming the converter is a lossless system and the voltage ripples are ignorable in a switching period. Based on the mode analysis and applying volt-second balance principle to inductors L1 and L2, steady-state voltage relationships among input voltage, output voltage, and capacitor voltages can be obtained as, IS1 peak = IL1P + IL2P, (15) IS2 peak = IL1P + IL2P. (16) (15) (16) (15) (15) (16) (16) By applying (5), (7), and (9) to (15) and (16), equa- tions (15) and (16) can be rewritten as follows, ( (VH −VL)D + (VH −VCb −VL)(1 −D) = 0, (VCb −VL)D −VL(1 −D) = 0, (3) (3) IS1 peak = IS2 peak = Io + VL(VH −VL) 2LefsVH , (17) (17) where D is the duty cycle of switch S1. where D is the duty cycle of switch S1. where Le = L1//L2. where Le = L1//L2. From (3), the voltage of capacitor Cb and voltage of capac- itor CL, i.e., the output voltage, can be derived as, 4) CRITICAL INDUCTANCE L1c AND L2c 1) OPERATION MODES Mode 1 [t0 < t < t1]: As shown in Fig. 3 (a), switch S1 is turned on at t = t0 and switch S2 is turned off. The input 140295 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems depicted in Fig. 2 (a). Main equations among the components during this time interval can be deduced as Then, current ripples of the inductors can be deduced as, B. STEP-UP MODE OF THE PROPOSED CONVERTER The proposed converter in step-up mode is shown in Fig. 1(c), whose equivalent circuits in CCM mode are depicted in Fig. 5, and the characteristic waveforms are illustrated in Fig. 4(a).    iL1(t) = IL1V + vH −vL −vCb L1 t, iL2(t) = IL2V + vL L2 t, iCb(t) = −(IL1V + vH −vL L1 t), iCH(t) = IL1V + vH −vL L1 t −io. (27) (27) 5) OUTPUT VOLTAGE RIPPLE ANALYSIS IN CCM According to the characteristics of the capacitor, voltage ripples of the capacitor can be calculated as, (9) vrip C = 1 C Z t02 t01 iC(t)dt, (21) (10) (21) where Io is the load current. The average current of each inductor can be derived as, where t01 is the time that the current flowing through it changes from positive to negative, and t02 is the time that the current flowing through the capacitor changes from negative to positive. ¯IL1 = DIo, (11) ¯IL2 = (1 −D)Io. (12) (11) (12) (12) VOLUME 8, 2020 VOLUME 8, 2020 140296 140296 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 4. Key waveforms of the proposed converter in the step-up mode: (a) CCM.; (b) BCM. FIGURE 4. Key waveforms of the proposed converter in the step-up mode: (a) CCM.; (b) BCM. For capacitor CL, the voltage ripple can be calculated as, quantities of the components during this time interval can be obtained, vCCM CL−rip = 1 CL Z t02 t01 iCL(t)dt, (22) (22)    iL1(t) = IL1P −vH −vL L1 t, iL2(t) = IL2P −vCb −vL L2 t, iCb(t) = IL2P −vCb −vL L2 t, iCL(t) = IL1P −vH −vL L1 t −io. (26) where t01 and t02 can be calculated as, (26) t01 = Le(Io −IL1V −IL2V ) VH −VL = DTs 2 , (23) t02 = Le(Io −IL1P −IL2P) VL = −(1 −D)Ts 2 . (24) (23) Mode 2 [t1 < t < t2]: As shown in Fig. 5 (b), switch S1 is turned off and the switch S2 is turned on. During this mode, VL charges L1, L2 and Cb while releasing energy to the load. So, iL1, iL2 and iCb start to increase. The main equations relating the components during this time interval can be deduced as, From (21)–(24), the voltage ripple can be calculated as, From (21)–(24), the voltage ripple can be calculated as, vCCM CL−rip = VL(VH −VL) 8LeCLVHf 2s . (25) (25) 5) OUTPUT VOLTAGE RIPPLE ANALYSIS IN CCM According to (21), voltage ripples of CH can be calcul 5) OUTPUT VOLTAGE RIPPLE ANALYSIS IN CCM According to (21), voltage ripples of CH can be calculated as, ( VCb = VH, VL = DVH. (29) ) According to (21), voltage ripples of CH can be calculated as, According to (21), voltage ripples of CH can be calcu (29) vCCM CH−rip = 1 CH Z t02 t01 iCH(t)dt = (VH −VL)3 8L1CHV 2 Hf 2s , (42) (42) According to (29), the voltage conversion ratio of the proposed converter can be expressed as, 2) CURRENTS OF INDUCTORS Combining (26), (27), (29) and (6), the maximum and mini- mum values of the inductor currents can be calculated as, IL1P = Io + VH −VL 2L1 (1 −D)Ts, (31) IL1V = Io −VH −VL 2L1 (1 −D)Ts, (32) IL2P = 1 −D D Io + VH −VL 2L2 (1 −D)Ts, (33) IL2V = 1 −D D Io −VH −VL 2L2 (1 −D)Ts. (34) (31) (32) (32) 1) OPERATION MODES Mode 1 [t0 < t < t1]: As shown in Fig. 5 (a), S1 is turned on and S2 is turned off. Input source VL and L1 and L1 supply power to the load, so iL1 decreases. At the same time, L2 and Cb release energy to the load, so iL2 and iCb also decrease. Therefore, the following equation set relating the electric Based on the mode analysis and applying volt-second bal- ance principle to inductors L1 and L2, voltage relationships in steady state among the input voltage, output voltage, and 140297 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 5. Equivalent circuits in step-up mode: (a) mode 1; (b) mode 2. C. COMPARION OF THE PROPOSED NPIC CONVERTER AND OTHER BI-DIRECTIONAL DC-DC CONVERTERS Gstep−up = VH VL = 1 D. (30) (30) Comparisons between the NPIC converter proposed in this paper and other commonly used and previously proposed bidirectional converters are shown in TABLE 1. Compared with these converters, the proposed NPIC converter has the advantages of continuous input current in the step-down mode, simple structure with the fewest number of additional electronic devices. 3) VOLTAGE AND CURRENT STRESSES OF POWER DEVICES 3) VOLTAGE AND CURRENT STRESSES OF POWER DEVICES Based on the previous discussions, voltage stresses of the switches VS1 and VS2 are equal to the voltage of the capacitor Cb i.e., VCb, which is shown in (29). According to Fig. 4(a), the peak value of the currents flowing through switches S1 and S2 can be derived as, IS1 peak = IL1P + IL2P, (37) IS2 peak = IL1P + IL2P. (38) (37) (38) By applying (30), (31), (33) to (37) and (38), we can have By applying (30), (31), (33) to (37) and (38), we can have IS1 peak = IS2 peak = 1 DIo + (VH −VL)2 2LefsVH , (39) (39) where Le = L1//L2. 4) CALCULATION OF CRITICAL INDUCTANCE L1c AND L2c When IL1V = 0, critical inductance of inductor L1 can be derived as, 4) CALCULATION OF CRITICAL INDUCTANCE L1c AND L2c When IL1V = 0, critical inductance of inductor L1 can be derived as, 4) CALCULATION OF CRITICAL INDUCTANCE L1c AND L2c When IL1V = 0, critical inductance of inductor L1 can be derived as, L1c = (VH −VL)(1 −D)Ts 2Io = RH(VH −VL)2 2V 2 Hfs . (40) (40) Similarly, critical inductance of inductor L2 can be derived as, FIGURE 5. Equivalent circuits in step-up mode: (a) mode 1; (b) mode 2. as, L2c = RHVL(VH −VL) 2V 2 Hfs . (41) capacitor voltages can be obtained as, (41) ( (VL −VH)D + (VL + VCb −VH)(1 −D) = 0, (VCb −VL)D −VL(1 −D) = 0. (28) When L1 and L2 are equal to their critical values, the converter operates in the BCM mode. Fig. 4 (b) shows some typical waveforms in BCM operation. (28) From (28), the voltage of capacitor Cb and the voltage of capacitor CL, i.e., the output voltage, can be derived as, III. SIMULATION VERIFICATION In order to verify the effectiveness of the proposed converter, the converter topology is simulated in PSIM software envi- ronment with useful parameters shown in TABLE 2. (33) Step-down simulation results are demonstrated in Fig. 6 (a) and (b), which comprise the driving signals (vgs1 and vgs2), input current (iin), output voltage (vo), inductor and switch currents (iL1, iL2, iS1 and iS2), switch voltage stresses (vds1 and vds2), respectively. Since inductor L1 is in series with the power source, current of L1 is equal to the input current, Then, average currents of L1 and L2 can be derived as Then, average currents of L1 and L2 can be derived as ¯IL1 = Io, (35) ¯IL2 = 1 −D D Io. (36) (35) 140298 VOLUME 8, 2020 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems TABLE 1. Performance comparison of various bi-directional dc-dc converters. TABLE 2. Simulation parameters. i.e., iL1 = iin. Fig. 7 shows the input current waveform of the proposed NPIC converter and the conventional buck/boost converter with the same simulation parameters. Apparently the proposed NPIC converter is able to regulate and draw non-pulsating input current into the converter system, demon- strating its superiority its conventional counterparts. The step-up simulation results are demonstrated in Fig. 6 (c) and (d), which comprise the driving signals (vgs1 and vgs2), input current (iin), output voltage (vo), inductor and switch currents (iL1, iL2, iS1 and iS2), switch voltage stresses (vds1 and vds2), respectively. According to Fig. 6 (c) and (d), this NPIC converter can obtain the same voltage gain as buck/boost bidirectional converter when working in the step-up mode. TABLE 3. Experimental platform. TABLE 4. Experimental parameters. with the physical prototype and experimental en shown in Fig. 8 and TABLE 3, and the paramete the experiment are shown in TABLE 4. A STEP DOWN MODE TABLE 1. Performance comparison of various bi-directional dc-dc converters. TABLE 1. Performance comparison of various bi-directional dc-dc converters. 1. Performance comparison of various bi-directional dc-dc converters. TABLE 3. Experimental platform. TABLE 4. Experimental parameters. TABLE 3. Experimental platform. TABLE 2. Simulation parameters. TABLE 2. Simulation parameters. TABLE 4. Experimental parameters. i.e., iL1 = iin. Fig. 7 shows the input current waveform of the proposed NPIC converter and the conventional buck/boost converter with the same simulation parameters. III. SIMULATION VERIFICATION Apparently the proposed NPIC converter is able to regulate and draw non-pulsating input current into the converter system, demon- strating its superiority its conventional counterparts. The step-up simulation results are demonstrated in Fig. 6 (c) and (d), which comprise the driving signals (vgs1 and vgs2), input current (iin), output voltage (vo), inductor and switch currents (iL1, iL2, iS1 and iS2), switch voltage stresses (vds1 and vds2), respectively. According to Fig. 6 (c) and (d), this NPIC converter can obtain the same voltage gain as buck/boost bidirectional converter when working in the step-up mode. with the physical prototype and experimental environment shown in Fig. 8 and TABLE 3, and the parameters used in the experiment are shown in TABLE 4. IV. EXPERIMENTAL VERIFICATION Experimental results are demonstrated in Figs. 9∼11, which consist of the driving signals (vgs1), output voltages (vL), inductor currents (iL1 and iL2), switch currents (iS1 and iS2) To further validate the effectiveness of the proposed con- verter, a prototype of the converter is built in this study, VOLUME 8, 2020 140299 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 6. Simulation results for the proposed NIPC converter: (a) CCM in step-down mode; (b) BCM in step-down mode; (c) CCM in step-up mode; (d) BCM in step-up mode. f h d d FIGURE 8. Prototype with the experimental environment. FIGURE 9. Experimental waveforms of the driving signal, output voltage, and the currents of inductors L1, L2 in step-down mode: (a) CCM; (b) BCM. FIGURE 10. Experimental waveforms of the currents of switches S1 and S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 8. Prototype with the experimental environment. FIGURE 9. Experimental waveforms of the driving signal, output voltage, and the currents of inductors L1, L2 in step-down mode: (a) CCM; (b) BCM. FIGURE 8. Prototype with the experimental environment. FIGURE 8. Prototype with the experimental environment. FIGURE 9. Experimental waveforms of the driving signal, output voltage, and the currents of inductors L1, L2 in step-down mode: (a) CCM; (b) BCM. FIGURE 9. Experimental waveforms of the driving signal, output voltage, and the currents of inductors L1, L2 in step-down mode: (a) CCM; (b) BCM. FIGURE 10. Experimental waveforms of the currents of switches S1 and S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 6. Simulation results for the proposed NIPC converter: (a) CCM in step-down mode; (b) BCM in step-down mode; (c) CCM in step-up mode; (d) BCM in step-up mode. FIGURE 7. Input current of the proposed NPIC converter and conventional buck/boost converter in step-down mode. FIGURE 10. Experimental waveforms of the currents of switches S1 and S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 11. Experimental waveforms of the voltage stresses of the switch S1 and S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 7. Input current of the proposed NPIC converter and conventional buck/boost converter in step-down mode. FIGURE 11. Experimental waveforms of the voltage stresses of the switch S1 and S2 in step-down mode: (a) CCM; (b) BCM. and switch voltage stresses (vds1 and vds2), respectively. As can be seen from Fig. V. CONCLUSION In this study, a novel bidirectional dc-dc converter converter with no inherent pulsating input current in step-down mode has been proposed. The proposed converter has lesser number of active and passive components. And in the step-down mode, the input current of the proposed converter in both CCM mode and BCM mode is greater than 0. In the step-up mode can retain the desired voltage gain. With the increase of the output power, the efficiency of the proposed converter promotes gradually. As it can be seen that the analysed effi- ciency is about 90-91.5% in the step-up mode and 91-94% in the step-down mode. This converter is a good candidate for applications like battery storage systems, which require continuous input current. Compared to traditional boost/buck bidirectional converters whose input current is inherently intermittent or pulsating, the proposed NPIC converter is able to produce a continuous non-pulsating input current in the step-down mode, overcomes the limitations of conventional converters and can better reserve the lifetime of electrochem- ically functioned dc sources such as batteries and fuel cells. The operating principle and steady-state performance of the proposed NPIC current have been first demonstrated in detail, which have then been corroborated through both simulation and experimentation, verifying the functionality and unique features of the proposed NPIC converter topology. It has also been proven in this study that the proposed converter has low output voltage ripples, high efficiency and the ability to remain the desired voltage gain in the step-up mode. FIGURE 13. Experimental waveforms of the currents of switches S1 and S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 14. Experimental waveforms of the voltage stresses of the switch S1 and S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 14. Experimental waveforms of the voltage stresses of the switch S1 and S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 15. Efficiency of proposed converter with different operation power. REFERENCES [1] G. Zhang, Z. Li, B. Zhang, and W. A. Halang, ‘‘Power electronics con- verters: Past, present and future,’’ Renew. Sustain. Energy Rev., vol. 81, pp. 2028–2044, Jan. 2018. [2] F. Xue, R. Yu, and A. Q. Huang, ‘‘A 98.3% efficient GaN isolated bidirectional DC–DC converter for DC microgrid energy storage system applications,’’ IEEE Trans. Ind. Electron., vol. 64, no. 11, pp. 9094–9103, Nov. 2017. FIGURE 15. Efficiency of proposed converter with different operation power. [3] D. Ma, W. Chen, L. V. CONCLUSION Shu, X. Qu, X. Zhan, and Z. Liu, ‘‘A multiport power electronic transformer based on modular multilevel converter and mixed-frequency modulation,’’ IEEE Trans. Circuits Syst. II, Exp. Briefs, vol. 67, no. 7, pp. 1284–1288, Jul. 2020, doi: 10.1109/TCSII.2019.2931529. results, further proving the non-pulsating input current fea- ture of the proposed NPIC converter. [4] G. Zhang, J. Yuan, Z. Li, S. S. Yu, S.-Z. Chen, H. Trinh, and Y. Zhang, ‘‘Forming a reliable hybrid microgrid using electric spring coupled with non-sensitive loads and ESS,’’ IEEE Trans. Smart Grid, vol. 11, no. 4, pp. 2867–2879, Jul. 2020. IV. EXPERIMENTAL VERIFICATION 9, input currents (iin = iL1) of the converter under both CCM and BCM modes are greater than 0. Obviously, the proposed converter can maintain a continuous and non-pulsating input current for the converter system. It is thus safe to state that the experimental results agree well with the theoretical analyses and stimulation VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 12. Experimental waveforms of the driving signal, output voltage, and currents of the inductors L1, L2 in step-up mode: (a) CCM; (b) BCM. FIGURE 13. Experimental waveforms of the currents of switches S1 and S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 12. Experimental waveforms of the driving signal, output voltage, and currents of the inductors L1, L2 in step-up mode: (a) CCM; (b) BCM. mentation that the proposed NPIC converter is able to retain the desired voltage gain in the step-up mode while producing non-pulsating input current in the step-down mode. Fig. 15 shows efficiency curves of the proposed converter under different operation modes and power output, which demonstrates that the proposed converter has high efficiency. when the output power varies from 25 W to 125 W, the efficiency of the proposed converters is over 90%. FIGURE 12. Experimental waveforms of the driving signal, output voltage, and currents of the inductors L1, L2 in step-up mode: (a) CCM; (b) BCM. FIGURE 12. Experimental waveforms of the driving signal, output voltage, and currents of the inductors L1, L2 in step-up mode: (a) CCM; (b) BCM. [1] G. Zhang, Z. Li, B. Zhang, and W. A. Halang, ‘‘Power electronics con- verters: Past, present and future,’’ Renew. Sustain. Energy Rev., vol. 81, pp. 2028–2044, Jan. 2018. [2] F. Xue, R. Yu, and A. Q. Huang, ‘‘A 98.3% efficient GaN isolated bidirectional DC–DC converter for DC microgrid energy storage system applications,’’ IEEE Trans. Ind. Electron., vol. 64, no. 11, pp. 9094–9103, Nov. 2017. B. STEP-UP MODE In the step-up mode, experimental results are demonstrated in Figs. 12∼14, including driving signals (vgs1), output voltages (vH), inductor currents (iL1 and iL2), switch currents (iS1 and iS2) and switch voltage stresses (vds1 and vds2), respectively. It can be seen from Figs. 12∼14 that the converter can achieve a stable output voltage. It can be concluded through experi- [5] A. Tong, L. Hang, G. Li, X. Jiang, and S. Gao, ‘‘Modeling and analysis of a dual-active-bridge-isolated bidirectional DC/DC converter to minimize RMS current with whole operating range,’’ IEEE Trans. Power Electron., vol. 33, no. 6, pp. 5302–5316, Jun. 2018. [6] F. Zhang and Y. Yan, ‘‘Novel forward–flyback hybrid bidirectional DC–DC converter,’’ IEEE Trans. Ind. Electron., vol. 56, no. 5, pp. 1578–1584, May 2009. 140301 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems GUIDONG ZHANG (Member, IEEE) was born in Guangdong, China, in 1986. He received the B.Sc. degree from the Xi’an University of Technology, in 2008, and the two Ph.D. degrees from the South China University of Technology and FernUniver- sität, Hagen, in 2014 and 2015, respectively. He is currently an Associate Professor with the School of Automation, Guangdong University of Technology, Guangzhou. His research inter- est includes power electronics topology and their applications. [7] A. K. Rathore and U. Prasanna, ‘‘Analysis, design, and experimental results of novel snubberless bidirectional naturally clamped ZCS/ZVS current-fed half-bridge DC/DC converter for fuel cell vehicles,’’ IEEE Trans. Ind. Electron., vol. 60, no. 10, pp. 4482–4491, Oct. 2013. GUIDONG ZHANG (Member, IEEE) was born in Guangdong, China, in 1986. He received the B.Sc. degree from the Xi’an University of Technology, in 2008, and the two Ph.D. degrees from the South China University of Technology and FernUniver- sität, Hagen, in 2014 and 2015, respectively. [8] G. Chen, Y. Deng, L. Chen, Y. Hu, L. Jiang, X. He, and Y. Wang, ‘‘A family of zero-voltage-switching magnetic coupling nonisolated bidi- rectional DC–DC converters,’’ IEEE Trans. Ind. Electron., vol. 64, no. 8, pp. 6223–6233, Aug. 2017. He is currently an Associate Professor with the School of Automation, Guangdong University of Technology, Guangzhou. His research inter- est includes power electronics topology and their applications. He is currently an Associate Professor with the School of Automation, Guangdong University of Technology, Guangzhou. His research inter- est includes power electronics topology and their applications. [9] L. Zhao, J. Chen, T. Chen, Y. Shi, Z. B. STEP-UP MODE degree in high-voltage technology and equipment from the Huazhong University of Science and Technology, Wuhan, Hubei, in 1989, the M.S. degree in control sci- ence and engineering from Xi’an Jiaotong Univer- sity, Xi’an, Shanxi, China, in 2002, and the Ph.D. degree in power electronics from the South China University of Technology, Guangzhou, Guang- dong, China, in 2009. She is currently a Professor at Foshan University, China. Her research interests include power electronic systems and devices, switching power supply, and circuit analysis technology and its applications. LILI QU was born in Hubei, China, in 1968. She received the B.S. degree in high-voltage technology and equipment from the Huazhong University of Science and Technology, Wuhan, Hubei, in 1989, the M.S. degree in control sci- ence and engineering from Xi’an Jiaotong Univer- sity, Xi’an, Shanxi, China, in 2002, and the Ph.D. degree in power electronics from the South China University of Technology, Guangzhou, Guang- dong, China, in 2009. LILI QU was born in Hubei, China, in 1968. She received the B.S. degree in high-voltage technology and equipment from the Huazhong University of Science and Technology, Wuhan, Hubei, in 1989, the M.S. degree in control sci- ence and engineering from Xi’an Jiaotong Univer- sity, Xi’an, Shanxi, China, in 2002, and the Ph.D. degree in power electronics from the South China University of Technology, Guangzhou, Guang- dong, China, in 2009. She is currently a Professor at Foshan University, China. Her research interests include power electronic systems and devices, switching power supply, and circuit analysis technology and its applications. [17] C.-C. Lin, L.-S. Yang, and G. Wu, ‘‘Study of a non-isolated bidirectional DC–DC converter,’’ IET Power Electron., vol. 6, no. 1, pp. 30–37, 2013. [18] H. Ardi, R. R. Ahrabi, and S. N. Ravadanegh, ‘‘Non-isolated bidirectional DC–DC converter analysis and implementation,’’ IET Power Electron., vol. 7, no. 12, 2014, pp. 3033–3044. [19] G. Zhang, J. Yuan, S. S. Yu, N. Zhang, Y. Wang, and Y. Zhang, ‘‘Advanced four-mode-modulation-based four-switch non-inverting buck–boost con- verter with extra operation zone,’’ IET Power Electron., vol. 13, no. 10, pp. 2049–2059, Aug. 2020. g She is currently a Professor at Foshan University, China. Her research interests include power electronic systems and devices, switching power supply, and circuit analysis technology and its applications. [20] M.-C. Cheng, C.-T. Pan, J.-H. Teng, and S.-W. Luan, ‘‘An input current ripple-free flyback-type converter with passive pulsating ripple canceling circuit,’’ IEEE Trans. Ind. Appl., vol. 53, no. B. STEP-UP MODE 2, pp. 1210–1218, Mar. 2017. [21] G. Zhang, S. Zou, S. S. Yu, S.-Z. Chen, B. Zhang, D. Qiu, and Y. Zhang, ‘‘Enhanced one-cycle control for multicell power converters,’’ IEEE Trans. Power Electron., vol. 35, no. 8, pp. 8846–8856, Aug. 2020. [22] X. Liu and H. Li, ‘‘An electrolytic-capacitor-free single-phase high-power fuel cell converter with direct double-frequency ripple current control,’’ IEEE Trans. Ind. Appl., vol. 51, no. 1, pp. 297–308, Jan. 2015. SAMSON SHENGLONG YU (Member, IEEE) received the master’s degree (Hons.) in electrical and electronic engineering and the Ph.D. degree in electrical power engineering from The Uni- versity of Western Australia (UWA), Perth, WA, Australia, in 2014 and 2017, respectively. SAMSON SHENGLONG YU (Member, IEEE) received the master’s degree (Hons.) in electrical and electronic engineering and the Ph.D. degree in electrical power engineering from The Uni- versity of Western Australia (UWA), Perth, WA, Australia, in 2014 and 2017, respectively. [23] Y. Ohnuma, K. Orikawa, and J.-I. Itoh, ‘‘A single-phase current-source PV inverter with power decoupling capability using an active buffer,’’ IEEE Trans. Ind. Appl., vol. 51, no. 1, pp. 531–538, Jan. 2015. [24] W.-T. Fan, K. K.-F. Yuen, and H. S.-H. Chung, ‘‘Power semiconduc- tor filter: Use of series-pass device in switching converters for filtering input current harmonics,’’ IEEE Trans. Power Electron., vol. 31, no. 3, pp. 2053–2068, Mar. 2016. From 2017 to 2019, he served as a Postdoc- toral Research Fellow at UWA. He is currently an Assistant Professor at Deakin University, Mel- bourne, VIC, Australia. His research interests include power system analysis, renewable energy integration and forecasting, and power electronics and its applications and control. He received the first and second Best Paper Awards in the IEEE Australia Paper Competition, in 2016 and 2017, the Best Reviewer Award for the IEEE TRANSACTIONS ON SMART GRID, in 2018, and the Featured Article in Chaos: An Interdisciplinary Journal for Nonlinear Science, in 2019. [25] V. Beldjajev, T. Lehtla, and J. Zakis, ‘‘Impact of component losses on the efficiency of the LC-filter based dual active bridge for the isolation stage of power electronic transformer,’’ in Proc. 8th Int. Conf.-Workshop Compat. Power Electron., Jun. 2013, pp. 132–137. [26] O. Garcia, P. Zumel, A. de Castro, and A. Cobos, ‘‘Automotive DC-DC bidirectional converter made with many interleaved buck stages,’’ IEEE Trans. Power Electron., vol. 21, no. 3, pp. 578–586, May 2006. [27] M. Ilic and D. B. STEP-UP MODE Fan, and Z. Zhuang, ‘‘Zero-voltage and zero-current-switching dual-transformer-based full-bridge converter with current doubler rectifier,’’ IEEE Trans. Power Electron., early access, May 25, 2020, doi: 10.1109/TPEL.2020.2997017. [10] J. Huang, Y. Wang, Z. Li, and W. Lei, ‘‘Unified triple-phase-shift control to minimize current stress and achieve full soft-switching of isolated bidirectional DC–DC converter,’’ IEEE Trans. Ind. Electron., vol. 63, no. 7, pp. 4169–4179, Jul. 2016. [11] X. Huang, F. C. Lee, Q. Li, and W. Du, ‘‘High-frequency high-efficiency GaN-based interleaved CRM bidirectional buck/boost converter with inverse coupled inductor,’’ IEEE Trans. Power Electron., vol. 31, no. 6, pp. 4343–4352, Jun. 2016. NA JIN was born in Zhanjiang, Guangdong, China, in 1996. She received the B.Sc. degree from the Guangdong Normal University of Technology, Guangzhou, China, in 2019. She is currently pur- suing the M.Sc. degree in electrical engineering with the School of Automation, Guangdong Uni- versity of Technology, Guangzhou. NA JIN was born in Zhanjiang, Guangdong, China, in 1996. She received the B.Sc. degree from the Guangdong Normal University of Technology, Guangzhou, China, in 2019. She is currently pur- suing the M.Sc. degree in electrical engineering with the School of Automation, Guangdong Uni- versity of Technology, Guangzhou. [12] D. Baolei, L. Tao, H. Jun, J. Yang, and W. Xiao, ‘‘High-efficiency buck–boost converter and its control strategy suitable for wide voltage range,’’ J. Eng., vol. 2019, no. 10, pp. 7293–7297, Oct. 2019. [13] I.-D. Kim, S.-H. Paeng, J.-W. Ahn, E.-C. Nho, and J.-S. Ko, ‘‘New bidi- rectional ZVS PWM sepic/zeta DC-DC converter,’’ in Proc. IEEE ISIE, Jun. 2007, pp. 555–560. Her current research interest includes power electronics topology and their applications. [14] B. Zhu, F. Ding, and D. M. Vilathgamuwa, ‘‘Coat circuits for DC–DC converters to improve voltage conversion ratio,’’ IEEE Trans. Power Elec- tron., vol. 35, no. 4, pp. 3679–3687, Apr. 2020, doi: 10.1109/TPEL.2019. 2934726. [15] E. Babaei and M. E. Seyed Mahmoodieh, ‘‘Analysis and investigation of energy transmission process in different operating modes of Sepic converter,’’ IET Power Electron., vol. 7, no. 4, pp. 819–828, Apr. 2014. [16] H. Ardi, A. Ajami, F. Kardan, and S. N. Avilagh, ‘‘Analysis and imple- mentation of a nonisolated bidirectional DC–DC converter with high voltage gain,’’ IEEE Trans. Ind. Electron., vol. 63, no. 8, pp. 4878–4888, Aug. 2016. LILI QU was born in Hubei, China, in 1968. She received the B.S. B. STEP-UP MODE Maksimovic, ‘‘Interleaved zero-current-transition buck converter,’’ IEEE Trans. Ind. Appl., vol. 43, no. 6, pp. 1619–1627, Nov./Dec. 2007. 140302 140302 VOLUME 8, 2020
https://openalex.org/W2017900394
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English
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In-Depth Transcriptome Analysis Reveals Novel TARs and Prevalent Antisense Transcription in Human Cell Lines
PloS one
2,010
cc-by
4,786
Experimental outline In this study we investigate the transcriptome of three cell lines, A431, U-2 OS and U251, by applying the massive SOLiD DNA sequencing technology facilitating sense/antisense identification of reads. The cell lines were chosen to represent three different lineages; epithelial, mesenchymal and glia cells. A total of 10 to 15 million high quality 50-basepair reads were obtained for each cell line. The reads were mapped onto the human reference genome (hg18), after which reads were aggregated for each gene. An expression value was calculated based on the number of reads per kilobase gene and million reads in each sample (RPKM) [12]. Analysis of the gene expression pattern demonstrated that 66% to 69% of all genes are expressed in each cell line of which 85% to 88% were shared for all three cell lines (figure S1). Several studies [3] have shown that antisense transcription is prevalent and likely to have a regulatory function. Studies indicate that 20% to 90% of all human protein-coding genes can generate transcripts with potential to form sense-antisense pairs [4–6] and that these generally are arranged in a tail-to-tail pattern. Recently, short fragments of RNA have been detected in the antisense direction in regions just upstream protein-coding genes [7–9]. In parallel to experimental discovery of regulatory RNAs, compu- tational methods are being developed to identify conserved structural RNA elements likely to be involved in transcriptional and trans- lational control [10]. These approaches aim to make in silico predic- tions of regulatory sites in the human genome that can be validated by the on-going massive transcriptome sequencing (RNA-Seq) efforts on cells, tissues and organs [11], however, more development is needed to make these algorithms more accurate and efficient. Abstract Competing Interests: The authors have declared that no competing interests exist. ¤a Current address: Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden ¤b Current address: Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospita lines. We show that approximately 20% of all protein-coding genes have antisense transcription coupled to them and that antisense transcription is prevalent in introns. Introduction Less than 2% of the human genome encodes for proteins, yet a large fraction, recently estimated to 60% to 90% of the genome can be transcribed [1]. The functions of the majority of these novel uncharacterized transcriptionally active regions (TARs) are currently unknown, but they are believed to be of regulatory importance. For example, Ebisuya and colleagues showed that ‘‘transcriptional ripples’’ can propagate along the genome and mediate regulation of genes several tens of kilobases away [2]. Daniel Klevebring¤a, Magnus Bjursell¤b, Olof Emanuelsson, Joakim Lundeberg* Di i i f G T h l S h l f Bi h l Alb N U i i C R l I i f T h l S kh l S d Daniel Klevebring¤a, Magnus Bjursell¤b, Olof Emanuelsson, Joakim Lundeberg* Division of Gene Technology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden on of Gene Technology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden PLoS ONE | www.plosone.org Abstract Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We find that the correlation between RNA-seq data and microarray data is dependent on the gene length, with longer genes showing a better correlation. We detect high antisense transcriptional activity from promoter, terminator and intron regions of protein- coding genes and identify a vast number of previously unidentified TARs, including putative novel EGFR transcripts. This shows that in-depth analysis of the transcriptome using RNA-seq is a valuable tool for understanding complex transcriptional events. Furthermore, the development of new algorithms for estimation of gene expression from RNA-seq data is necessary to minimize length bias. Citation: Klevebring D, Bjursell M, Emanuelsson O, Lundeberg J (2010) In-Depth Transcriptome Analysis Reveals Novel TARs and Prevalent Antisense Transcription in Human Cell Lines. PLoS ONE 5(3): e9762. doi:10.1371/journal.pone.0009762 Editor: Fatah Kashanchi, George Washington University, United States of America Received December 3, 2009; Accepted February 22, 2010; Published March 25, 2010 Received December 3, 2009; Accepted February 22, 2010; Published March 25, 2010 Copyright:  2010 Klevebring et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Swedish Scientific Council, and the Knut and Alice Wallenberg Foundation. 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. Funding: This work was supported by the Swedish Scientific Council, and the Knut and Alice Wallenberg Foundation. 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. Investigation of antisense expression The vast majority of all reads originate from the sense strand of protein-coding genes (figure 2A and figure S3). A large fraction of the reads also originate from the introns of protein-coding genes, but when normalized to the length of the introns, the relative expression levels of introns are very low (figure 2B and figure S3, S4, S5). We also note that many reads map to regions distant from protein-coding genes (here denoted as ‘‘Other’’), which to some extent can be expected since this includes many long non-protein- coding genes. Previous studies have described a class of short transcripts (20–90 nucleotides) that originate from the antisense strand in the promoter regions of genes [7–9,15]. We investigated tag densities in promoter and terminator regions (defined as 1000 base pairs upstream and downstream of genes, respectively) and are unable to detect an increased density upstream of genes. This is expected since our extraction method does not capture fragments shorter than roughly 200 nucleotides. In the terminator regions, however, the relative antisense tag density is higher than that in exons and promoter regions (figure 2B and figure S3). This indicates that transcription of long RNAs in terminator regions could represent a regulatory mechanism for termination of transcription. We investigated the sense-to-antisense ratio for Even though most novel TARs seem to be lowly expressed, we find a few interesting instances among these. Several clusters are detected downstream of a tRNA pseudogene on chromosome 3. We believe that this is the result of transcription which has been initiated upstream of the pseudogene and continues downstream (Figure 4A). The pseudogene itself has 100% sequence identity to another region in the genome (chr5:79,982,623–79,982,691), and since only reads that map uniquely to the genome were used in this analysis, this gene appears not to be expressed. Very high expression of EGFR is one of the hallmarks for the A431 cell line [16]. In figure 4B, a prolonged exon of the epidermal growth factor receptor (EGFR) is shown, along with two small clusters several tens of kilobases away. Whether or not the two small Figure 1. Scatter plot of RNA-seq (x-axis) versus microarray log2(fold change) (y-axis) for all protein coding genes (A), genes shorter than 2000 nts (B) and genes longer than 10 000 nts (C). Colors dots indicate the most similar microarray probe (black), the mean probe ratio (red) and median ratio (green). Identification of novel TARs To identify novel TARs, we merged reads from all three cell lines and created clusters from overlapping and nearby reads. After subtraction of known genes and non-coding RNA genes, we identify approximately 40,000 novel TARs, of which most are short (figure 3A). In fact, only 1360 TARs are longer than 500 base pairs and only 508 are longer than 1000 base pairs. Expression values for all TARs were calculated using the same approach as for protein-coding genes. This showed that most TARs are lowly expressed and covered by few reads. In A431, only approximately 10% (4144 TARs) are detected by 10 reads or more (figure 3B). The corresponding number in protein-coding genes is 33%, but this number is likely biased by the fact that protein-coding genes are generally longer than the putative novel TARs (data not shown). The method for RNA extraction used in this study excludes fragments shorter than approximately 200 nucleotides. This suggests that the majority of TARs identified in this study are in fact 200 nucleotides or longer and that deeper sequencing is needed to cover the entire TARs in order to define their boundaries. Comparison of RNA-seq and microarray gene expression data To validate the results obtained from RNA-seq, we compared the data to gene expression data from the A431 and U251 cell lines obtained using microarrays (no data was available for U-2 In this study, we use massive DNA sequencing to investigate RNA longer than 200 nucleotides from three human cancer cell March 2010 | Volume 5 | Issue 3 | e9762 1 Human Antisense Transcription OS). Since the microarray platform only generates relative expression values, the correlation between the RNA-seq data and the microarray data was calculated using the log2 value of the ratio between A431 and U251, which in the RNA-seq case yields one value per Ensembl-gene. Since one gene can be represented by several microarray probes, we used three different methods to convert these to a single value that could be compared to the RNA-seq data (mean, median and best probe, see Materials and Methods for details). The Spearman correlation was determined to 0.55, 0.55 and 0.64 for the three methods respectively, values in the same range as those described earlier [13]. Oshlack and Wakefield recently showed that the variance estimation of the RPKM measure is dependent on the gene length [14]. Thus, we hypothesized that the correlation between microarray data and RNA-seq data would share this dependence, since the log2-fold change in RNA-seq will have lower variance for longer genes that for shorter genes. This assumption turned out to be correct; for genes shorter than 2000 bases, the correlation was 0.48 to 0.52 depending on method, while for genes longer than 10 kb, this range was 0.59 to 0.71 (figure 1B–C and figure S2). different regions of the genome. In protein-coding exons, 98% to 99.5% of the reads originate from the sense strand, indicating that antisense transcripts are present at very low levels (figure 2C and figure S3). Interestingly, the sense-to-antisense ratio is markedly increased for promoter and terminator regions. In promoter regions, about 12% of the reads originate from the antisense strand, and in terminator regions, the fraction is 16%. In introns, the corresponding number increases to approximately 50% (figure 2C and figure S3). Investigation of antisense expression Longer genes correlate better with microarray data than short genes. doi:10.1371/journal.pone.0009762.g001 Figure 1. Scatter plot of RNA-seq (x-axis) versus microarray log2(fold change) (y-axis) for all protein coding genes (A), genes shorter than 2000 nts (B) and genes longer than 10 000 nts (C). Colors dots indicate the most similar microarray probe (black), the mean probe ratio (red) and median ratio (green). Longer genes correlate better with microarray data than short genes. doi:10.1371/journal.pone.0009762.g001 March 2010 | Volume 5 | Issue 3 | e9762 PLoS ONE | www.plosone.org 2 Human Antisense Transcription Human Antisense Transcription Figure 2. (A) Pie chart describing read mappings to different genomic regions. Almost half of all reads map to known genes (5 0 UTRs, CDS and 3 0 UTRs). A large fraction of reads map to regions outside promoters, known genes or terminator regions (red). A very low fraction of all reads map to antisense to protein coding genes. (B) Relative expression densities in different genomics regions. For the antisense strand, a small increase can be seen in promoter and terminator regions. Error bars are one standard deviation calculated across all three samples. (C) Fraction of reads that map to different regions in the genome. In introns, roughly half of all reads map to the antisense strand. Sense in light gray, antisense in dark gray. doi:10.1371/journal.pone.0009762.g002 Figure 2. (A) Pie chart describing read mappings to different genomic regions. Almost half of all reads map to known genes (5 0 UTRs, CDS and 3 0 UTRs). A large fraction of reads map to regions outside promoters, known genes or terminator regions (red). A very low fraction of all reads map to antisense to protein coding genes. (B) Relative expression densities in different genomics regions. For the antisense strand, a small increase can be seen in promoter and terminator regions. Error bars are one standard deviation calculated across all three samples. (C) Fraction of reads that map to different regions in the genome. In introns, roughly half of all reads map to the antisense strand. Sense in light gray, antisense in dark gray. doi:10.1371/journal.pone.0009762.g002 clusters are in fact novels exons remains to be investigated. In figure 4C, transcription is detected from both strands of a 1.3 Mb- region surrounding Peroxisome Proliferator-activated Receptor c Coactivator-1 a (PPARGC1A) on chromosome 4. Investigation of antisense expression Expression from this regions is detected at high levels in A-431 cells, but is almost completely shut off in U-2 OS and U-251 cells. This provides an intriguing example of complex transcription, and could indicate gene regulation through antisense transcript expression. This gene has been implicated in diabetes where lower expression has been linked to insulin resistance and DNA damage [17]. What functional role the antisense transcript plays remains to be elucidated. increased precision in expression level measurements for longer genes due to the fact that a higher number of reads will map to longer genes than short, as described earlier [14]. In this study, this means that the correlation between DNA microarrays and RNA- seq ranges from approximately 0.48 to 0.71 depending on which gene length and microarray probe selection method is used. However, this points to a larger statistical issue when using RNA- seq data to assess differential expression, since long genes will bias for example lists of differentially expressed genes between samples, and thus influence the power of gene set enrichment analysis negatively. Future research in this area will certainly have to address this issue, for example by improved statistical methods or by limiting the analysis to reads mapping to the 3 0 part of the gene with length equal to the shortest gene included in the analysis. Discussion We show that for approximately 20% of all human protein- coding genes, there is at least weak antisense transcription to exonic sequences. We also show that many of the antisense signatures overlap between the investigated cell lines (figure S3b). During recent years, several studies have indicated that 20–90% of In the current study we have investigated the transcriptional levels of three human cancer cell lines using RNA-seq. We show that the correlation between DNA microarray data and RNA-seq data depends on gene length, and that the reason for this is the Figure 3. (A) Histogram of detected TAR lengths. Many regions appear as shorter than 200 base pairs which is likely caused by the fact that they are very lowly expressed. The RNA extraction method captures fragments longer than 200 nucleotides, indicating that the majority of the detected TARs are in fact longer than we detect. (B) Bar plot describing number of reads in the detected TARs. Roughly 5 000 of the 40 000 TARs are detected by more than 10 reads. Error bars are one standard deviation calculated across all three samples. doi:10.1371/journal.pone.0009762.g003 Figure 3. (A) Histogram of detected TAR lengths. Many regions appear as shorter than 200 base pairs which is likely caused by the fact that they are very lowly expressed. The RNA extraction method captures fragments longer than 200 nucleotides, indicating that the majority of the detected TARs are in fact longer than we detect. (B) Bar plot describing number of reads in the detected TARs. Roughly 5 000 of the 40 000 TARs are detected by more than 10 reads. Error bars are one standard deviation calculated across all three samples. doi:10.1371/journal.pone.0009762.g003 PLoS ONE | www.plosone.org March 2010 | Volume 5 | Issue 3 | e9762 3 Human Human Antisense Transcription PLoS ONE | www.plosone.org 4 March 2010 | Volu March 2010 | Volume 5 | Issue 3 | e9762 PLoS ONE | www.plosone.org Human Antisense Transcription Human Antisense Transcription Figure 4. (A) Transcription downstream of a known tRNA pseudogene. Several TARs are detected in all three cell lines. The pseudogene itself has perfect identity to another region in the genome, so reads derived from is will not map unambiguously are are discarded. (B) Several TARs are detected downstream the gene EGFR. A group of them likely describe a prolonged 3 0 UTR. Two TARs further downstream could represent new exons. Materials and Methods A431, U-2 OS and U251 cells were grown as described earlier [21]. Cells were harvested and RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) following the manufac- turer’s instructions, and 15 m g of total RNA was used as input material for the SOLiD Whole Transcriptome kit (Applied Biosystems Inc., Foster City, CA) and 14 372 246, 10 547 681 and 11 449 673 reads (each 50 nucleotides) passed quality filters including filtering against adaptors. The reads were mapped to chromosomes 1–22, X and Y of the human genome (hg18) using Corona lite with default parameters (Life Technologies/Applied Biosystems). Identification of novel transcriptionally active regions To identify putative novel transcriptionally active regions, we clustered reads (using the online-version of Galaxy, [24] allowing for reads to be 15 bases apart and require at least three reads to be present to form a cluster. These first clusters were then merged across cell lines. We then subtracted clusters that overlap with known genes (as defined by Ensembl genes) as well as non-coding RNA genes (RNA genes, UCSC Genome Browser). Supporting Information Figure S1 Overlap of sense and antisense expression between the cell lines. Found at: doi:10.1371/journal.pone.0009762.s001 (0.23 MB PDF) Found at: doi:10.1371/journal.pone.0009762.s001 (0.23 MB PDF) Figure S2 Correlation to microarray data, binned per gene length in intervals of 2000 bps. See main text for discussion. Found at: doi:10.1371/journal.pone.0009762.s002 (11.70 MB TIF) Figure S2 Correlation to microarray data, binned per gene length in intervals of 2000 bps. See main text for discussion. Found at: doi:10.1371/journal.pone.0009762.s002 (11.70 MB TIF) Figure S3 Information on read mappings for additional cell lines. (D, G) Fraction of reads mapping to different regions. (E, H) Relative tag density in different regions. (F, I) Fraction reads mapping to the sense and antisense strand for different regions. See main text for discussion. Found at: doi:10.1371/journal.pone.0009762.s003 (0.20 MB PDF) Figure S3 Information on read mappings for additional cell lines. (D, G) Fraction of reads mapping to different regions. (E, H) Relative tag density in different regions. (F, I) Fraction reads mapping to the sense and antisense strand for different regions. See main text for discussion. Sense versus antisense expression regions To investigate the sense and antisense expression in different genomic regions, we calculated the number of reads that map to each region of interest. Some regions (coding regions, introns, 5 0 UTRs and 3 0 UTRs of protein-coding genes) were downloaded as BED-files from the UCSC table browser. Promoter and terminator regions were defined as 1 kb upstream or downstream of a protein-coding gene, respectively, similar to what has been used earlier [7]. If a neighbouring gene resides within the promoter or terminator region, the overlap with this gene was removed from the promoter or terminator region. For each region type, we calculated the expression density by counting number of reads that map entirely within the region type and normalized to the total length of the regions and the total amount of sense or antisense reads. This procedure yields one relative tag density value for each region type, sense and sample. We also calculated the sense-to-antisense ratio for each regions type and sample. The ENCODE project showed that transcription was frequent even outside of protein-coding genes [1], and with the recent emerge of new sequencing technologies, vast numbers of new transcriptionally active units have been detected. These TARs are situated in a non-random pattern along the chromosomes, indicating that they are not general background transcription. Some also show patterns of differential expression (figure 4). As more in-depth transcriptome studies deposit their data into publically available warehouses, such as Gene Expression Omnibus (http:www.ncbi.nlm.nih.gov/geo), more regions like these will likely be detected and characterized. It will be of great importance to functionally characterize these novel non-protein- coding transcripts and their potential role in gene regulation. Discussion The gene and TARs are detected at higher levels in A431 than the other samples, which agrees with previous knowledge [16]. (C) Massive transcription from a region around PPARGC1A. Transcription is detected from the sense strand several hundred kilobases downstream of the gene, as well as from the antisense strand several hundred kilobases upstream in A431. Transcription from this region is almost completely shut off in U251 and U-2 OS. d i 10 1371/j l 0009762 004 doi:10.1371/journal.pone.0009762.g004 doi:10.1371/journal.pone.0009762.g004 for the ratio A431 versus U251. Since one gene can be interrogated with several microarray probes, three different methods were used transform the microarray expression data to one value per gene; the mean of all probes, the median of all probes or the probe with the value closest to the RNA-seq data. We used Spearman’s rho to quantify the correlation between the two platforms. all human genes can generate antisense transcripts that can mediate regulation of the sense transcript [4–6]. Our study falls into the lower end of that interval, possibly indicating that deeper sequencing is required to investigate this phenomenon further. We also investigate antisense transcription in different regions of the genome. He and colleagues demonstrated that antisense tran- scription was prevalent upstream of transcription start sites, and Preker and colleagues showed that these transcripts are polyad- enylated and short (20–90 nucleotides) [7,18]. We do not identify such a pattern, which is likely explained by the fact that our study targets transcripts longer than 200 nucleotides. After clustering reads, we identify many novel TARs, most of which are shorter than 200 base pairs. This is likely due to the fact that they are generally lowly expressed, and a deeper sequencing of these samples would likely reveal the remaining parts of these TARs. Interestingly, we see approximately equal levels of transcription from both strands of introns of protein-coding genes. Non-protein coding intronic transcripts have been shown to be enriched in genes related to transcription regulation and interact with promoters to mediate regulation [19,20]. References 14. Oshlack A, Wakefield MJ (2009) Transcript length bias in rna-seq data confounds systems biology. Biol Direct 4: 14. 1. Birney E, Consortium EP, Stamatoyannopoulos JA, Dutta A, Guigo´ R, et al. (2007) Identification and analysis of functional elements in 1% of the human genome by the encode pilot project. Nature 447: 799–816. 15. Core LJ, Waterfall JJ, Lis JT (2008) Nascent rna sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322: 1845–1848. 2. Ebisuya M, Yamamoto T, Nakajima M, Nishida E (2008) Ripples from neighbouring transcription. Nat Cell Biol. 16. Lin CR, Chen WS, Kruiger W, Stolarsky LS, Weber W, et al. (1984) Expression cloning of human egf receptor complementary dna: gene amplification and three related messenger rna products in a431 cells. Science 224: 843–8. 3. Lapidot M, Pilpel Y (2006) Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms. EMBO Rep 7: 1216–22. 17. Lai CQ, Tucker KL, Parnell LD, Adiconis X, Garcı´a-Bailo B, et al. (2008) Ppargc1a variation associated with dna damage, diabetes, and cardiovascular diseases: the boston puerto rican health study. Diabetes 57: 809–16. 4. Chen J, Sun M, Kent WJ, Huang X, Xie H, et al. (2004) Over 20% of human transcripts might form sense-antisense pairs. Nucleic Acids Research 32: 4812–20. p y 18. Preker P, Nielsen J, Kammler S, Lykke-Andersen S, Christensen MS, et al. (2008) Rna exosome depletion reveals transcription upstream of active human promoters. Science 322: 1851–4. 5. Yelin R, Dahary D, Sorek R, Levanon EY, Goldstein O, et al. (2003) Widespread occurrence of antisense transcription in the human genome. Nat Biotechnol 21: 379–86. 19. Nakaya HI, Amaral PP, Louro R, Lopes A, Fachel AA, et al. (2007) Genome mapping and expression analyses of human intronic noncoding rnas reveal tissue-specific patterns and enrichment in genes related to regulation of transcription. Genome Biol 8: R43. 6. Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, et al. (2005) Antisense transcription in the mammalian transcriptome. Science 309: 1564–6. 7. He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW (2008) The antisense transcriptomes of human cells. Science 322: 1855–7. 20. Louro R, Smirnova AS, Verjovski-Almeida S (2009) Long intronic noncoding rna transcription: Expression noise or expression choice? Genomics 93: 291–298. 8. Seila AC, Calabrese JM, Levine SS, Yeo GW, Rahl PB, et al. (2008) Divergent transcription from active promoters. Science 322: 1849–1851. 21. Acknowledgments We would like to thank Dr. Emma Lundberg for providing cells and Uppsala Genome Center for technical assistance and helpful discussions. Also, Dr. Rickard Sandberg and Daniel Ramskld are acknowledged for valuable discussion on data analysis and provision of analysis scripts. p Found at: doi:10.1371/journal.pone.0009762.s004 (1.41 MB PDF) Found at: doi:10.1371/journal.pone.0009762.s004 (1.41 MB PDF) Figure S5 Smooth scatterplots of log10(antisense-rpkm) between samples. Spearman’s rho correlation coefficient is here slightly lower than that in the sense-case (supplementary figure S4). A reason for this could be that the majority of antisense transcripts are lowly expressed. It is also possible that these antisense transcripts have regulatory function and differ more than the bulk of mRNAs expressed in a cell. Comparison with microarray data Two-color DNA microarray data for the cell lines A431 and U251 was provided by Gry et al., and was pre-processed as described elsewhere [22]. The quality of the arrays has previously been addressed by comparison with MAQC data [23]. To allow for comparison to with RNA-seq data, RPKM expression levels were calculated for every Ensembl gene (http:www.ensembl.org) as described elsewhere [12], and a log2-fold change was calculated Found at: doi:10.1371/journal.pone.0009762.s003 (0.20 MB PDF) Found at: doi:10.1371/journal.pone.0009762.s003 (0.20 MB PDF) Figure S4 Smooth scatterplots of log10(rpkm) between samples along with Spearman’s rho correlation. The correlation is .87 to PLoS ONE | www.plosone.org March 2010 | Volume 5 | Issue 3 | e9762 March 2010 | Volume 5 | Issue 3 | e9762 5 Human Antisense Transcription .88 between all samples. This indicates that most genes have similar levels across all samples. .88 between all samples. This indicates that most genes have similar levels across all samples. Author Contributions Conceived and designed the experiments: JL. Performed the experiments: DK. Analyzed the data: DK MKB OE. Wrote the paper: DK MKB OE JL. p Found at: doi:10.1371/journal.pone.0009762.s005 (1.41 MB PDF) Found at: doi:10.1371/journal.pone.0009762.s005 (1.41 MB PDF) References Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, et al. (2007) Toward a confocal subcellular atlas of the human proteome. Molecular & Cellular Proteomics 7: 499–508. 9. Taft RJ, Glazov EA, Cloonan N, Simons C, Stephen S, et al. (2009) Tiny rnas associated with transcription start sites in animals. Nat Genet 41: 572–8. 10. Havgaard JH, Torarinsson E, Gorodkin J (2007) Fast pairwise structural rna alignments by pruning of the dynamical programming matrix. PLoS Comput Biol 3: 1896–908. 22. Gry M, Rimini R, Stro¨mberg S, Asplund A, Ponte´n F, et al. (2009) Correlations between rna and protein expression profiles in 23 human cell lines. BMC Genomics 10: 365. 11. Lindberg J, Lundeberg J (2009) The plasticity of the mammalian transcriptome. Genomics. 23. Klevebring D, Gry M, Lindberg J, Eidefors A, Lundeberg J (2009) Automation of cdna synthesis and labelling improves reproducibility. Journal of Biomedicine and Biotechnology 2009: 1–8. 12. Mortazavi A, Williams BA, Mccue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by rna-seq. Nat Meth. 8 p. 24. Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, et al. (2005) Galaxy: a platform for interactive large-scale genome analysis. Genome Res 15: 1451–5. quantifying mammalian transcriptomes by rna-seq. Nat Meth. 8 p. 13. Fu X, Fu N, Guo S, Yan Z, Xu Y, et al. (2009) Estimating accuracy of rna-seq and microarrays with proteomics. BMC Genomics 10: 161. PLoS ONE | www.plosone.org March 2010 | Volume 5 | Issue 3 | e9762 6
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Rethinking University Presidencies in Canadian Higher Education
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https://johepal.com Journal of Higher Education Policy And Leadership Studies JHEPALS (E-ISSN: 2717-1426) Rethinking University Presidencies in Canadian Higher Education Lawrence A. White Faculty of Education, Unicaf University, CANADA Email: larry.ookpik@gmail.com https://orcid.org/0000-0003-4459-4499 https://johepal.com Journal of Higher Education Policy And Leadership Studies JHEPALS (E-ISSN: 2717-1426) Rethinking University Presidencies in Canadian Higher Education Lawrence A. White Faculty of Education, Unicaf University, CANADA Email: larry.ookpik@gmail.com https://orcid.org/0000-0003-4459-4499 https://johepal.com Cite article as: White, L. A. (2023). Rethinking university presidencies in Canadian higher education. Journal of Higher Education Policy and Leadership Studies, 4(3), 157- 166. https://dx.doi.org/10.61186/johepal.4.3.157 Leadership Studies JHEPALS (E-ISSN: 2717-1426) Rethinking University Presidencies in Canadian Higher Education Lawrence A. White Faculty of Education, Unicaf University, CANADA Email: larry.ookpik@gmail.com https://orcid.org/0000-0003-4459-4499 Article Received Article Accepted Published Online 2023/05/27 2023/09/07 2023/09/30 Rethinking University Presidencies in Canadian Higher Education Published Online 2023/09/30 Article Accepted 2023/09/07 Published Online 2023/09/30 Article Accepted 2023/09/07 Cite article as: White, L. A. (2023). Rethinking university presidencies in Canadian higher education. Journal of Higher Education Policy and Leadership Studies, 4(3), 157- 166. https://dx.doi.org/10.61186/johepal.4.3.157 White, L. A. Journal of Higher Education Policy And Leadership Studies (JHEPALS) E-ISSN: 2717-1426 Volume: 4 Issue: 3 pp. 157-166 DOI: 10.61186/johepal.4.3.157 Rethinking University Presidencies in Canadian Higher Education E-ISSN: 2717-1426 Volume: 4 Issue: 3 pp. 157-166 DOI: 10.61186/johepal.4.3.157 Keywords: Higher Education Leadership; University Presidents; University Culture; Career Complexity; Leadership Roles Abstract This article discusses the evolving role of university presidents in Canada. The university president is accountable for the successful operation of a university and achieving its strategic goals. The role has become complex due to factors such as the absence of a standard definition of a university, decentralization of funding, and increased competition for student enrollment and research funding. Universities have grown considerably, leading to more diverse and activist populations, larger budgets, and the need for more people to manage internal and external relationships. University presidents must now balance the interests of various stakeholders such as faculty, boards, alumni, governments, and learners. They must also plan for and address the challenges created by system modernization and enterprise technologies, internationalization, campus health and security, changing governments, shifting policy directions, climate change, social justice, and neoliberalism. This article argues that university presidents must possess strong communication skills, impeccable management acumen, and the ability to relate to the evolving realities of learners. This article concludes that the role of university president has evolved from being presiding figures to being accountable leaders who must navigate the complex demands of the modern university system. Lawrence A. White * Lawrence A. White * Keywords: Higher Education Leadership; University Presidents; University Culture; Career Complexity; Leadership Roles *Corresponding author’s email: larry.ookpik@gmail.com *Corresponding author’s email: larry.ookpik@gmail.com E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 157 E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 Rethinking University Presidencies “Being president of a university is no way for an adult to make a living. Which is why so few adults actually attempt to do it. It is to hold a mid-nineteenth-century ecclesiastical position on top of a late-twentieth-century corporation”. A. Bartlett Giametti, 19th President of Yale University, 1978-1986 The World Changed Unexpectedly, the COVID-19 pandemic occasioned the single largest disruption of education ever experienced resulting in the closure of learning spaces in some 190 countries and affecting 1.6 billion learners (United Nations, 2020), as well as educators and families. It tested and continues to test the resilience, fortitude, and ingenuity of educational leaders worldwide. As the world becomes more globalized and interconnected, as recognition grows regarding planetary limits to our civicidal (Hancock, 2019) commodification fetish (Marx, 1887), as violence and environmental disaster lead to mass migration and refugeeism (Talbot, 2013), as populism threatens acceptance of science as reality (Wall et al., 2017) and distorts reality through the “weaponisation of information” (Ireton & Posetti, 2018, p. 15), as the world reckons with institutions and processes grounded in putative privilege for some (Carr, 2016; McIntosh, 1988), and as the gap between marginalized populations and the rest of the world grows (Corbett, 2008; Leal Filho et al., 2020), the world turns to education, and its leaders, for solutions (Kwauk, 2020; Wals & Benavot, 2017). In fact, the OECD (2018) suggests that education may be the difference between people embracing or being defeated by future challenges (Common Worlds Research Collective, 2020). Educational leadership continues to grow increasingly complex as a profession as does the pressure on an educational leader to perform and succeed (Cafley, 2015). This paper explores the evolving and increasingly complex role of university presidents focusing predominantly on the characteristics and attributes fostering efficacy. Specifically, this paper considers post-secondary education leadership in a Canadian context through both historical and future visionary lenses. Evolution of a Complex Role Establishing the Dominion of Canada in 1867 under the British North America Act created, perhaps, the earliest and longest lasting of challenges facing university presidents. To assure a relatively even distribution of power and authority between federal and provincial governments, Section 93 of The Act gave the provinces authority over education. To confound matters further, responsibility for academic research remained with the federal government. Consequently, presidential advocacy efforts for funding are decentralized. Likewise, there is neither a standard definition of ‘university’ across Canada (Usher, 2018) nor a standard system of accreditation creating a network of unique educational systems between which student and credit transferability is not streamlined (Paul, 2011). That said, decentralization also created stability for university presidents by preventing national interference in and requiring the collaboration of 13 jurisdictions to make changes to the mandate for universities (Paul, 2011). Canada remains the only federated country without a national Ministry of Education. Influenced greatly by French and British colonialism, the majority of Canadian universities prior to 1960 were faith based (Paul, 2011; Usher, 2018). Since c.1960, however, most universities are secular, many revising their purpose to take advantage of changes to ongoing provincial funding requiring a public, secular governance model. In Canada, the separation of state and church in education is now well established. In the latter half of the twentieth century, the university sector in Canada experienced tremendous growth both in the number of institutions and the proportion of the population engaged in university study (Paul, 2011). Growth in demand and numbers of competitors increases the pressure, challenging educational leaders to increase both depth and breadth of academic programming and to find ways to distinguish their university from others. Absent a complementary increase in funding, growth also created challenges such as larger class sizes, greater student/teacher ratios, the growing use of sessional and other part-time instructors (Paul, 2011), and the seemingly ubiquitous task of ephemeralization. In the early 2000s, more than a few colleges and other institutes made the strategic transformation to become universities (Usher, 2018). These transitions in status create greater direct competition between universities for student enrollment, both domestic and international, and for faculty expertise. At the same time, more universities now vie for limited funding available for research. University Presidents The university president is the chief executive officer of the organization and is formally accountable to a Board of Governors or Senate for the fiscal and reputational operation of a university as well as for the successful achievement of strategic plans, goals, and objectives. Michael et al. (2001, p. 332) describe the university president as “the most visible embodiment of institutional mission, vision and culture”. Paul (2011) concurs and views the role as the leader and manager of a university’s big picture while Woodsworth likens the role to a translator: "communicating between cultures and across borders, conveying the sense of the academic enterprise to external stakeholders and board members, and interpreting the business-related concerns and preoccupations of a responsible board to the internal community" (2013, p. 134). Journal of Higher Education Policy And Leadership Studies (JHEPALS) 158 Evolution of a Complex Role In their previous iterations as colleges rooted in more technical and applied learning, the new universities also create dilemmas of culture and self- identity for others that viewed their central roles as research institutions by blurring the lines between academic research and career preparedness. In Canada, this often materializes as conflicts of perspective and motivation among faculty who view research as their mandate and career preparedness as specifically not their responsibility (Spencer, 2014). Unions for post-secondary faculty and staff, akin to those throughout education in Canada, are particularly effective in representing these and other interests on behalf of their members, increasing the complexity and criticality of communication and interpersonal relations for university presidents. In the past, university communities comprised faculty with longevity and a more homogeneous full-time student body that resided close to campus. Times were simpler; organizations less complex. University presidents, usually men with previous Canadian post- secondary executive experience, presided over their institutions more than they managed E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 159 Rethinking University Presidencies or led (Paul, 2011). The president of a small university might know all students, staff, and faculty; teach one or two courses; represent the university in the community and with government; and attend or host community events. The role was somewhat low-key because “universities were not generally in the public eye” (Paul, 2011, p. 12). Contemporary universities have much larger, more diverse, and activist student, staff, and faculty populations. Some have operating budgets in excess of $500 million and all have multiple departments staffed with people to manage internal and external relationships. As Paul (2011) delineates, university presidents are now accountable to: (a) faculty with interests in academic freedom, profile, prestige, and reputation; (b) a board of governors or senate that values integrity, financial stability, impeccable management acumen, and stellar communication skills; (c) alumni seeking strong and stable, but growing, community relationships; (d) governments needing change agents to weave academic culture with fiscal performance and the successful achievement of strategic goals; and (e) learners who, increasingly, have growing obligations (with university studies being just one of many) and needing a leader who can relate to their evolving realities. Adding in system modernization and enterprise technologies; internationalization and globalization; campus health, well- being, safety, and security; politics, changing governments, and shifting policy directions; changing and increasingly less reliable funding mechanisms; evolving best practices in learning and teaching; and competing and conflicting demands from almost every possible direction, it is clear that presidential workloads are expanding and that expectations for success are more numerous, diverse, and acute than ever (Khalideen, 2014; Fullan & Leithwood, 2012; Paul, 2011). The continuously mounting and intensifying responsibility, and concomitant complexity, required to lead an increasingly public institution led to the development of a satirical job posting: Wanted: A miracle worker who can do more with less, pacify rival groups, endure chronic second-guessing, tolerate low levels of support, process large volumes of paper, and work double shifts (75 nights a year out). He or she will have carte blanche to innovate, but cannot spend much money, replace any personnel, or upset any constituency. (Evans, 1995, p. 23) Wanted: A miracle worker who can do more with less, pacify rival groups, endure chronic second-guessing, tolerate low levels of support, process large volumes of paper, and work double shifts (75 nights a year out). Rethinking University Presidencies He or she will have carte blanche to innovate, but cannot spend much money, replace any personnel, or upset any constituency. (Evans, 1995, p. 23) Challenges of Culture Perhaps the single greatest challenge facing a university president, especially one who may have recently been appointed from outside the organization, is developing an understanding about and participating effectively and respectfully within the culture of the university. It can be a delicate balance between being culturally aware and respectful, and finding ways to move a new agenda for change positively forward, including finding opportunities to voice a few key strategic initiatives as frequently as possible (Paul, 2011). Ultimately, the fit of a president within the culture of a university often determines success going forward (Cafley, 2015; Paul, 2011). Every stakeholder, internal and external, has a personal perspective on fit and the efficacy of the person in the role. To help navigate the challenge of innumerable perspectives that, I would argue, is not dissimilar to simultaneous, diverse, continuous, on-the-job performance evaluations, Bennis (2009) identifies six core competencies required of a university president: (a) have a strong sense Journal of Higher Education Policy And Leadership Studies (JHEPALS) 160 Journal of Higher Education Policy And Leadership Studies (JHEPALS) Presidencies Looking Forward Presidential roles are influenced significantly by current events, especially if those events arise from systemic social oppression or systemic consumerism. Freire (2000) advocates empowering individual consciousness to overcome oppression in education. In Canada, this is best exemplified by three significant social uprisings: (a) the calls to action arising from the Truth and Reconciliation Commission’s final report (see Truth and Reconciliation Commission of Canada, 2015); (b) the rise of Black Lives Matter and its founding principles of diversity, intersectionality, empathy, and engagement (see Potvin, 2020); and (c) the #MeToo movement that challenged post-secondary leaders to acknowledge that an absence of policy and action may have increased vulnerabilities, predominantly those of female students and staff, on campuses across the country (see MacArthur, 2019). Similarly, Orr (1992, 2020) and others (Common Worlds Research Collective, 2020; Leal Filho et al., 2020; Reid, 2019; Suzuki, 1998) advocate taking action to mitigate the “long emergency” (Orr, 2020, p. 13) created by our tendency to overlook anthropocentric causes of the climate crisis facing the planet. Both the global #FridaysForFuture movement demanding adults believe climate science and take action against climate change (see Bowman, 2019; Pickard, 2019), and the fossil fuel divestment movement in Canadian academia (see Maina et al., 2020) are affecting policy directions and, therefore, affecting the qualities and characteristics of an effective university president going forward. I argue that, in order to be both relevant and successful in leading universities, presidents of the 21st century must be responsive to these and other growing public demands for changes in policy and action, particularly regarding systemic issues. Future presidents will be those who can capitalize on university culture, build on past accomplishments of the institution, implement an agenda focused on teaching learners how to learn, foster agency in learners, and ensure they have a solid foundation of skills to function and adapt to the 21st century workplace and climate (Bates, 2019; OECD, 2018). In order to produce graduates who are active citizens that know how to “navigate through uncertainty” (OECD, 2018, p. 5), presidents must be able to anticipate ambiguous future global and community needs and to champion this direction within the university community and among external partners. Furthermore, this should promote the strengths and opportunities that come with diversity, inclusivity, collaboration, and respect for the valuable contributions each person brings. Journal of Higher Education Policy And Leadership Studies (JHEPALS) 160 White, L. A. of mission, (b) foster support for the mission among others, (c) establish relationships that are flexible and adaptable, (d) engender optimism and trust, (e) develop leadership qualities in others, and (f) achieve results. Cafley (2015) combines the first two of Bennis’ competencies, suggesting that these and success of the organization might be better served by working toward these collaboratively with the university community. Campbell (2003/2016) describes fit within organizational culture as the basis for a solid, positive, successful working relationship with its president. E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 Presidencies Looking Forward Presidents must be prepared to change mindsets; specifically, to promote and instill a cultural proficiency mindset across the university community (Lindsey et al., 2019). That is, they must come with some form of vision for changing the general perception of cultural differences as problematic to one of intercultural interaction that celebrates diversity and, E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 161 E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 Rethinking University Presidencies academically, to one that recognizes the value of cultural uniqueness as a foundation from which to create learning experiences. Such presidents would be confident enough in themselves, their beliefs, values, and worldviews to be robustly self-reflective and sufficiently self-disruptive and open to reconsidering, and changing, deep-rooted meanings. They must model critical thinking, creative problem solving, and communication transparently, respectfully, and collaboratively. They may wish to surround themselves with a senior executive team that shares and believes in the mission and strategic vision but that may have perspectives on pathways to success that differ and which, together, facilitate, foster, and nurture the development of emergent citizens who are prepared for the challenges of any eventuality (Islam et al., 2006). Learning from the COVID-19 experience, university presidents must come with a plan to work with the university community and with the executive team, specifically, to strengthen the capacities for risk and resilience of the institution, to reimagine education and the right to equitable learning, to remove barriers to learning, and to support the professional development of all faculty and staff (United Nations, 2020). E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 161 Human Participants p Human participants were not consulted or otherwise involved in this research. Funding Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Conclusion From climate to technology, politics to academics, and from global health to individual people, the education landscape today is shifting and evolving at an ever-increasing pace. Canadian Prime Minister Justin Trudeau commented at the 2018 World Economic Forum in Davos, Switzerland, that “the pace of change has never been this fast, yet it will never be this slow again” (World Economic Forum, 2018, 5:34). The roles and spheres of influence of universities have expanded greatly in recent decades. No longer can the role of a university president appear as a concise list of management and leadership duties on a single page job posting. “Leadership within a system of education that has been comfortable, predictable and ordered, to one that is new, uncertain and chaotic is no small task” (Khalideen, 2014, p. 44). Instead, in the coming decades, university presidents will be required to work and interact with stakeholders simultaneously at the micro, meso, and macro levels and to develop autonomy and agency across a team, indeed across an entire community, in order to support them in this work (Fullan & Leithwood, 2012). They must demonstrate an overtly high degree of social respect, emotional intelligence, spiritual wisdom, integrity, and maturity (Astin & Astin, 2000) as well as sufficient self-awareness, self-efficacy, and self- disruptive openness to consider changing their own deep-rooted belief systems and worldviews. The future requires a university leader who genuinely understands, in a deeply personal way, the value of respectful and equitable collaboration in the co-creation of knowledge (Harris et al., 2019) and “learning to become with the world” (Common Worlds Research Collective, 2020). I conclude with a word of caution. In moving in these directions and in having these expectations of university leaders, we run the risk of expecting them to be “all things to all people” (Evans, 1995, para. 8). 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United Nations. https://www.un.org/sites/un2.un.org/files/sg_policy_brief_covid19_and_education_august_ 2020.pdf 20 Usher, A. (2018). The state of post-secondary education in Canada, 2018. Higher Education Strategy Associates. https://higheredstrategy.com/wp- content/uploads/2018/08/HESA SPEC 2018 final pdf Rethinking University Presidencies Rethinking University Presidencies Mr. Lawrence A. White is an advanced graduate student pursuing a Doctor of Education (EdD) at Unicaf University. His current research interests lie at the intersection of curriculum and pedagogical design and development with supporting learner mental health. In a grounded theory study rooted in lived experience and pedagogical journey mapping, he hopes to identify new pedagogical approaches that mindfully reduce or remove academic stressors that students invariably experience on their academic journeys in the hope of reducing challenges to mental well-being and improving academic success. White also holds a Master of Distance Education (MDE) and a Bachelor of Environmental Studies (BES). He worked for 20+ years in various increasingly senior leadership roles in Canadian colleges and universities throughout his career, is active with several global research projects related to mental health, and volunteers with the Global Mental Health Peer Network. He now lives in Yellowknife, Northwest Territories, Canada. Mr. Lawrence A. White is an advanced graduate student pursuing a Doctor of Education (EdD) at Unicaf University. His current research interests lie at the intersection of curriculum and pedagogical design and development with supporting learner mental health. In a grounded theory study rooted in lived experience and pedagogical journey mapping, he hopes to identify new pedagogical approaches that mindfully reduce or remove academic stressors that students invariably experience on their academic journeys in the hope of reducing challenges to mental well-being and improving academic success. White also holds a Master of Distance Education (MDE) and a Bachelor of Environmental Studies (BES). He worked for 20+ years in various increasingly senior leadership roles in Canadian colleges and universities throughout his career, is active with several global research projects related to mental health, and volunteers with the Global Mental Health Peer Network. He now lives in Yellowknife, Northwest Territories, Canada. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format for non-commercial purposes only, and only so long as attribution is given to the creator. Journal of Higher Education Policy And Leadership Studies (JHEPALS) 166 Journal of Higher Education Policy And Leadership Studies (JHEPALS) content/uploads/2018/08/HESA_SPEC_2018_final.pdf Wall, T., Hindley, A., Hunt, T., Peach, J., Preston, M., Hartley, C., & Fairbank, A. (2017). Work-based learning as a catalyst for sustainability: A review and prospects. Higher Education, Skills and Work-Based Learning, 7(2), 211-224. https://doi.org/10.1108/HESWBL-02-2017-0014 Wals, A. E. J., & Benavot, A. (2017). Can we meet the sustainability challenges? The role of education and lifelong learning. 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М.М. Алимурзаева1,2, С.М. Изудинова2, А.М. джафарова1,2, Р.А. Халилов1 1Дагестанский государственный университет, Махачкала, Дагестан 2Медико-санитарная часть МВД по Республике Дагестан, Махачкала, Дагестан Автор, ответственный за переписку: Альбина Мехьядиновна Джафарова, albina19764@mail.ru Аннотация. Цель: изучение влияния новой коронавирусной инфекции на ряд стандартных биохимических пока­ зателей крови у больных разного возраста. Материалы и методы: исследовано 514 пациентов трёх возрастных групп (I группа — 25-35лет, II группа — 36-50 лет, III группа — 51-75 лет) с диагнозом «Внебольничная пневмония, вызван­ ная инфекцией COVID-19». Определение всех биохимических параметров крови больных производили на биохимиче­ ском анализаторе Indiko. Результаты: исследование показателей азотистого метаболизма показало, что у больных всех возрастных групп содержание общего белка и мочевины не претерпевает существенных изменений, однако при этом происходит повышение концентрации креатинина и мочевой кислоты, наиболее выраженное у пациентов III группы. Исследование активностей АСТ, АЛТ в крови больных показало существенное их повышение у всех возрастных кате­ горий, более значимое — у лиц II и III возрастных групп. При этом с возрастном увеличиваются активность а-амилазы, концентрации билирубина и снижаются уровни железа, достигая патологических значений. Содержание маркера вос­ паления (СРБ) демонстрирует наиболее выраженную зависимость от возраста больных: у больных I группы оно уве­ личивается в 6,9 раз, у II — в 12,3 раза, у лиц старше 51 года — в 17,25 раз. Вывод: с увеличением возраста больных COVID-19 отклонения многих биохимических показателей от соответствующих каждой группе контрольных уровней становятся более выраженными и достигают значений, существенно отклоняющихся от референсных. К COVID 19 б б Ключевые слова: COVID-19, возраст больных, кровь, биохимические параметры. Финансирование. Исследование не имело спонсорской поддержки. , р , р , р р Финансирование. Исследование не имело спонсорской поддержки. Для цитирования: Алимурзаева М.М., Изудинова С.М., Джафарова А.М., Халилов Р.А. Возрастные особенности из­ менений биохимических параметров крови при COVID-19. Медицинский вестник Юга России. 2023;14(2):90-96. DOI 10.21886/2219-8075-2023-14-2-90-96 Оригинальная статья УДК 616.9 https://doi.org/10.21886/2219-8075-2023-14-2-90-96 Оригинальная статья УДК 616.9 https://doi.org/10.21886/2219-8075-2023-14-2-90-96 УДК 616.9 https://doi.org/10.21886/2219-8075-2023-14-2-90-96 Медицинский вестник Юга России 2023; 14(2):90-96 © М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов, 2023 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ 3.1.22 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ 3.1.22 Материалы и методы Ретроспективное исследование проводили на базе Медико-санитарной части МВД по РД (г. Махачкала). В период начала пандемии коронавирусной инфекции (апрель-май 2020 г.) в госпитале было организовано отде­ ление для пациентов с диагнозом «внебольничная пнев­ мония, вызванная инфекцией COVID-19» (подтверж­ дённая обнаружением РНК SARS-CoV-2 или, в редких случаях — только данными КТ). Исследованы биохими­ ческие показатели крови больных на момент госпитали­ зации в стационар на 6,5±2,5 день болезни. Критериями включения в группы для проведения сравнительного ана­ лиза были 1-й день госпитализации больных, мужской пол, средняя степень тяжести процесса, поражение лёг­ ких, по данным КТ, от 20% до 50%. Критериями исключе­ ния из исследования служили наличие в анамнезе пере­ несённого инфаркта миокарда и инсульта, нестабильной стенокардии, сердечной недостаточности II-IV степени, эндокринных заболеваний. Вирус может проникать в клетки эпителия желудка, тонкой и толстой кишки. Имеются сведения о специфи­ ческом поражении сосудов (эндотелия), миокарда, по­ чек и других органов. Установлено, что диссеминация SARS-CoV-2 из системного кровотока или через пла­ стинку решетчатой кости может привести к поражению головного мозга. Обнаружена возможность специфиче­ ского поражения лимфоцитов и гиперактивности ма­ крофагов [3]. Исследовано три группы разных возрастных кате­ горий: I группа — 109 пациентов возрастом 25-35 лет, II группа — 310 пациентов возрастом 36-50 лет, III груп­ па — 95 пациентов возрастом 51-75 лет. В качестве кон­ троля исследованы биохимические параметры мужчин тех же возрастных групп, не болеющих COVID-19 и не имеющих выраженных патологий со стороны сердечно­ сосудистой системы, органов пищеварения, эндокринной системы, выделительной системы. Забор венозной крови производился утром натощак в первые сутки поступле­ ния больных в специальные вакуумные пробирки с акти­ ватором свертывания. Определение всех биохимических параметров в сыворотке крови производили на биохими­ ческом анализаторе Indiko (Thermal Fisher, США). При критическом течении COVID-19 вследствие дис- регуляции синтеза провоспалительных и антивоспали­ тельных цитокинов развивается патологическая реакция, называемая «цитокиновым штормом». Гиперактивация иммунного ответа при COVID-19 часто ограничивается лёгочной паренхимой, прилегающей альвеолярной лим­ фоидной тканью и ассоциируется с развитием острого респираторного дисстресс-синдрома (ОРДС). У пациен­ тов с критическим течением развивается васкулярная эн­ дотелиальная дисфункция, коагулопатия, тромбозы. Ци- токиновый шторм при COVID-19, как правило, приводит к полиорганной недостаточности и может стать причи­ ной летального исхода [4]. Статистическую обработку данных производили с помощью однофакторного дисперсионного анали­ за (ANOVA) с использованием пакета прикладных про­ грамм Statistica 8.0 (StatSoft, Inc., США). Нормальность распределения оценивали критерием Шапиро-Уилка. До­ стоверность различий между нормально распределенны­ ми данными определяли с помощью критерия Фишера на уровне значимости p < 0.05. Medical Herald of the South of Russia 2023; 14(2):90-96 Введение Цель исследования — изучение влияния новой коро­ навирусной инфекции на ряд стандартных биохимиче­ ских показателей крови у больных разного возраста В 2019 г. произошла вспышка острой респираторной инфекции, вызванной вирусом Sars-Cov-2 семейства ко­ роновирусов, которая быстро распространилась по все­ му миру и привела к глобальной пандемии Covid 19. На­ чальным этапом заражения является проникновение SARS-CoV-2 в клетки-мишени, имеющие рецепторы ан- гиотензинпревращающего фермента II типа (АСЕ-2) [1]. Рецепторы АСЕ-2 экспрессированы на поверхности раз­ личных клеток органов дыхания, пищевода, кишечни­ ка, сердца, надпочечников, мочевого пузыря, головно­ го мозга (гипоталамуса) и гипофиза, а также эндотелия и макрофагов. Однако основной и быстро достижимой мишенью SARS-CoV-2 являются альвеолярные клетки II типа лёгких, что определяет развитие диффузного аль­ веолярного повреждения [2]. INFECTIOUS DISEASES 3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19) ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS For citation: Alimurzaeva M.M., Izudinova S.M., Dzhafarova A.M., Khalilov R.A. Age aspects of the effects of the new corona- virus infection (COVID 19) on certain biochemical blood parameters. Medical Herald of the South of Russia. 2023;14(2):90-96. DOI 10.21886/2219-8075-2023-14-2-90-96 Материалы и методы Данные в таблице приведены в следующем виде: среднее (M) ± ошибка среднего (m). Высокая летальность и громадные социально­ экономические последствия эпидемии COVID-19 требу­ ют анализа имеющихся наблюдений и разработки мето­ дов эффективной терапии и профилактики осложнений. Известно, что возраст и сопутствующая патология могут быть факторами, предопределяющими тяжёлое течение COVID-19. Прежде всего к группе риска относятся по­ жилые или лица с хроническими заболеваниями. Однако и у молодых людей без хронических заболеваний также могут возникнуть потенциально летальные осложнения, такие как молниеносный миокардит и диссеминирован­ ная внутрисосудистая коагулопатия (ДВС-синдром) [5]. Таким образом, риск инфицирования и тяжёлого течения болезни или летального исхода затрагивает всех и имеет­ ся во всех возрастных группах. В связи с этим возникает необходимость анализа клинико-лабораторных особен­ ностей течения новой коронавирусной инфекции у боль­ ных различных возрастных категорий. M.M. Alimurzaeva1,2, S.M. Izudinova2, A.M. Dzhafarova1,2, R.A. Khalilov1 Dagestan State University, Makhachkala, Dagestan 2Medical and sanitary unit of the Ministry of Internal Affairs for the Republic of Dagestan, Makhachkala, Dagestan Corresponding author: Albina M. Dzhafarova, albina19764@mail.ru Abstract. Objective: to study the effects of a new coronovirus infection on a number of standard biochemical blood parameters in patients of different ages. Materials and methods: 514 patients of 3 age groups (group 1st — 25-35 years old, group 2nd — 36-50 years old and group 3rd — over 51 years old) with a diagnosis of community-acquired pneumonia caused by COVID-19 infection were studied. All biochemical parameters of the blood of patients were determined using an Indiko biochemical analyzer. Results: the study of nitrogen metabolism indicators showed that in patients of all age groups, the content of total protein and urea does not undergo significant changes, however, there is an increase in the concentration of creatinine and uric acid, which is most pronounced in patients of the group 3rd. The study of the activities of AST, ALT in the blood of patients showed a significant increase in all age categories, more significant in persons of the groups 2nd and 3rd. At the same time, the activity of a-amylase, the concentration of bilirubin increase with age, and the levels of iron decrease, reaching pathological values. The content of the inflammation marker — CRP shows the most pronounced dependence on the age of patients: in patients of the group 1st, it increases by 6.9 times, in the 2nd group — by 12.3 times, in patients over 51 years old — by 17.25 once. Conclusion: with an increase in the age of patients, the deviations of many biochemical parameters from the control levels corresponding to each group become more pronounced and reach values that deviate significantly from the reference K d COVID 19 age of patients blood biochemical parameters Keywords: COVID-19, age of patients, blood, biochemical paramet Keywords: COVID-19, age of patients, blood, biochemical parameters. Finansing. The study did not have sponsorship. 90 INFECTIOUS DISEASES 3.1.22 Результаты У больных различных возрастных категорий были исследованы биохимические показатели азотистого ме­ таболизма, характеризующие синтетическую функцию печени, распад белков и нуклеиновых кислот с образова­ нием небелковых низкомолекулярных азотсодержащих конечных продуктов. Исследования показало, что у всех возрастных групп содержание общего белка и мочевины не претерпевает существенных изменений (табл. 1). 91 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИ М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ 3.1.22 Таблица / Table 1 Содержание маркеров азотистого метаболизма в крови больных COVID-19 The content of markers of nitrogen metabolism in the blood of patients with COVID-19 Возраст пациентов, лет Age of patients Общий белок (г/л) Total protein (g/l) Мочевина (мМоль/л) Urea (pmol/l) Креатинин (мкМ/л) Creatinine (pmol/l) Мочевая кислота (мкМ/л) Uric acid (pmol/l) 25-35 Контроль Control (n=32) 73,68±0,84 4,73±0,23 75,04±0,93 259,50±15,04 Больные Sick (n =83) 71,12±0,74 4,89±0,14 96,16±2,05* 358,0±23,67* 36-50 S 3 " (n =36) 71,85±0,83 5,17±0,29 90,41±2,13 259,0±15,04 Б , Г “ (n=288) 69,83±0,65 4,91±0,15 100,82±3,89* 371,14±21,53* 51-75 Контроль Control (n =33) 75,66-±1,22 5,47±0,28 79,57±1,05 255,60±22,20 Больные Sick (n =66) 68,85±1,36 6,80±0,95 115,14±6,59*+ 410,60±31,17* Референтные интервалы Reference intervals 65-85 3,2-7,3 74-110 210-420 Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — II группой. Note: p<0,05 compared to * — the control group, + group 1st, # — group 2nd. Содержание маркеров азотистого метаболизма в крови больных COVID-19 The content of markers of nitrogen metabolism in the blood of patients with COVID-19 Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — II группой. Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — II группой. Note: p<0,05 compared to * — the control group, + group 1st, # — group 2nd. Note: p<0,05 compared to * — the control group, + group 1st, # — group 2nd. Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — Медицинский вестник Юга России 2023; 14(2):90-96 Медицинский вестник Юга России 2023; 14(2):90-96 Медицинский вестник Юга России Note: p<0,05 compared to * — the control group, + group 1st, # — group 2nd. INFECTIOUS DISEASES 3.1.22 Из рисунка 1, видно, что у больных I воз­ растной категории содержание СРБ увеличивается в 6,9 раз (p<0,05), у II — в 12,3 раза (p<0,05), у лиц старше 51 года — в 17,25 раз (p<0,05). Высокие значения СРБ у исследованных нами больных с коронавирусной инфек­ цией, особенно старшего возраста, указывают на про­ грессирующее развитие острой воспалительной реакции. Во время инфекционных заболеваний помимо клас­ сических маркеров воспаления также могут происхо­ дить изменения и в обмене железа. Из рисунка 2 видно, что содержание железа уменьшается у I группы на 54,2% (p<0,05), а у II — на 74,2% (p<0,05), достигая уровня пата- логически низких значений. Такое же выраженное сни­ жение содержания железа пнаблюдается у пациентов из III группы — 66,7%. Таким образом, у больных старших возрастных категорий концентрации данного микроэле­ мента становятся существенно ниже предела референт­ ных значений. Коронавирусная инфекция инициирует у больных всех возрастных категорий выраженную воспалитель­ ную реакцию. Об этом свидетельствует повышение уров­ ня С-реактивного белка (СРБ) — классического маркера воспаления. Из рисунка 1, видно, что у больных I воз­ растной категории содержание СРБ увеличивается в 6,9 раз (p<0,05), у II — в 12,3 раза (p<0,05), у лиц старше 51 года — в 17,25 раз (p<0,05). Высокие значения СРБ у исследованных нами больных с коронавирусной инфек­ цией, особенно старшего возраста, указывают на про­ грессирующее развитие острой воспалительной реакции. Так у I группы активность АСТ выше в 1,9 раз, а АЛТ — в 2,1 раз по сравнению со здоровыми донорами того же возраста (p<0,05). У пациентов старших возраст­ ных групп наблюдается ещё более выраженное повыше­ ние активности данных ферментов: у II группы АСТ уве­ личивается в 2,7 раз, а АЛТ — в 2,60 раз, а III — АСТ в 2,6 раза, АЛТ — в 2,5 раза (p<0,05). Анализ активности АЛТ и АСТ у больных старших возрастных групп по­ зволил предположить существенное нарушение прони­ цаемости цитоплазматических (маркер — АЛТ) и мито­ хондриальных мембран (маркер — АСТ) клеток печени. Интересно то, что содержание билирубина в I группе не изменяется, что указывает на сохранение функциональ­ ной активности гепатоцитов и эпителия желчевыводя­ щих путей. Однако у II группы концентрация общего би­ лирубина увеличивается в 3 раза, а прямого — в 2,4 раза Во время инфекционных заболеваний помимо клас­ сических маркеров воспаления также могут происхо­ дить изменения и в обмене железа. Результаты Note: p<0 05 compared to * the controlgroup + group 1st # group 2nd Таблица / Table Активность клеточных ферментов и содержание билирубина в крови больных COVID-19 The activity of cellular enzymes and the content of bilirubin in the blood of patients with COVID-19 Таблица / Table 2 Активность клеточных ферментов и содержание билирубина в крови больных COVID-19 The activity of cellular enzymes and the content of bilirubin in the blood of patients with COVID-19 Возраст пациентов, лет Age of patients АСТ (Е/л) AST (U/l) АЛТ (Е/л) ALT (U/l) Общий билирубин (мкмоль/л) Total bilirubin (pmol/l) Прямой билирубин (мкмоль/л) Direct bilirubin (pmol/l) а-Амилаза (Е/л) a-amylase (U/l) 25-35 Контроль Control (n=32) 24,83 ±1,83 24,72 ±9,50 10,31 ±4,08 4,90 ±0,39 46,40 ±3,69 Больные Sick (n =83) 39,72 ±2,95 * 52,43 ±5,98 * 11,13 ±5,37 5,32 ±0,32 69, 00 ±6,80 * 36-50 Контроль Control (n =36) 26,58 ±1,63 29,72 ±1,62 10,52 ±0,65 5,26 ±0,47 57,71 ±5,67 Больные Sick (n=288) 71,65 ±5,03 *+ 77,58 ±4,30 *+ 31,82 ±2,24 *+ 12,61 ±0,84 *+ 86,26 ±5,78 51-75 Контроль Control (n =33) 28,85 ±2,61 35,23 ±3,49 9,21 ±0,61 4,61 ±0,40 60,20 ±3,05 Больные Sick (n =66) 75,21 ±8,86 *+ 89,05 ±13,09 *+ 23,33 ±1,56 *+ 12,46 ±2,06 *+ 111,75 ±4,09 *+ # Референтные интервалы Reference intervals < 40 Е/л < 40 Е/л 3,4-20,5 < 9,2 < 100 Табли Активность клеточных ферментов и содержание билирубина в крови больных COVID-19 The activity of cellular enzymes and the content of bilirubin in the blood of patients with COVID-19 92 92 INFECTIOUS DISEASES 3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19) ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS Medical Herald of the South of Russia 2023; 14(2):90-96 INFECTIOUS DISEASES 3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19) ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19) ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS При этом наблюдается повышение креатинина и мо­ чевой кислоты, уровни которых зависят от возраста па­ циентов. Так, у I группы концентрации данных продук­ тов азотистого метаболизма увеличиваются на 28,2% и 38,2% (p<0,05), у I — на 50% и 43,3% (p<0,05), а у III — на 44,3% и 61% (p<0,05) относительно соответствующих контрольных групп. Интересно то, что сравнительный анализ содержания креатинина и мочевой кислоты у больных II и III возрастных групп не выявил статистиче­ ски значимых различий. Следует отметить, что, несмо­ тря на существенное повышение содержание креатини­ на и мочевой кислоты у больных всех возрастных групп, данные параметры всё же остается в пределах референс- ных значений. (p<0,05), а у III — в 2,52 раз и в 2,7 раз соответственно (p<0,05). Интересно то, что сравнительный анализ всех указанных выше параметров (АСТ, АЛТ, билирубина) между разными группами больных выявил, что стати­ стически значимые различия имеют место только между I группой и каждой из 2-х других групп, в то время как между II и III группами различия несущественны. (p<0,05), а у III — в 2,52 раз и в 2,7 раз соответственно (p<0,05). Интересно то, что сравнительный анализ всех указанных выше параметров (АСТ, АЛТ, билирубина) между разными группами больных выявил, что стати­ стически значимые различия имеют место только между I группой и каждой из 2-х других групп, в то время как между II и III группами различия несущественны. У больных COVID-19 I и II групп наблюдается повы­ шение (на 48,7% и 49,5 % (p<0,05)) активности а-амилазы, однако оно укладывается в диапазон референсных значе­ ний данного показателя. Значительное (на 85,6%) и вы­ ходящие за пределы референсных значений увеличение активности этого фермента наблюдается у III возрастной категории (p<0,05). Исследование активности клеточных ферментов — ха­ рактерных маркеров цитолиза различных клеток, в част­ ности гепатоцитов (АСТ, АЛТ) и клеток поджелудочной железы (а-амилазы) — показало, что во всех возрастных группах содержание данных ферментов в сыворотке кро­ ви резко возрастает (табл. 2). Коронавирусная инфекция инициирует у больных всех возрастных категорий выраженную воспалитель­ ную реакцию. Об этом свидетельствует повышение уров­ ня С-реактивного белка (СРБ) — классического маркера воспаления. INFECTIOUS DISEASES 3.1.22 Из рисунка 2 видно, что содержание железа уменьшается у I группы на 54,2% (p<0,05), а у II — на 74,2% (p<0,05), достигая уровня пата- логически низких значений. Такое же выраженное сни­ жение содержания железа пнаблюдается у пациентов из III группы — 66,7%. Таким образом, у больных старших возрастных категорий концентрации данного микроэле­ мента становятся существенно ниже предела референт­ ных значений. В, О О I — 25-35 лет II — 36-50 лет III — 51-75 лет 1st — 25-35 years old 2nd — 36-50 years old 3rd — 51-75 years old контроль control больные patients p<0,05 по сравнению с * — контрольной группой, + — I группой, # — II группой. p<0,05 compared to * — the control group, + — group 1st, # — group 2nd. Рисунок 1. Содержание СРБ в сыворотке крови больных COVID-19 различных возрастных групп Figure 1. The content of CRP in the blood serum of patients with COVID-19 of different age groups Рисунок 1. Содержание СРБ в сыворотке крови больных COVID-19 различных возрастных групп Figure 1. The content of CRP in the blood serum of patients with COVID-19 of different age groups 93 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ 3.1.22 я с;о S S о" 5о £а. сГ I — 25-35 лет II — 36-50 лет III — 51-75 лет 1st — 25-35 years old 2nd — 36-50 years old 3rd — 51-75 years old контроль control больные patients p<0,05 по сравнению * — с контрольной группой, + — I группой, # — II группой. p<0,05 compared to * — the control group, + — group 1st, # — group 2nd. Рисунок 2. Содержание железа в сыворотке крови больных COVID-19 различных возрастных групп Figure 2. The content of iron in the blood serum of patients with COVID-19 of different age groups М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ 3.1.22 Рисунок 2. Содержание железа в сыворотке крови больных COVID-19 различных возрастных групп Figure 2. The content of iron in the blood serum of patients with COVID-19 of different age groups Сравнительный анализ содержания СРБ и железа в крови больных разного возраста выявил, что статисти­ чески значимые различия имеют место только между I группой и каждой из других групп, в то время как между II и II группой различия несущественны. INFECTIOUS DISEASES 3.1.22 скелетной мускулатуры и других органов. Повышение её активности в сыворотке крови может быть связано с вы­ ходом фермента при цитолизе клеток миокарда, печени и др. Поскольку активности и АСТ и АЛТ у больных Co- vid-19 увеличиваются в равной степени, то это указывает на некроз гепатоцитов. Медицинский вестник Юга России 2023; 14(2):90-96 INFECTIOUS DISEASES 3.1.22 липазы у 1-2 и 17 % больных с нетяжёлым и тяжёлым те­ чением соответственно. Хотя любое серьёзное заболева­ ние может быть ассоциировано с гипергликемией, свя­ занной со стрессом. Yang J. K. и соавт. ранее описали, что пациенты с SARS (вызванным SARS-CoV, «двоюродным братом» SARS-CoV-2), которые никогда не получали глю­ кокортикоиды, имели значительно более высокий уро­ вень глюкозы в плазме крови натощак по сравнению с пациентами с пневмонией без SARS. В другом исследова­ нии в качестве вероятного механизма развития «острого диабета» у пациентов с SARS было предложено опосредо­ ванное SARS-CoV повреждение ^-клеток поджелудоч­ ной железы [10]. старшей возрастной группы. Предполагается, что сниже­ ние уровня железа происходит за счёт поглощения и де­ понирования его активированными гистиоцитами (ма­ крофагами). Железо накапливается в макрофагах в виде ферритина, перенос которого от ферритина к трансфер- рину нарушается, в результате концентрация ферритина в крови увеличивается в 3 и более раз, что мы и наблюда­ ем у пациентов больных COVID-19 [3]. Medical Herald of the South of Russia 2023; 14(2):90-96 ЛИТЕРАТУРА / REFERENCES 1. Lan J, Ge J, Yu J, Shan S, Zhou H, et al. Structure of the SARS- CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581(7807):215-220. https://doi.org/10.1038/s41586-020-2180-5 cell in culture. Int J Biol Macromol. 2011;49(1):79-84. doi: 10.1016/j.ijbiomac.2011.03.018 cell in culture. Int J Biol Macromol. 2011;49(1):79-84. doi: 10.1016/j.ijbiomac.2011.03.018 cell in culture. Int J Biol Macromol. 2011;49(1):79-84. doi: 10.1016/j.ijbiomac.2011.03.018 cell in culture. Int J Biol Macromol. 2011;49(1):79- doi: 10.1016/j.ijbiomac.2011.03.018 7. Zheng KI, Gao F, Wang XB, Sun QF, Pan KH, et al. Letter to the Editor: Obesity as a risk factor for greater severity of COVID-19 in patients with metabolic associated fatty liver disease. Metabolism. 2020;108:154244. doi: 10.1016/j.metabol.2020.154244 2. Perrella A, Trama U, Bernardi FF, Russo G, Monastra L, et al. Editorial - COVID-19, more than a viral pneumonia. Eur Rev Med Pharmacol Sci. 2020;24(9):5183-5185. https://doi.org/10.26355/eurrev_202005_21216 8. Liu PP, Blet A, Smyth D, Li H. The Science Underlying COVID-19: Implications for the Cardiovascular System. Circulation. 2020;142(1):68-78. doi: 10.1161/CIRCULATIONAHA.120.047549 p g 3. Полушин Ю.С., Шлык И.В., Гаврилова Е.Г., Паршин Е.В., Гинзбург А.М. Роль ферритина в оценке тяжести COVID-19. Вестник анестезиологии и реаниматологии. 2021;18(4):20-28. Polushin Yu.S., Shlyk I.V, Gavrilova E.G., Parshin E.V, Ginzburg A.M. The Role of Ferritin in Assessing COVID-19 Severity. Messenger of anesthesiology and resuscitation. 2021;18(4):20-28. (In Russ.) https://doi.org/10.21292/2078-5658-2021-18-4-20-28 9. Liu F, Long X, Zhang B, Zhang W, Chen X, Zhang Z. ACE2 Expression in Pancreas May Cause Pancreatic Damage After SARS-CoV-2 Infection. Clin Gastroenterol Hepatol. 2020;18(9):2128-2130.e2. doi: 10.1016/j.cgh.2020.04.040 https://doi.org/10.21292/2078-5658-2021-18-4-20-28 10. Yang JK, Feng Y, Yuan MY, Yuan SY, Fu HJ, et al. Plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with SARS. Diabet Med. 2006;23(6):623-8. doi: 10.1111/j.1464-5491.2006.01861.x 4. Chan JF, Zhang AJ, Yuan S, Poon VK, Chan CC, et al. Simulation of the Clinical and Pathological Manifestations of Coronavirus Disease 2019 (COVID-19) in a Golden Syrian Hamster Model: Implications for Disease Pathogenesis and Transmissibility. Clin Infect Dis. 2020;71(9):2428-2446. doi: 10.1093/cid/ciaa325 11. Андреева Е.А. С-реактивный белок в оценке пациентов с респираторными симптомами до и в период пандемии COVID-19. РМЖ. 2021;6:14-17. Andreeva E.A. C-reactive protein in the assessment of patients with respiratory symptoms before and during the COVID-19 pandemic. RMJ. 2021;6:14-17. (in Russ.). eLIBRARY ID: 46433896 5. Madjid M, Safavi-Naeini P, Solomon SD, Vardeny O. Potential Effects of Coronaviruses on the Cardiovascular System: A Review. JAMA Cardiol. 2020;5(7):831-840. doi: 10.1001/jamacardio.2020.1286 6. Aragao DS, Cunha TS, Arita DY, Andrade MC, Fernandes AB, et al. Выводы Коронавирусная инфекция, вызванная SARS-CoV2, приводит к существенным изменениям ряда стандарт­ ных биохимических параметров крови (креатинина, мо­ чевой кислоты, АСТ, АЛТ, а- амилазы) даже у людей мо­ лодого возраста (25-35 лет). С повышением возраста больных отклонения многих биохимических показателей от соответствующих каждой группе контрольных уров­ ней становятся более выраженными и достигают значе­ ний, существенно отклоняющихся от референсных. При этом сравнительные межгрупповые исследования пока­ зали, что обнаруженные различия между многими био­ химическими показателями больных II (36-50 лет) и III (51-75 лет) возрастной групп не являются статистически достоверными. Таким образом, статистический анализ лабораторных параметров крови больных из различных возрастных групп показал, что возраст является значи­ мым предиктором тяжёлого течения COVID-19, но не аб­ солютным, поскольку риски неблагоприятного развития заболевания могут затрагивать людей разного возраста. В соответствии с результатами нашего исследования в крови больных короновирусной инфекцией существен­ но повышается содержание СРБ и снижается уровень железа. СРБ — острофазный белок, уровни которого в крови резко повышаются при острых респираторных заболеваниях и используются для их диагностики. Мо­ ниторинг маркеров воспаления, в том числе СРБ, в рам­ ках биохимического анализа крови при COVID-19 вклю­ чён в национальные рекомендации многих стран, в том числе в России. При этом, по данным ресурса UpToDate, концентрация СРБ, ассоциированная с тяжёлым течени­ ем COVID-19, превышает 100 мг/л [11]. Таким образом, больные старшей возрастной группы, содержание СРБ в крови которых является критическим, являются наибо­ лее подверженной рискам тяжёлого течения COVID-19. Исследование указывает на значительное снижение уровня железа в сыворотке крови, особенно у больных Обсуждение Khalilov AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19) ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS 1. Lan J, Ge J, Yu J, Shan S, Zhou H, et al. Structure of the SARS- CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581(7807):215-220. https://doi.org/10.1038/s41586-020-2180-5 Обсуждение Можно предположить разные механизмы поврежде­ ния печени: в результате прямого цитопатического дей­ ствия вируса, токсических эффектов комплексной те­ рапии COVID-19-инфекции. Важное воздействие на тяжесть течения заболевания оказывают также хрониче­ ские гепатиты разной этиологии, жировой гепатоз, ток­ сическое и аутоиммунное повреждение, а также цирроз печени. В печени также выявляются признаки, характер­ ные для вирусного поражения (апоптоз и регенерацию гепатоцитов) и дистрофию (жировую и баллонную) ге- патоцитов [7]. Причём, чем более тяжёлое течение забо­ левания было отмечено, тем чаще выявлялись признаки поражения печени. Исследование стандартных биохимических параме­ тров крови, характеризующих состояние различных ор­ ганов и систем организма, свидетельствует о том, что ко­ ронавирусная инфекция, вызванная Sars-Cov-2, приводит к развитию целого ряда структурно-функциональных на­ рушений. При этом наблюдается следующая закономер­ ность: чем старше возраст больных, тем более выражены патологические эффекты вирусной экспансии. Анализ конечных продуктов азотистого метаболизма показал повышение содержания креатинина и мочевой кислоты в сыворотке крови больных COVID-19. это мо­ жет указывать на нарушение клиренса данных низкомо­ лекулярных веществ почками, вызванными структурно­ функциональными изменениями нефронов. Известно, что одним из важнейших органов-мишеней COVID-19 могут быть почки, что, вероятно, связано с широким представи­ тельством АСЕ-2 в организме человека, который является точкой входа SARS-CoV-2. В почках АСЕ-2 присутствует в подоцитах, мезангиальных клетках, париетальном эпи­ телии капсулы Боумена, нефроцитах проксимальных ка­ нальцев и клетках собирательных трубочек [6]. Повышение активности АСТ может быть также и мар­ кером повреждения кардиомиоцитов. По данным обще­ мировой статистики, у 19 % пациентов, госпитализиру­ емых с COVID-19, наблюдаются признаки поражения сердца (боли в грудной клетке, гипотензия, аритмия, при­ знаки сердечной недостаточности), также отмечаются слу­ чаи брадиаритмии, острого коронарного синдрома [8]. Более высокая активность амилазы в сыворотке крови больных COVID-19 может быть обусловлена нарушени­ ями проницаемости клеток поджелудочной железы. Из­ вестно, что ACE-2 усиленно экспрессируется в поджелу­ дочной железе, причем уровень мРНК в поджелудочной железе выше, чем в лёгких. экспрессия ACE2 наблюдается как в клетках экзокринной части поджелудочной железе, так и эндокринных клетках островков [9]. Повреждение экзокринной части поджелудочной железы проявляет­ ся повышением уровня сывороточной амилазы и/или Исследование показало, что у всех групп больных ко­ роновирусной инфекцией происходит в разной степени выраженное повышенные уровни активности фермен­ тов АСТ и АЛТ. Оба фермента распространены во всех тканях. Поскольку наибольшая концентрация АЛТ от­ мечается в печени, то данный фермент является специ­ фическим маркёром заболеваний этого органа. АСТ со­ держится в тканях сердца, печени, почек, нервной ткани, 94 INFECTIOUS DISEASES 3.1.22 INFECTIOUS DISEASES INFECTIOUS DISEASES 3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19) ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS Медицинский вестник Юга России 2023; 14(2):90-96 Information about the authors ф р ц р Алимурзаева Марьям Мурадовна, магистр 2-го года обучения Дагестанского государственного уни­ верситета, фельдшер-лаборант Медико-санитарная часть МВД по Республике Дагестан, Махачкала, Россия; alimurzayeva95@mail.ru. ф р р Алимурзаева Марьям Мурадовна, магистр 2-го года обучения Дагестанского государственного уни­ верситета, фельдшер-лаборант Медико-санитарная часть МВД по Республике Дагестан, Махачкала, Россия; alimurzayeva95@mail.ru. Maryam M. Alimurzaeva, master of the 2nd year of study of the Dagestan State University, medical assistant-laboratory assistant of the Medical and Sanitary Department of the Ministry of Internal Affairs for the Republic of Dagestan, Makhachkala, Russia; alimurzayeva95@mail.ru. Sagibat M. Izudinova, laboratory assistant of Medical and sanitary unit of the Ministry of Internal Affairs for the Republic of Dagestan, Makhachkala, Russia; izudinovas@gmail.com. Изудинова Сагибат Магомедовна, врач-лаборант, Медико-санитарная часть МВД по Республике Дагестан, Махачкала, Россия; izudinovas@gmail.com. Sagibat M. Izudinova, laboratory assistant of Medical and sanitary unit of the Ministry of Internal Affairs for the Republic of Dagestan, Makhachkala, Russia; izudinovas@gmail.com. Изудинова Сагибат Магомедовна, врач-лаборант, Медико-санитарная часть МВД по Республике Дагестан, Махачкала, Россия; izudinovas@gmail.com. джафарова Альбина Мехьядиновна, доцент кафедры биохимии и биофизики Дагестанского государственного университета, врач-лаборант, Медико-санитарная часть МВД по Республике Дагестан, Махачкала, Россия; albi- na19764@mail.ru; https://orcid.org/0000-0001-7744-859x Albina M. Dzhafarova, Associate Professor of the Department of Biochemistry and Biophysics of the Dagestan State University, laboratory assistant at the Medical and Sanitary Unit of the Ministry of Internal Affairs for the Republic of Dagestan, Makhachkala, Russia; albina19764@ mail.ru; https://orcid.org/0000-0001-7744-859x Халилов Рустам Абдуразакович, заведующий ка­ федрой биохимии и биофизики Дагестанского государ­ ственного университета, Махачкала, Россия; dagbiofak@ mail.ru; https://orcid.org/0000-0001-9369-1645 Rustam A. Khalilov, Head of the Department of Biochemistry and Biophysics, Dagestan State University, Makhachkala, Russia. E-mail: dagbiofak@mail.ru; https:// orcid.org/0000-0001-9369-1645 Rustam A. Khalilov, Head of the Department of Biochemistry and Biophysics, Dagestan State University, Makhachkala, Russia. E-mail: dagbiofak@mail.ru; https:// orcid.org/0000-0001-9369-1645 Authors' contribution Алимурзаева Марьям Мурадовна — получение и ана­ лиз данных; Alimurzaeva Maryam Muradovna — data acquisition and analysis; Izudinova Sagibat Magomedovna — review of publications on the topic of the article; Izudinova Sagibat Magomedovna — review of publications on the topic of the article; Изудинова Сагибат Магомедовна — обзор публикаций по теме статьи; Джафарова Альбина Мехьядиновна — интерпретация данных, написание текста рукописи; Dzhafarova Albina Mekhyadinovna — interpretation of data, writing the text of the manuscript; Dzhafarova Albina Mekhyadinovna — interpretation of data, writing the text of the manuscript; Халилов Рустам Абдуразакович — разработка дизай­ на исследования, окончательное утверждение версии для публикации. Khalilov Rustam Abdurazakovich — study design development, final approval of the version for publication. Khalilov Rustam Abdurazakovich — study design development, final approval of the version for publication. ЛИТЕРАТУРА / REFERENCES Purification and characterization of angiotensin converting enzyme 2 (ACE2) from murine model of mesangial 95 95 Medical Herald of the South of Russia 2023; 14(2):90-96 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов ВОЗРАСТНЫЕ ОСОБЕННОСТИ И ЗМ ЕН ЕН И й БИОХИМИЧЕСКИХ ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ 3.1.22 ИНФЕКЦИОННЫЕ БОЛЕЗНИ 3.1.22 Conflict of interest Авторы заявляют об отсутствии конфликта интересов. Authors declares no conflict of interest. Поступила в редакцию / Received: 16.02.2023 Доработана после рецензирования / Revised: 28.03.2023 Принята к публикации / Accepted: 11.04.2023 Поступила в редакцию / Received: 16.02.2023 Доработана после рецензирования / Revised: 28.03.2023 Принята к публикации / Accepted: 11.04.2023 96 96
https://openalex.org/W2943055093
https://europepmc.org/articles/pmc6529697?pdf=render
English
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NUPR1 acts as a pro-survival factor in human bone marrow-derived mesenchymal stem cells and is induced by the hypoxia mimetic reagent deferoxamine
Journal of clinical biochemistry and nutrition
2,019
cc-by
5,737
Kazuhito Matsunaga,1,# Koichi Fujisawa,1,2,# Taro Takami,1,* Guzel Burganova,1,3 Nanami Sasai,1,4 Toshihiko Matsumoto,1,5 Naoki Yamamoto1 and Isao Sakaida1,2 Kazuhito Matsunaga,1,# Koichi Fujisawa,1,2,# Taro Takami,1,* Guzel Burganova,1,3 Nanami Sa Toshihiko Matsumoto,1,5 Naoki Yamamoto1 and Isao Sakaida1,2 1Department of Gastroenterology and Hepatology, 2Center for Regenerative Medicine, 4Department of Laboratory Science and 5Department of Oncology and Laboratory Medicine, Yamaguchi University School of Medicine, Minami Kogushi 111, Ube, Yamaguchi 7558505, Japan 3Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russia 5 (Received 19 December, 2018; Accepted 25 December, 2018; Published online 23 March, 2019) requires a specific equipment and cells may be temporarily exposed to non-hypoxia states during medium exchange. Thus, the possi- bility to employ hypoxia-mimetic reagents, such as deferoxamine (DFO), an iron chelator, has drawn much attention.(6) Iron deple- tion by DFO mimics hypoxia by inhibiting iron-dependent prolyl-4 hydroxylase activity and, thereby, HIF1a hydroxylation and degradation. DFO preconditioning in Wharton’s jelly-derived neural-like cells has been shown to improve tolerance and thera- peutic potential, and enhance the effectiveness of cell therapy.(7) It has been reported that MSC preconditioning with DFO, before transplantation, increases the homing capacity of MSCs and endo- thelial progenitor cells(8) and improves cell therapy.(9) Moreover, hypoxic preconditioning potentiates the trophic and anti-apoptotic effects of MSCs on hepatocytes(10) or brain cells.(11) On the other hand, additional investigation is necessary to clarify the molecular basis of DFO effects. We have compared the changes in MSC metabolism induced by DFO and hypoxia, and found that low concentrations of DFO may be appropriate for MSC precondi- tioning.(12) Copyright © 2019 JCBN 2019 This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unre- stricted use, distribution, and reproduction in any medium, pro- vided the original work is properly cited. Differences in the culturing conditions of mesenchymal stem cells used in regenerative medicine may affect their differentiation ability, genome instability, and therapeutic effects. In particular, bone marrowderived mesenchymal stem cells cultured under hypoxia are known to proliferate while maintaining an undiffer entiated state and the use of deferoxamine, a hypoxia mimetic reagent, has proven to be a suitable strategy to maintain the cells under hypoxic metabolic state. Here, the deferoxamine effects were investigated in mesenchymal stem cells to gain insights into the mechanisms regulating stem cell survival. A 12h deferox amine treatment reduced proliferation, oxygen consumption, mitochondrial activity, and ATP production. Microarray analysis revealed that deferoxamine enhanced the transcription of genes involved in glycolysis and the HIF1α pathway. #Equally contributed. *To whom correspondence should be addressed. Email: ttakami@yamaguchiu.ac.jp Kazuhito Matsunaga,1,# Koichi Fujisawa,1,2,# Taro Takami,1,* Guzel Burganova,1,3 Nanami Sasai,1,4 Toshihiko Matsumoto,1,5 Naoki Yamamoto1 and Isao Sakaida1,2 Among the earliest changes, transcriptional variations were observed in HIF1α, NUPR1, and EGLN, in line with previous reports showing that short deferoxamine treatments induce substantial changes in mesenchymal stem cells glycolysis pathway. NUPR1, which is induced by stress and involved in autophagymediated survival, was upregulated by deferoxamine in a concentrationdependent manner. Consistently, NUPR1 knockdown was found to reduce cell proliferation and increase the proapoptotic effect of stauro sporine, suggesting that deferoxamineinduced NUPR1 promotes mesenchymal stem cell survival and cytoprotective autophagy. Our findings may substantially contribute to improve the effec tiveness of mesenchymal stem cellbased regenerative medicine. Nuclear protein 1 (NUPR1) (also known as p8) is a molecule that has recently caught much attention owing to its association with chemotherapy resistance in a wide range of cancer types. Its suppression of cell cycle via p53 and cytoprotective autophagy induction have been previously reported.(13) To date, NUPR1 has mainly been studied in cancer cells, where it supports drug resis- tance. However, the underlying mechanisms have not yet been clarified in detail. Moreover, although an involvement of NUPR1 in the survival of mesenchymal stem cells has been speculated, no clues are available, to date, regarding its function in MSCs. Original Article Original Article J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 209–216 doi: 10.3164/jcbn.18112 ©2019 JCBN Results Furthermore, fluorescent immunohistochemical staining of cells treated with 10 mM DFO and CQ showed an increase in the LC3-positive puncta, suggesting DFO-induced autophagy enhancement (Fig. 3B). An analysis of NUPR1 expression revealed a DFO concentration-dependent increase 48 h after DFO treat- ment (Fig. 3C). In light of the observed DFO-induced NUPR1 up- regulation, we next verified whether similar expression changes could be induced by hypoxia and found that these conditions also increased NUPR1 expression (Fig. 3D). In addition, enhanced NUPR1 expression was detected after a 2-h cell incubation with HBSS, an amino acid starvation medium known to enhance autophagy (Fig. 3E). y y g y y y ( ) Oxygen consumption rate (OCR) measurements. OCR measurements were performed using a Seahorse Biosciences XF96 Extracellular Flux Analyzer. Cells were seeded at 6,000/well in XF96 microplates (Seahorse Biosciences, Santa Clara, CA). After a 24-h incubation, the growth medium was replaced with XF Assay Medium (Seahorse Biosciences) supplemented with 25 mM glucose (Sigma-Aldrich, Darmstadt, Germany). OCR measurements were made over 5-min periods following a 3-min mix period. The following components were sequentially added to the cells: 1 mg/mL oligomycin (Sigma-Aldrich), 300 nM carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; Sigma-Aldrich), and 2 mM rotenone (MP Biomedicals, Tokyo, Japan). The spare respiratory capacity and coupling efficiency were calculated according to the Seahorse Bioscience instructions and the basal OCR was normalized to the cell number. Immunofluorescence Analysis. MSCs were cultured in chamber slides, and then fixed in 4% paraformaldehyde. Immuno- fluorescence analysis was performed by using the Opal Multiplex Immunohistochemistry Kit (Perkin Elmer, Norwalk, CT) as per manufacturer’s instructions. NUPR1 knockdown reduces MSC proliferation and decreases drug resistance. Next, to directly explore the role of NUPR1 in MSC proliferation, its expression was suppressed by siRNA knockdown. The knockdown was efficient and resulted in clear LC3-II upregulation (Fig. 4A). Cell proliferation was slightly reduced in NUPR1-knocked down cells, but in the pres- NUPR1 knockdown reduces MSC proliferation and decreases drug resistance. Next, to directly explore the role of NUPR1 in MSC proliferation, its expression was suppressed by siRNA knockdown. The knockdown was efficient and resulted in clear LC3-II upregulation (Fig. 4A). Cell proliferation was slightly reduced in NUPR1-knocked down cells, but in the pres- Statistical analysis. The homogeneity of variance was assessed by the F-test. Results DFO suppresses mitochondrial oxygen consumption and lowers the level of intracellular ATP. In order to investigate the effects of DFO on cell proliferation, BMSCs were treated with different drug concentrations, for 12 and 48 h, followed by cell culturing in DFO-free medium. Proliferation was then evaluated by IncuCyte HD imaging system. We found that treat- ment for 48 h with 3 mM DFO, a concentration that is effective for medium preconditioning, and for 12 h with 50 mM DFO, resulted in comparable effects on cell proliferation (Fig. 1A). Next, the amount of ATP per cell was measured and found to be substantially reduced at DFO concentrations above 100 mM (Fig. 1B). To investigate the reason behind the ATP depletion, the OCR (oxygen consumption rate) was measured with a flux analyzer, revealing a detectable decrease of both basal and maximal respiration with 10 mM DFO, and a drastic reduction with 100 mM and 300 mM (Fig. 1C). Cell and cell culture. Bone marrow cells were purchased from (Lonza, Basel, Switzerland) and MSCs were cultured in DMEM medium, and cells at passages 3–8 were used. The MSC phenotype, CD11b(-), CD90(+), CD73(+), and CD105(+), was confirmed by flow cytometry. y y y Proliferation assay. To measure proliferation, 1,000 MSCs were seeded in each well of a 96-well plate, DFO was then added at different concentrations, and the cellular area was evaluated with an IncuCyte HD imaging system (Essen BioScience, London, UK). DFO was purchased from Novartis Pharma K.K. (Tokyo, Japan). Apoptosis assay. After siRNA treatment, 3,000 cells were seeded in each well of a 96-well plate and cultured for 3 days. Next, cells were treated with staurosporine (0.3 mM) and 6 h later the cell number was measured with a CyQUANT® cell pro- liferation assay (Thermo Fisher Scientific, Waltham, MA). Cells were then evaluated with a Caspase-3 Assay Kit (Promega, Tokyo, Japan) and caspase-3 activity per cell was measured. The results were obtained from three independent experiments with 6 replicates each. m m ( g ) DFO treatment alters the expression of genes for energy metabolism. After treatment of MSCs with 50 mM DFO for 12 h, RNA was extracted for microarray analysis. Functional classification of genes was performed using the IPA software, and pathways involved in sugar metabolism, such as glycolysis and gluconeogenesis, HIF1a-mediated and VEGF-mediated signaling, were classified as Canonical pathways (Fig. 2A). Results Exam- ination of the glycolysis pathway revealed an upregulation of the enzymes glucose 6 phosphate isomerase, 6-phosphofructokinase, fructose-bisphosphate aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, and phosphopyruvate hydratase (Fig. 2B). In the HIF1a pathway, due to HIF1a degradation inhibition by DFO, HIF1a was downregulated, and HIF1a target genes, namely GLUT, LDH and VEGF, were upregulated (Fig. 2C). Among the upstream DFO-induced transcriptional changes, HIF1a was mostly activated, and hypoxia-induced EPAS1 and PDGF were also upregulated. Notably, NUPR1, which is known to be involved in mitochondrial stress, was activated. On the other hand, EGLN (hypoxia-inducible factor prolyl hydroxylase) and curcumin were among the suppressed upstream factors (Fig. 2D). Western blot analysis. Western blotting was performed according to a standard method.(14) In brief, cell lysis buffer contained 62.5 mM Tris-HCl (pH 6.8), 4% SDS, and 200 mM dithiothreitol. Cell lysates were electrophoresed on 12% acryl- amide gels. Anti b-actin antibody (Sigma, Darmstadt, Germany), anti-p62 antibody (Abcam, Tokyo, Japan), anti-LC3 antibody (Life Technologies, Waltham, MA), and anti-NUPR1 antibodies (ProteinT, Chicago, IL) were used. Total RNA isolation. Total RNA was isolated from cells using TRIzol Reagent (Life Technologies) and purified using SV Total RNA Isolation System (Promega) according to the manufac- turer’s instructions. RNA samples were quantified by an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and RNA quality was confirmed with an Experion System (Bio-Rad Laboratories, Hercules, CA). ) SAGE. Ion Ampliseq Transcriptome Human Gene Expression Kit (Life Technologies) was used to create an Ion Proton next- generation sequencer library of analysis beads. An Ion PI IC 200 Kit (Life Technologies) and an Ion PI Chip Kit v2 BC were used for sequencing, using an Ion Proton next-generation sequencer. SAGE results were analyzed by Ingenuity Pathways Analysis (IPA). O ti t (OCR) t OCR DFO treatment enhances NUPR1 expression and auto phagy. In a previously reported microarray analysis, we found that NUPR1 expression was increased in MSCs treated with 3 mM DFO for 48 h.(12) Moreover, NUPR1 was found to be involved in cytoprotective autophagy. Thus, we examined the impact of DFO on autophagy. Interestingly, LC3-II was upregulated and p62 was slightly downregulated, and the amplitude of these effects was dependent on DFO concentration. Furthermore, both LC3-II and p62 were upregulated by DFO, in a concentration-dependent manner, after a 16-h treatment with chloroquine (CQ), an autophagy inhibitor, indicating the activation of autophagy (Fig. 3A). Key Words: mesenchymal stem cells, transcriptome, hypoxia, iron chelator, autophagy This study demonstrated the survival-promoting effect of treat- ments with low DFO concentrations for relatively short times (as low as 12 h) and investigated DFO-induced gene expression changes in MSCs. In addition, DFO-induced stress resistance mechanisms mediated by NUPR1 were explored. We expect that our findings will substantially contribute to the improvement of MSC survival and the advancement of MSC-based regenerative medicine. Introduction Human bone marrow-derived mesenchymal stem cells (BMSCs) possess the ability to differentiate into adipocytes, chondro- cytes, and osteocytes, and their application in regenerative medicine is highly anticipated. However, the culturing conditions may affect the quality of BMSCs and, thereby, their therapeutic performance. As the oxygen concentration in the bone marrow is about 1–7%, BMSCs cultured under 21% oxygen undergo stress, which may result in genetic instability. Repeated culturing may also result in genetic instability, cell aging, and decreased stemness mainte- nance. Interestingly, it has been reported that these problems can be overcome by hypoxic culturing.(1–3) Furthermore, it is known that hypoxia preconditioning promotes mesenchymal cell pro- liferation and is utilized to increase MSC survival after transplan- tation.(4,5) Although hypoxic culturing can be advantageous, it H H doi: 10.3164/jcbn.18112 ©2019 JCBN Results DFO was administered for 12 h (brown profile) or 48 h (blue ndicate the termination time of DFO treatments (brown arrow, d cells were seeded in 96well plates and, on the next day, ATP ed based on the cell number deduced from CyQUANT® cell pro t t i th Fl A l Si th d ll d d Fig. 1. Evaluation of DFO effects on proliferation, ATP, and oxygen consumption of BMSCs. (A) A comparison of the effects of different DFO concentrations and treatment times on cell proliferation. In each well one thousand cells were seeded, DFO was added at different concentrations on the next day, and proliferation was evaluated every two hours with IncuCyte. DFO was administered for 12 h (brown profile) or 48 h (blue profile), and then the medium was replaced with DFOfree medium. The arrows indicate the termination time of DFO treatments (brown arrow, 12 h; blue arrow, 48 h). (B) The effects of DFO on ATP production. Three thousand cells were seeded in 96well plates and, on the next day, ATP production was measured 12 h after DFO treatment. The ATP level was normalized based on the cell number deduced from CyQUANT® cell pro liferation assay (*p<0.05). (C) An evaluation of the OCR of cells after a 12h DFO treatment using the Flux Analyzer. Six thousand cells were seeded in 96well plates and OCR was measured on the next day, 12 h after DFO treatment. OCR was measured using the Flux Analyzer before and after the administration of oligomycin, FCCP, antimycin, and rotenone, as indicated. It is thought that DFO suppression of cell proliferation occurs because HIF1a accumulates as a result of reduced PDH activity, required for HIF1a degradation. In the pathway analysis of gene expression, glycolysis was ranked first, because of the overexpres- sion of 5 of the 6 enzyme-encoding genes involved in glycolysis. Moreover, sirtuin, HIF1a, and VEGF signaling pathways, involved in glycolytic and mitochondrial metabolism, were also ranked, thus confirming the presence of DFO-induced changes even after a 12-h treatment. DFO-induced variations occurred in a few upstream regulators. HIF1a and NUPR1 were upregulated, while EGLN and curcumin related genes were downregulated. As EGLN requires iron for enzyme activities, its inhibition upon DFO-induced iron deprivation was expected. Results It has been reported that curcumin possesses iron chelation ability(15) and reduces iron in mice and rats.(16) Moreover, its application as a suppressor of oxidative stress has been proposed.(15) Although NUPR1 upregula- tion was an upstream effect of the DFO treatment, it was maintained after cell treatment with 10 mM DFO for 48 h.(12) NUPR1 is thought to be involved in chemotherapy resistance in cancer cells and is induced by environmental stresses such as nutrient starvation and hypoxia. Moreover, NUPR1 is known to be ence of DFO the decline became greater (Fig. 4B). Next, as NUPR1 has been reported to be associated with drug resistance in cancer cells, staurosporine-induced apoptosis was evaluated. NUPR1 knockdown increased caspase-3 activity in MSCs treated with staurosporine (Fig. 4C). J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 211 ©2019 JCBN K. Matsunaga et al. Results The results were analyzed by either the Student’s t test or Welsh’s two-factor t tests, and the data are presented as the mean ± SD, with the significance threshold established at p<0.05. doi: 10.3164/jcbn.18112 ©2019 JCBN 210 Fig. 1. Evaluation of DFO effects on proliferation, ATP, and oxygen consumption of BMSCs. (A) A comparison of the effects of different DFO concentrations and treatment times on cell proliferation. In each well one thousand cells were seeded, DFO was added at different concentrations on the next day, and proliferation was evaluated every two hours with IncuCyte. DFO was administered for 12 h (brown profile) or 48 h (blue profile), and then the medium was replaced with DFOfree medium. The arrows indicate the termination time of DFO treatments (brown arrow, 12 h; blue arrow, 48 h). (B) The effects of DFO on ATP production. Three thousand cells were seeded in 96well plates and, on the next day, ATP production was measured 12 h after DFO treatment. The ATP level was normalized based on the cell number deduced from CyQUANT® cell pro liferation assay (*p<0.05). (C) An evaluation of the OCR of cells after a 12h DFO treatment using the Flux Analyzer. Six thousand cells were seeded in 96well plates and OCR was measured on the next day, 12 h after DFO treatment. OCR was measured using the Flux Analyzer before and after the administration of oligomycin, FCCP, antimycin, and rotenone, as indicated. ption of BMSCs. (A) A comparison of the effects of different DFO sand cells were seeded, DFO was added at different concentrations yte. DFO was administered for 12 h (brown profile) or 48 h (blue ws indicate the termination time of DFO treatments (brown arrow, and cells were seeded in 96well plates and, on the next day, ATP ized based on the cell number deduced from CyQUANT® cell pro treatment using the Flux Analyzer. Six thousand cells were seeded nt. OCR was measured using the Flux Analyzer before and after the on of BMSCs. (A) A comparison of the effects of different DFO d cells were seeded, DFO was added at different concentrations e. doi: 10.3164/jcbn.18112 ©2019 JCBN Discussion Red arrows indicate genes with a statistically significant upregulation in known nalysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate met C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are in ed and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to h (D) T 5 d b 5 l B i di l di d b i d hil bl i di Fig. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal display of p value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional classifica tion of gene clusters with altered expression (glycolysis). Red arrows indicate genes with a statistically significant upregulation in known pathway analysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate metabolites. (C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are indicated. Red and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to the fold change. (D) Top 5 and bottom 5 upstream regulators. Brown indicates upstream regulators predicted to be activated, while blue indicates upstream regulators predicted to be inhibited. Fig. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal display of p value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional classifica tion of gene clusters with altered expression (glycolysis). Red arrows indicate genes with a statistically significant upregulation in known pathway analysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate metabolites. (C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are indicated. Red and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to the fold change. (D) Top 5 and bottom 5 upstream regulators. Brown indicates upstream regulators predicted to be activated, while blue indicates upstream regulators predicted to be inhibited. Discussion Previous reports have demonstrated that hypoxia precondi- tioning enhances MSC retention during intramuscular injec- tion.(7,9) We have also shown that DFO, a highly safe drug that has long been used to treat iron overload, can be an alternative to oxygen depletion as it mimics hypoxia.(12) We found that relatively short incubations (12 h) with 50 mM DFO resulted in cell pro- liferation rates comparable to those observed after 48-h treatments with 3 mM DFO, i.e., the concentration normally used for pre- conditioning. Furthermore, ATP depletion, OCR decrease, inhibi- tion of mitochondrial oxidative phosphorylation, as well as changes in glycolytic metabolism, were also observed after 12-h treatments. Therefore, we concluded that shorter preconditioning could be as effective as the conventional 48-h treatments. J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 211 ©2019 JCBN 212 doi: 10.3164/jcbn.18112 ©2019 JCBN involved in cytoprotective autophagy(13) and possesses cell cycle suppression ability via p53. Therefore, the effects of DFO on functions under stress, and prevents cell death through organelle turnover.(17) Although it has been reported that DFO treatment Fig. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal display of p value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional classifica tion of gene clusters with altered expression (glycolysis). Red arrows indicate genes with a statistically significant upregulation in known pathway analysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate metabolites. (C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are indicated. Red and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to the fold change. (D) Top 5 and bottom 5 upstream regulators. Brown indicates upstream regulators predicted to be activated, while blue indicates upstream regulators predicted to be inhibited. g. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal d value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional c on of gene clusters with altered expression (glycolysis). J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 213 ©2019 JCBN K. Matsunaga et al. Discussion involved in cytoprotective autophagy(13) and possesses cell cycle suppression ability via p53. Therefore, the effects of DFO on autophagy were explored in MSCs. In fact, it was found that combined cell treatments with DFO and CQ increased the intra- cellular levels of LC3-II and p62, indicating autophagy enhance- ment. Moreover, NUPR1 was upregulated by DFO treatment, hypoxia, and amino acid starvation. By regenerating metabolic precursors and eliminating unwanted substances in the cell, autophagy functions as a survival mechanism that maintains cell functions under stress, and prevents cell death through organelle turnover.(17) Although it has been reported that DFO treatment induces protein aggregation and aggresome in NIH3T3 fibro- blasts, as well as the autophagic gene p62/SQSTM1,(18) a report has indicated that DFO may suppress autophagy caused by iron overload.(19) Thus, autophagy may result in different outcomes depending on environmental and/or physiopathological cell condi- tions. The possible involvement of NUPR1 in cytoprotective autophagy has drawn much attention.(13) Here, its function was J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 213 ©2019 JCBN K. Matsunaga et al. Fig. 3. DFOinduced changes in NUPR1 expression and autophagy. (A) DFOinduced expression of autophagyrelated proteins. Proteins were collected 16 h after the addition of chloroquine (CQ), and evaluated by western blotting. (B) DFOinduced autophagy visualized by immuno histochemical staining. The white bar indicates 50 mm. (C) DFOinduced changes in NUPR1 expression. (D) Hypoxiainduced changes in NUPR1 expression. (E) NUPR1 expression changes induced by cell culturing in amino aciddeficient medium (HBSS). Fig. 3. DFOinduced changes in NUPR1 expression and autophagy. (A) DFOinduced expression of autophagyrelated proteins. Proteins were collected 16 h after the addition of chloroquine (CQ), and evaluated by western blotting. (B) DFOinduced autophagy visualized by immuno histochemical staining. The white bar indicates 50 mm. (C) DFOinduced changes in NUPR1 expression. (D) Hypoxiainduced changes in NUPR1 expression. (E) NUPR1 expression changes induced by cell culturing in amino aciddeficient medium (HBSS). investigated by gene knockdown, resulting in inhibition of cell proliferation. This finding was consistent with the function of NUPR1 in MSCs as a pro-survival factor. Author Contributions Conceived and designed the experiments: KM, KF, TT and IS. Performed the experiments: KM, KF, TT, GB and NS. Analyzed the data: KM, KF and TT. Contributed reagents/materials/analysis tools: TM and NY. Wrote the paper: KM, KF and TT. In this study, NUPR1 depletion enhanced staurosporine- induced apoptosis, indicating the importance of NUPR1 in drug resistance, in line with previously reported results.(22) However, its role in drug resistance is yet to be clarified in detail. Pancreatic cancer cells have been reported to be resistant to starvation and gemcitabine treatment owing to the NUPR1/RELB/IER3 survival pathway, and CDKN1A/p21 phosphorylation and relocalization from the nucleus to the cytoplasm are important for breast cancer cell resistance to chemotherapeutic agents such as doxorubicin.(23) In this study, the phosphorylation and localization of p21 were Discussion However, the degree of this proliferation block was lower than the reported effects in cancer cells.(20) The increase in LC3-II upon NUPR1 knockdown was consistent with a previous study reporting that NUPR1 is involved in the maintenance of the autolysosomal efflux in A549 cells and that NUPR1 depletion deregulates autophagic flux and impairs autolysosomal clearance.(21) In addition, it has been reported that NUPR1 depletion leads to massive cytoplasmic vacuolization and premature senescence. However, the latter events were not observed in MSCs in this study (data not shown). As in these cells the block of proliferation induced by NUPR1 knockdown is less pronounced compared to cancer cells, MSCs might have a lower dependency on NUPR1 and phenotypes such as premature senescence might be less likely to occur. Further work will be necessary to solve these issues. also evaluated but no clear changes were observed (data not shown). Recently, the usefulness of NUPR1 in cells other than cancer cells has also been studied, and it has been reported that, in keratinocytes, NUPR1 is involved in UV-induced stress response.(24) Our findings, including the observation of glycolysis-dominant metabolic changes in MSCs treated with DFO for 12 h, suggested that it is possible to apply shorter times of hypoxic precondi- tioning, compared to traditional protocols. Furthermore, as DFO treatment, similarly to hypoxia and starvation, upregulates NUPR1, and this may lead to improved cell survival, our results may contribute to the design of optimized methods of MSC pre- paration for regenerative medicine. Acknowledgments We would like to thank Ms. Mariko Yamada, Ms. Risa Mochizuki and Ms. Kumie Ota for their technical assistance. doi: 10.3164/jcbn.18112 ©2019 JCBN 214 g. 4. Changes induced by NUPR1 knockdown. (A) Western blot analysis in MSCs after siRNAinduced NUPR1 knockdown. (B) Effects of NUPR1 nockdown on MSC proliferation. Proliferation was measured every 2 h with IncuCyte (n = 6). (C) Effects of NUPR1 knockdown on staurosporine nduced apoptosis. Caspase3 activity is shown 6 h after treatment with staurosporine was shown. Three thousand cells were seeded in 96well lates and Caspase3 activity was measured 6 h after staurosporine treatment. Caspase activity was normalized based on the cell number deduced rom analysis with CyQUANT® cell proliferation assay. * indicates statistically significant difference compared with siCONTROL #1, p<0.01. Fig. 4. Changes induced by NUPR1 knockdown. (A) Western blot analysis in MSCs after siRNAinduced NUPR1 knockdown. (B) Effects of NUPR1 knockdown on MSC proliferation. Proliferation was measured every 2 h with IncuCyte (n = 6). (C) Effects of NUPR1 knockdown on staurosporine induced apoptosis. Caspase3 activity is shown 6 h after treatment with staurosporine was shown. Three thousand cells were seeded in 96well plates and Caspase3 activity was measured 6 h after staurosporine treatment. Caspase activity was normalized based on the cell number deduced from analysis with CyQUANT® cell proliferation assay. * indicates statistically significant difference compared with siCONTROL #1, p<0.01. Fig. 4. Changes induced by NUPR1 knockdown. (A) Western blot analysis in MSCs after siRNAinduced NUPR1 knockdown. (B) Effects of NUPR1 knockdown on MSC proliferation. Proliferation was measured every 2 h with IncuCyte (n = 6). (C) Effects of NUPR1 knockdown on staurosporine induced apoptosis. Caspase3 activity is shown 6 h after treatment with staurosporine was shown. Three thousand cells were seeded in 96well plates and Caspase3 activity was measured 6 h after staurosporine treatment. Caspase activity was normalized based on the cell number deduced from analysis with CyQUANT® cell proliferation assay. * indicates statistically significant difference compared with siCONTROL #1, p<0.01. in-Aid for Challenging Exploratory Research (24659369) from the Japan Society for the Promotion of Science. Financial Support in-Aid for Challenging Exploratory Research (24659369) from the Japan Society for the Promotion of Science. This study was supported by The Project Promoting The Research and Development (R&D) Center on Regenerative Medicine in Yamaguchi Prefecture. This study was also supported by Grant-in-Aid for Young Scientists (B) (18K15815), and Grant- Conflict of Interest No potential conflicts of interest were disclosed. J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 215 ©2019 JCBN e0178011. 1 Dos Santos F, Andrade PZ, Boura JS, Abecasis MM, da Silva CL, Cabral JM. Ex vivo expansion of human mesenchymal stem cells: a more effective cell proliferation kinetics and metabolism under hypoxia. J Cell Physiol 2010; 223: 27–35. 1 Dos Santos F, Andrade PZ, Boura JS, Abecasis MM, da Silva CL, Cabral JM. Ex vivo expansion of human mesenchymal stem cells: a more effective cell proliferation kinetics and metabolism under hypoxia. J Cell Physiol 2010; 223: 27–35. e0178011. 7 Nouri F, Salehinejad P, Nematollahi-Mahani SN, Kamarul T, Zarrindast MR, Sharifi AM. Deferoxamine preconditioning of neural-like cells derived from human Wharton’s jelly mesenchymal stem cells as a strategy to promote their tolerance and therapeutic potential: an in vitro study. Cell Mol Neurobiol 2016; 36: 689–700. 2 Hoch AI, and Leach. Concise review: optimizing expansion of bone marrow mesenchymal stem/stromal cells for clinical applications. Stem Cells Transl Med 2014; 3: 643–652. 8 Cheng XW, Kuzuya M, Kim W, et al. Exercise training stimulates ischemia- induced neovascularization via phosphatidylinositol 3-kinase/Akt-dependent hypoxia-induced factor-1 alpha reactivation in mice of advanced age. Circu- lation 2010; 122: 707–716. 3 Sugrue T, Lowndes NF, Ceredig R. Hypoxia enhances the radioresistance of mouse mesenchymal stromal cells. Stem Cells 2014; 32: 2188–2200. 4 Beegle J, Lakatos K, Kalomoiris S, et al. Hypoxic preconditioning of mesenchymal stromal cells induces metabolic changes, enhances survival, and promotes cell retention in vivo. Stem Cells 2015; 33: 1818–1828. 9 Najafi R, Sharifi AM. Deferoxamine preconditioning potentiates mesenchymal stem cell homing in vitro and in streptozotocin-diabetic rats. Expert Opin Biol Ther 2013; 13: 959–972. 5 Rosová I, Dao M, Capoccia B, Link D, Nolta JA. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells 2008; 26: 2173–2182. 5 Rosová I, Dao M, Capoccia B, Link D, Nolta JA. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells 2008; 26: 2173–2182. 10 Qin HH, Filippi C, Sun S, Lehec S, Dhawan A, Hughes RD. Hypoxic pre- conditioning potentiates the trophic effects of mesenchymal stem cells on co- cultured human primary hepatocytes. Stem Cell Res Ther 2015; 6: 237. 6 Oses C, Olivares B, Ezquer M, et al. Preconditioning of adipose tissue- derived mesenchymal stem cells with deferoxamine increases the production of pro-angiogenic, neuroprotective and anti-inflammatory factors: potential application in the treatment of diabetic neuropathy. K. Matsunaga et al. e0178011. PLoS One 2017; 12: 6 Oses C, Olivares B, Ezquer M, et al. Preconditioning of adipose tissue- derived mesenchymal stem cells with deferoxamine increases the production of pro-angiogenic, neuroprotective and anti-inflammatory factors: potential application in the treatment of diabetic neuropathy. PLoS One 2017; 12: 11 Chu K, Jung KH, Kim SJ, et al. Transplantation of human neural stem cells protect against ischemia in a preventive mode via hypoxia-inducible factor- 1alpha stabilization in the host brain. Brain Res 2008; 1207: 182–192. J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 215 ©2019 JCBN K. Matsunaga et al. 19 Zheng Q, Zhao Y, Guo J, et al. Iron overload promotes mitochondrial frag- mentation in mesenchymal stromal cells from myelodysplastic syndrome patients through activation of the AMPK/MFF/Drp1 pathway. Cell Death Dis 2018; 9: 515. 12 Fujisawa K, Takami T, Okada S, et al. Analysis of metabolomic changes in mesenchymal stem cells on treatment with desferrioxamine as a hypoxia mimetic compared with hypoxic conditions. Stem Cells 2018; 36: 1226–1236. 13 Hamidi T, Cano CE, Grasso D, et al. Nupr1-aurora kinase A pathway provides protection against metabolic stress-mediated autophagic-associated cell death. Clin Cancer Res 2012; 18: 5234–5246. 20 Grasso D, Garcia MN, Hamidi T, et al. Genetic inactivation of the pancreatitis- inducible gene Nupr1 impairs PanIN formation by modulating Kras(G12D)- induced senescence. Cell Death Differ 2014; 21: 1633–1641. 14 Fujisawa K, Hara K, Takami T, et al. Evaluation of the effects of ascorbic acid on metabolism of human mesenchymal stem cells. Stem Cell Res Ther 2018; 9: 93. 21 Mu Y, Yan X, Li D, et al. NUPR1 maintains autolysosomal efflux by activating SNAP25 transcription in cancer cells. Autophagy 2018; 14: 654–670. 15 Jiao Y, Wilkinson J 4th, Christine Pietsch E, et al. Iron chelation in the biological activity of curcumin. Free Radic Biol Med 2006; 40: 1152–1160. 22 Malicet C, Giroux V, Vasseur S, Dagorn JC, Neira JL, Iovanna JL. Regula- tion of apoptosis by the p8/prothymosin alpha complex. Proc Natl Acad Sci U S A 2006; 103: 2671–2676. 16 Chin D, Huebbe P, Frank J, Rimbach G, Pallauf K. Curcumin may impair iron status when fed to mice for six months. Redox Biol 2014; 2: 563–569. 23 Vincent AJ, Ren S, Harris LG, et al. Cytoplasmic translocation of p21 mediates NUPR1-induced chemoresistance: NUPR1 and p21 in chemoresistance. FEBS Lett 2012; 586: 3429–3434. doi: 10.3164/jcbn.18112 ©2019 JCBN e0178011. 17 Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. 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Inclusive Financing’s Development and Urban-rural Income Gap—Empirical analysis based on provincial panel data
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Inclusive Financing's Development and Urban-rural Income Gap —Empirical analysis based on provincial panel data Xu Jing1 a*, Sun Mingyue2 b, Wang Hening3c Xu Jing1 a*, Sun Mingyue2 b, Wang Hening3c 123Shandong University of Science and Technology, Shandong, Jinan, 250031 123Shandong University of Science and Technology, Shandong, Jinan, 250031 123Shandong University of Science and Technology, Shandong, Jinan, 250031 Abstract: With the further development of China's economy, the income of urban and rural residents has increased. At present, China's economy has changed from a stage of high-speed growth to a stage of high-quality development. However, uneven and insufficient regional development is still a problem that requires great attention. Inclusive Financing's development provides residents with more access to financial services and is one of the important ways to build a well-off society and develop the economy in recent years. Based on the Peking University Digital Inclusive Financing Index, this paper studies the relationship between the development level of Inclusive Financing and the income gap between urban and rural residents by constructing panel data of 31 provinces, cities and regions in China and establishing a model for empirical analysis. The study found that the development of Inclusive Financing has a significant impact on the income gap between urban and rural areas, among which the improvement of the level of Inclusive Financing in the economically developed eastern region has a very obvious effect on narrowing the income gap between urban and rural areas. However, blindly developing Inclusive Financing in the underdeveloped central and western regions will also cause the further widening of the income gap between urban and rural areas. The conclusion of this paper has certain positive significance for narrowing the income gap between urban and rural areas and promoting the development of Inclusive Financing. The Third Plenary Session of the 18th CPC Central Committee in 2013 established "developing Inclusive Financing" as a national strategy. In 2015, the State Council issued the Plan for Promoting the Development of Inclusive Financing (2016-2020). In recent years, the development of Inclusive Financing in our country has been supported by the government and the public. Vigorously developing Inclusive Financing is an important embodiment of the financial industry's support for the construction of a modern economic system and the enhancement of its ability to serve the real economy. It is also an important way to ease the contradiction between the people's growing demand for financial services and the inadequate and unbalanced financial supply. *a698766474@qq.com b2819252853@qq.com, c1145720083@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/). Inclusive Financing's Development and Urban-rural Income Gap —Empirical analysis based on provincial panel data It is also an inevitable requirement for China to build a well-off society in an all-round way. E3S Web of Conferences 275, 03015 (2021) EILCD 2021 E3S Web of Conferences 275, 03015 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 icenses/by/4.0/). 1 Introduction How to further narrow the income gap between urban and rural areas is a hot issue studied by experts in the economic field in recent years. Although the income of urban and rural residents has been rising since the reform and opening up, the gap has been gradually widening, and the dual economic structure of urban and rural division has not changed. The report of the 19th National Congress of the Communist Party of China in 2017 pointed out that the gap between urban and rural regional development and income distribution is still large, some outstanding problems of insufficient development imbalance have not yet been solved, and the quality and efficiency of development are still not high, all of which need us to focus on solving. With the rapid development of science and technology, Digital Inclusive Financing has reduced the transaction costs of financial services through the advantages of digital technology, which is a new idea and new way for Inclusive Financing to develop innovation. With the use of information technology, the coverage of basic financial services continues to expand, so that low-income people can have more opportunities and more easy access to financial services, which is of great significance for the income growth of low-income people and thus narrowing the income gap between urban and rural areas. The concept of Inclusive Financing first appeared in the UN's vocabulary of promoting micro-credit in 2005. It is a financial system that can effectively and comprehensively serve all social strata and groups. The inclusive financial system believes that everyone should have access to financial services and everyone should participate in the economic development process, so as to realize the common prosperity of the society. And the inclusive financial system provides opportunities for low-income people, which is also of great significance in narrowing the income gap between urban and rural areas. However, the inclusive financial system is still facing E3S Web of Conferences 275, 03015 (2021) E3S Web of Conferences 275, 03015 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 many practical difficulties and challenges in its development. The uneven development in regions with economic levels has also affected the development of Inclusive Financing in some regions. However, the concept of digital Inclusive Financing, which has arisen in recent years, is facing greater controversy and restriction. role in promoting the narrowing of the urban-rural income gap at a high level, but it is not statistically significant. 2 Literature review and theoretical analysis In the existing literature, scholars have different views on whether Inclusive Financing and the digital Inclusive Financing developed in recent years can narrow the income gap between urban and rural areas. Some scholars build panel data and models for empirical analysis, and think that the development of Inclusive Financing can effectively narrow the income gap between urban and rural areas. Allen(2016) found that developing Inclusive Financing can expand employment and raise income level [1]. Zhang Xiaoyan (2016) measured the level of Inclusive Financing in China by using the relevant data of China's Internet finance, Inclusive Financing and urban-rural income gap from 2004 to 2014, and based on this, established a VECM model to empirically analyze the impact of Inclusive Financing's development on urban-rural income gap under the background of Internet finance. The research shows that there is a long-term equilibrium relationship between the development of Inclusive Financing and the income gap between urban and rural areas. Improving the development level of Inclusive Financing can significantly reduce the income gap between urban and rural areas, and the effect is lasting. There is also a mutual promotion effect between the two [2]. Zhang Zihao (2018) constructed provincial spatial panel data from 2013 to 2015, and used spatial panel econometric model to empirically analyze the impact of digital Inclusive Financing on China's urban-rural income gap. The results show that the income gap between urban and rural areas in all provinces, autonomous regions and municipalities in China shows a significant spatial dependence on the whole, and shows "high spatial agglomeration" and "low spatial agglomeration" locally. Digital Inclusive Financing can significantly promote the narrowing of the income gap between urban and rural areas, improve the welfare level of low-income groups and help underdeveloped areas accelerate their development. And the decomposition indicators of digital Inclusive Financing can significantly reduce the urban-rural income gap [3]. By combing the literature, some scholars believe that the impact of Inclusive Financing on residents' income is mainly due to the following reasons. First, the inclusive financial system covers a wider range. Especially after the development of Digital Inclusive Financing, groups with different income levels, especially low-income groups, have more access to financial services and have improved their awareness of financial products. Secondly, the development of Inclusive Financing has provided more people with financial services suitable for them. Many financial institutions have also innovated their products and businesses. 1 Introduction When the level of digital Inclusive Financing is greater than the threshold value, the income gap between urban and rural areas in the regions with low economic development level will effectively narrow with the improvement of the level of digital Inclusive Financing [4]. Xiong Deping (2020) selected the data and information of 31 provinces (autonomous regions and municipalities) in China from 2011 to 2018, constructed a fixed and random effect double regression model, and tested the impact of digital Inclusive Financing development on urban-rural income gap by using the empirical method of non-equilibrium effect test. According to the analysis of the results, the infrastructure construction in rural areas is relatively backward in less developed areas, the primitive accumulation of capital in urban areas is significantly higher than that in rural areas, and financial resources are concentrated in cities. On the whole, the income gap between urban and rural areas has not narrowed, but expanded [5]. 3.1 Data sources (1) Urbanization Level (PURB) In recent years, urbanization has expanded rapidly, and the urban-rural dual economic structure has widened the income gap between urban and rural areas. The urbanization level is measured by selecting the ratio of urban population to total population. This paper selects the panel data of 31 provinces, municipalities and autonomous regions in China from 2010 to 2019. Among them, the data related to digital Inclusive Financing comes from "Digital Inclusive Financing Index of Peking University (2011-2018)", which is compiled by using the massive data of Ant Financial Service on digital Inclusive Financing, and mainly consists of indicators such as account coverage, payment business, credit business and insurance business. Other data such as urban and rural residents' income, economic growth and per capita GDP mainly come from China Statistical Yearbook, website of National Bureau of Statistics, website of local bureau of statistics and Wind database. (2) The level of economic development (RGDP) The level of economic development in different regions will have an impact on the income of urban and rural residents. In this paper, the per capita GDP is selected to measure the level of economic development, and considering the dimension problem, the index is treated logarithmically. (3) fiscal expenditure (TAX) Fiscal expenditure can promote the development of different industries, thus affecting the income of urban and rural residents. When government expenditure tends to be urban, the income gap between urban and rural residents is likely to widen further. This paper measures this index by the proportion of fiscal expenditure to regional GDP. 2 Literature review and theoretical analysis Compared with the traditional bank savings income, residents are more inclined to invest in financial products. Third, with the development of digital Inclusive Financing, information technology has improved the efficiency and level of financial services. Through big data analysis and other means, the scheme analysis can be provided for different types of users, so as to reduce investment risks and financing costs for low-income groups, and thus encourage more residents to participate in the development of Inclusive Financing. To sum up, the narrowing of the income gap between urban and rural areas needs to be driven by economic development, but there are still differences on whether inclusive financial system can play a role in narrowing the income gap between urban and rural areas. Moreover, due to the imbalance of regional economic development in our country, whether the impact of Inclusive Financing's development is always positive or negative in regions with different economic levels needs to be tested and proved. At present, Digital Inclusive Financing has opened up an innovative way for the development of Inclusive Financing, but how to graft relevant technologies with different regions so as to have a positive impact on residents' income and further narrow the income gap is still a problem that needs to be focused on.. Some scholars believe that the level and stage of development in Inclusive Financing will have different effects on the income gap between urban and rural areas. Zhao Bingqi and others (2020) constructed the provincial panel data from 2011 to 2018, and selected a single threshold model to conduct an empirical analysis and research on the relationship between the two. They proposed that the development of digital Inclusive Financing will further widen the urban-rural income gap at a low level of digital Inclusive Financing, and play a 2 2 E3S Web of Conferences 275, 03015 (2021) EILCD 2021 E3S Web of Conferences 275, 03015 (2021) https://doi.org/10.1051/e3sconf/202127503015 3 Data sources and variable selection dimensions: coverage breadth, use depth and digitalization degree. 3. Control variables: 3. Control variables: 3. Control variables: (5) Education level (EDU) A Chi ' b d (5) Education level (EDU) A Chi ' b d At present, China's urban education level and teachers have obvious advantages compared with rural areas, so the education level also has an impact on urban and rural income. This paper selects the average number of students in colleges and universities per 100,000 population to measure the education level. 2. Core explanatory variable: Inclusive Financing Index (IFI) According to the report of Digital Inclusive Financing Index of Peking University (Phase II, 2011-2018) released by Digital Finance Research Center of Peking University in April 2019, the provincial digital Inclusive Financing index calculated by this method from 2011 to 2018 is selected as the explanatory variable of the model. And some missing data are supplemented by difference method. The index includes three first-level index (6) Employment status (EMP) (6) Employment status (EMP) The employment situation has a high correlation with the income of urban and rural residents. The high unemployment rate in rural areas and the low unemployment rate in urban areas will further widen the income gap between urban and rural areas. This paper selects the proportion of employed population to the total population to measure the employment situation. Table 1 Descriptive statistics of each variable index Variable Obs Mean Std. Dev. Min Max IG 310 84466 27677 10475 190064 IFI 310 184.6 103.4 6.800 419.2 PURB 310 0.561 0.134 0.227 0.896 RGDP 310 4.641 0.207 3.843 5.216 TAX 310 0.293 0.209 0.113 1.354 IS 310 0.900 0.0526 0.742 0.997 EDU 310 2535 830.5 1082 6196 EMP 310 0.254 0.116 0.125 0.761 3.3 Possible problems in the model (1) the endogenous problem This paper discusses the influence of the development level of Inclusive Financing on the income gap between urban and rural areas. The endogenous problem of the model mainly comes from the measurement error. Measurement error refers to the error between the values Table 1 Descriptive statistics of each variable index level of Inclusive Financing on the income gap between urban and rural areas. The endogenous problem of the model mainly comes from the measurement error. Measurement error refers to the error between the values 3.2 variable selection 1. Explained variable: urban-rural income gap (IG) This paper chooses the ratio of urban per capita disposable income to rural per capita disposable income to reflect the urban-rural income gap. The smaller the ratio, the smaller the income gap between urban and rural areas, whereas the more serious the income gap between urban and rural areas. According to the relevant data in China Statistical Yearbook, there were no statistics on per capita disposable income of rural residents before 2013, so the per capita net income of rural residents from 2010 to 2012 was selected for analysis. g (4) Industrial structure (IS) The industrial structure is measured by the ratio of the added value of the secondary and tertiary industries to GDP in various provinces and cities. 3.3 Possible problems in the model 3 E3S Web of Conferences 275, 03015 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 random disturbance, IG for income gap between urban and rural residents, IFI for development level of Inclusive Financing, PURB for urbanization level, RGDP for economic development level, TAX for fiscal expenditure, IS for industrial structure, EDU for education level and EMP for employment situation. of the explanatory variables used in the model and the true values. There are some errors in the measurement of Inclusive Financing level with Inclusive Financing index as an index. (2) Robustness When considering the impact of Inclusive Financing's development level on the urban-rural income gap, we should also consider the heterogeneity of different regions. For example, whether it is located in the eastern or central and western regions, and the economic development level of the region. In this paper, the robustness is tested by sub-sample regression. 4.2 Regression analysis of benchmark model In this paper, the panel data model is used for regression analysis, so we should judge whether to use mixed model, fixed effect model or random effect model. Therefore, this paper uses Hausmann test method to judge the model. The judging steps are as follows: firstly, Hausmann test method is used to judge whether to choose fixed effect model or random effect model. When P value is greater than 0.05, the original hypothesis is accepted and the random effect model is established; If the p value is less than 0.05, the original hypothesis is rejected and the fixed effect model is adopted. 4.1 model setting IGi,t=α1 + β1IFIi,t + β2PURBi,t + β3RGDPi, t + β4TAXi,t + β5ISi,t+β6EDUi,t+β7EMPi,t+εi,t t + β4TAXi,t + β5ISi,t+β6EDUi,t+β7EMPi,t+εi,t Among them, I stands for province, T for year, ε for Among them, I stands for province, T for year, ε for Table 2 Stepwise regression results of benchmark model (1) (2) (3) (4) (5) (6) m1 m2 m3 m4 m5 m6 VARIABLES IG IG IG IG IG IG IFI -0.0847 *** -0.0909 *** -0.0726 *** -0.0806 *** -0.0317 *** -0.0327 *** (0.008) (0.008) (0.008) (0.009) (0.006) (0.006) RGDP 2.4711 *** 2.4628 *** 2.6012 *** 2.6167 *** 3.0202 *** 3.0355 *** (0.063) (0.062) (0.060) (0.061) (0.040) (0.040) EMP 0.4419*** 0.2441* 0.2540* -0.2456 *** -0.2387 *** (0.148) (0.138) (0.138) (0.085) (0.084) EDU -0.3951 *** -0.3666 *** -0.0147 -0.0213 (0.054) (0.056) (0.037) (0.036) TAX 0.3057* 0.1409 0.1333 (0.164) (0.098) (0.096) PURB -3.3371 *** -3.1879 *** (0.150) (0.154) IS -0.7279 *** (0.220) Constant 11.7108*** 11.7413 *** 11.6514 *** 11.6904 *** 11.4494 *** 11.4544 *** Table 2 Stepwise regression results of benchmark model 4 E3S Web of Conferences 275, 03015 (2021) E3S Web of Conferences 275, 03015 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 (0.039) (0.039) (0.038) (0.043) (0.028) (0.028) Observations 310 310 310 310 310 310 R-squared 0.93 0.93 0.94 0.94 0.98 0.98 Number of pro1 31 31 31 31 31 31 Adj. R-squared 0.98 0.98 0.98 0.98 0.98 0.98 Standard errors in parentheses *** p<0.01, ** p<0.05, * p<0.1 economy and pull in the distance between rural and urban areas, thus effectively reducing the income gap between urban and rural areas. Industrial structure has a significant impact on narrowing the income gap between urban and rural areas. It shows that the development of secondary and tertiary industries can effectively improve the transformation and development of urban and rural industrial structure, especially in rural areas, where the industrial structure is weaker than that in cities. By focusing on the development of secondary and tertiary industries, the efficiency of industrial adjustment and optimization can be improved, which is closer to the urban industrial development and structure, thus raising the income level and narrowing the income gap. According to the regression results, the coefficient sign and significance level of the core explanatory variable are generally consistent, especially the coefficient sign and significance level of the core explanatory variable number Hewlett-Packard Finance are basically the same, indicating that the regression results are relatively robust. 4.1 model setting By establishing a panel data model, this paper studies and analyzes the direct relationship between the development level of Inclusive Financing and the income gap between urban and rural residents in 31 provinces, cities and regions of China. According to Hausman test results, the P value is less than 0.05, and the fixed effect model is adopted for solid selection. Table 2 shows the regression results of gradually adding control variables. In addition to Inclusive Financing's development level, urbanization level, economic development level, fiscal expenditure, industrial structure, education level and employment status will also affect the income gap between urban and rural areas. As shown in the table, the first to sixth columns are the regression results obtained by gradually adding the control variables of economic development level, employment status, education level, fiscal expenditure, urbanization level and industrial institutions according to the optimal significance. In order to study the relationship between the development level of Inclusive Financing and the income gap between urban and rural areas, the following model is established: The last column shows the most significant set of regression results, that is, the regression results after considering all the above-mentioned core variables and control variables. 4.1 model setting There is an obvious negative correlation between Inclusive Financing's development level and urban-rural income gap, which means that the improvement of Inclusive Financing's development level can narrow the urban-rural income gap, and the impact is very significant. By promoting the development of Inclusive Financing, more township residents can learn about various financial products and services, and increase their participation in investment and financial management, thereby increasing their income and further reducing the income gap with urban residents. At the same time, we can see that education level and government expenditure have little effect on reducing urban and rural income. Education level negatively affects the income gap between urban and rural areas, while government expenditure positively affects the income gap between urban and rural areas, but it is not statistically significant. Therefore, these two factors have no obvious influence on the income gap between urban and rural areas. Among the control variables, economic development level, employment status, urbanization level and industrial structure also have a very significant impact on the income gap between urban and rural areas. There is a positive relationship between the level of economic development and the income gap between urban and rural areas, which indicates that the improvement of economic level in different regions will lead to the further widening of the gap between urban and rural areas. For urban residents, the income growth brought by the improvement of economic level may be greater than that of rural residents, so it will increase the income gap between urban and rural areas. Employment status, urbanization level, industrial structure and urban-rural income gap are negatively correlated. From the results, the higher the employment coverage, the higher the income level of urban and rural residents will be. For cities, the employment rate is close to saturation most of the time, but for rural areas, many jobs are vacant, and the employment coverage is far less optimistic than that of cities. The increase of rural employment rate can effectively increase the income of rural residents, thus helping to narrow the income gap between urban and rural areas. The improvement of urbanization level can also narrow the income gap between urban and rural areas, which is consistent with the conclusion of Zhang Yiming et al. (2018), which shows that the improvement of urbanization level can drive the development of rural 5.1 Endogenous problems Although the regression results show that the development level of Inclusive Financing has a significant effect on the income gap between urban and rural areas, its endogenous problems may still have an impact on the significance, so this paper chooses endogenous test. Using the explanatory variable of the lag period as the instrumental variable to test the endogeneity, that is, the development level of Inclusive Financing in the lag period. On the one hand, there is a high correlation between lagging indicators and current indicators; on the other hand, lagging indicators affect urban-rural income gap through current digital Inclusive Financing indicators, so lagging indicators meet the correlation and exclusiveness, which is a more reasonable tool variable. Table 3 Estimation of tool variables (1) (2) (3) (4) (5) (6) m1 m2 m3 m4 m5 m6 VARIABLES IG IG IG IG IG IG IFI -0.1286*** -0.1361 *** -0.1106 *** -0.1194 *** -0.0423 *** -0.0429 *** Table 3 Estimation of tool variables IFI 5 5 E3S Web of Conferences 275, 03015 (2021) E3S Web of Conferences 275, 03015 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 (0.010) (0.010) (0.010) (0.011) (0.007) (0.007) RGDP 2.6389 *** 2.6413 *** 2.7683 *** 2.7853 *** 3.0929 *** 3.1023 *** (0.067) (0.066) (0.061) (0.062) (0.038) (0.038) EMP 0.5617*** 0.4570*** 0.4669*** -0.1803** -0.1771** (0.170) (0.152) (0.152) (0.091) (0.090) EDU -0.4337 *** -0.3981 *** -0.0467 -0.0564 (0.060) (0.062) (0.038) (0.038) TAX 0.3844** 0.3707*** 0.3474*** (0.176) (0.099) (0.098) PURB -3.3705 *** -3.2475 *** (0.156) (0.165) IS -0.5236** (0.241) Constant 11.9368*** 11.9725 *** 11.8449 *** 11.8880 *** 11.5024 *** 11.5055 *** (0.051) (0.052) (0.050) (0.054) (0.035) (0.035) Observations 279 279 279 279 279 279 Number of pro1 31 31 31 31 31 31 Adj. R-squared 0.98 0.98 0.98 0.98 0.98 0.98 Standard errors in parentheses *** p<0.01, ** p<0.05, * p<0.1 According to the test results, it can be seen that the significance of the core explanatory variables is consistent with the benchmark regression, and the improvement of Inclusive Financing level plays a very significant role in narrowing the income gap between urban and rural areas. Moreover, the most significant model selected from the benchmark regression is still the best in the endogeneity test. In this group, the development level of Inclusive Financing has more obvious influence on narrowing the income gap between urban and rural areas. 5.1 Endogenous problems Among the control variables, the economic development level and urbanization level are still significant, showing positive and negative correlation respectively. Although the significance of employment status and industrial institutions has declined, the overall results are consistent with the results of the benchmark regression model. The effect of education level on narrowing the income gap between urban and rural areas is still not obvious. However, in the test, government expenditure has played a significant role in narrowing the income gap between urban and rural areas. It can be seen that under the influence of some missing variables, the significance of government expenditure has changed. To sum up, the test situation basically coincides with the significant results of the benchmark regression model, so this paper controls the endogenous problem well. 5.2 Robustness test This paper considers the regional heterogeneity of Inclusive Financing's development level, that is, the development of Inclusive Financing in different regions will have a significant impact on narrowing the income gap between urban and rural areas. In this paper, we set up sub-samples of the whole country, the eastern region and the central and western regions for grouping regression to test the robustness. Table 4 Test results of different regional samples (1) (2) (3) M8 M9 M10 VARIABLES IG IG IG IFI -0.0327*** -0.0663*** 0.0199*** (0.006) (0.011) (0.005) RGDP 3.0355*** 3.2424*** 2.0248*** (0.040) (0.046) (0.070) EMP -0.2387*** -0.2433** -0.3358*** (0.084) (0.105) (0.109) EDU -0.0213 -0.0439 0.0010 (0.036) (0.078) (0.026) TAX 0.1333 0.0164 -0.2622*** Table 4 Test results of different regional samples 6 https://doi.org/10.1051/e3sconf/202127503015 E3S Web of Conferences 275, 03015 (2021) EILCD 2021 E3S Web of Conferences 275, 03015 (2021) (0.096) (0.226) (0.071) PURB -3.1879*** -2.0068*** -1.5933*** (0.154) (0.366) (0.162) IS -0.7279*** -3.6483*** 0.0169 (0.220) (1.032) (0.147) Constant 11.4544*** 11.6545*** 11.1810*** (0.028) (0.049) (0.027) Observations 310 110 200 R-squared 0.98 0.99 0.99 Number of pro1 31 11 20 Adj. R-squared 0.99 0.99 0.99 Standard errors in parentheses *** p<0.01, ** p<0.05, * p<0.1 quite special. The impact of fiscal expenditure changes from insignificant to significant, while the impact of industrial structure changes from significant to insignificant. The results in the eastern region and the whole country are opposite. This shows that the impact of fiscal expenditure in the central and western regions is very direct and effective. Because the economic development is at a low level, if there is some rural bias in fiscal expenditure, it will have a very obvious impact on narrowing the income gap between urban and rural areas. However, in the eastern region, where the economic level is developing at a high level, the impact of fiscal expenditure cannot produce such an effect in the central and western regions. By the same token, the transformation of industrial structure is more urgent and crucial for the eastern region with better economic development, and the eastern region has more preparation and resources for the development of industrial structure, so the upgrading of industrial structure can play a positive role in narrowing the income gap between urban and rural areas. However, the industrial structure in the central and western regions is unreasonable. 5.2 Robustness test For the central and western regions, the time for development and further transformation is not yet ripe, which will increase the pressure of economic development and lead to more problems, and may reduce the stability of rural economic development, which is not conducive to the improvement of rural residents' income, thus further widening the income gap between urban and rural areas. From the regression results, it can be seen that the development level of Inclusive Financing has a very significant impact on the urban-rural income gap in the whole country, the east and the central and western regions. For the whole country and the eastern region, they are negatively correlated, but in the central and western regions, they have different results and are positively correlated. This shows that from the perspective of the whole country and the eastern region, the overall economic development of Inclusive Financing is relatively moderate. With the improvement of Inclusive Financing level, the income gap between urban and rural areas can be effectively narrowed. According to the absolute value of the regression coefficient of variables, Inclusive Financing has the most significant effect in the eastern region, that is, developing Inclusive Financing in the eastern region can effectively narrow the income gap between urban and rural areas. The central and western regions are relatively low-income regions with low economic development level. Developing Inclusive Financing here may further widen the income gap between urban residents and rural residents. For the central and western regions, the economic structure is imperfect, the development is insufficient, and the conditions for contacting with financial services are lacking. If Inclusive Financing is developed hard, it is likely to lead urban and rural residents to participate in financial activities that do not match their own income level and bear higher financial risks. Moreover, the ability to resist risks in the central and western regions is weaker than that in the eastern regions, which makes the development of Inclusive Financing have an opposite impact. We judge that Inclusive Financing can effectively narrow the income gap between urban and rural areas on the basis of economic development to a certain extent, otherwise it will further widen the gap. 6.2 Relevant suggestions (1) Improve the development system of Inclusive Financing and further promote the digital Inclusive Financing. At present, Inclusive Financing is still in the development stage, and many urban and rural residents don't understand the true meaning and function of Inclusive Financing, which leads to the difficulty of promoting Inclusive Financing, especially in rural areas. Relevant departments should actively popularize the knowledge of Inclusive Financing and do a good job of top-down information transmission. With the development of information technology, the importance of the development of digital Inclusive Financing is increasing, and digital Inclusive Financing can effectively improve the coverage, expand the coverage and make it easier for residents to understand digital Inclusive Financing. The diversified financial products and services developed through digital Inclusive Financing have carried out risk management and control more rationally, reduced the financial investment risks faced by low-income people, helped more residents to invest in the construction of Inclusive Financing, experienced financial services, promoted the development of Inclusive Financing through personal experience, promoted the economic level of urban and rural areas, and further narrowed the income gap between urban and rural areas. (3) Pay attention to the innovation of financial services and promote the diversification of products. Innovative design of financial services and products can reduce the cost of financial services and products. Encourage relevant institutions to open business departments or outlets in rural areas, design and provide corresponding services for rural areas and residents, provide more diversified and personalized products, and set up functional institutions such as self-service banks. Increase the scope of rural collateral, and explore innovations such as rural forest rights, housing property rights and land contractual management rights as collateral. Transfer financial services through digital technology, explore low-cost and high-efficiency digital financial services, improve coverage breadth and depth through financial innovation, and increase agriculture-related financial business. (2) Optimize the development environment of rural Inclusive Financing. Compared with urban areas, rural areas have a lower level of economic development, and their education level and industrial development are in a relatively weak position. We should pay attention to the environmental construction of Inclusive Financing in rural areas, and open up residents' understanding of Inclusive Financing through various publicity and promotion channels. Table 5 Basic conclusions of empirical research Development level of Inclusive Financing Income gap between urban and rural areas whole country outstanding Zoom out (negative) eastern region outstanding Zoom out (negative) Midwest outstanding Expand (forward) departments to strengthen residents' confidence. Attention should be paid to the development of Internet information technology in rural areas. Technology is an important driving force and a support for the development of Inclusive Financing. Providing better Internet technology environment for rural residents can accelerate the popularization of Inclusive Financing in rural areas. The government should gradually change the intensity of fiscal expenditure from urban to rural areas, and increase financial support. Encourage and guide private capital to establish new financial institutions in rural areas. Encourage and help small and micro enterprises to obtain financial services, help residents in rural areas who do not need education to choose appropriate financial services or products, promote economic development in rural areas, and further narrow the income gap between urban and rural areas. 6.1 Conclusion From the control variables, the effects of regional economic development level, employment status and urbanization level on narrowing the urban-rural income gap are still very significant, while the effects of education level and industrial structure on narrowing the urban-rural income gap are still not obvious. With the continuous improvement of economic level, China's economy has turned into a high-quality development stage, but narrowing the income gap between urban and rural areas has always been the direction that needs efforts. In recent years, the rise of Inclusive Financing has provided new ideas for narrowing the income gap between urban and rural areas. Compared with the eastern region and the whole country, the results in the central and western regions are 7 E3S Web of Conferences 275, 03015 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 With the development and wide application of digital information technology, digital Inclusive Financing has gradually entered the public's field of vision and played an important role in narrowing the income gap between urban and rural areas. Based on the panel data of 31 provinces and cities in China, this paper establishes a model for empirical analysis and studies, and draws the following conclusions: with access to financial products and services, improve the income level and narrow the income gap between urban and rural areas. (2) From the regional situation, the development of Inclusive Financing plays the most significant role in narrowing the gap between the rich and the poor in the economically developed eastern region. (3) In the less developed central and western regions, the improvement of Inclusive Financing level may further widen the income gap between urban and rural areas, which is not conducive to the development of rural economy. (1) The development level of Inclusive Financing has a significant effect on narrowing the income gap between urban and rural areas, and developing Inclusive Financing can effectively narrow the income gap between urban and rural areas. To provide more residents 6.2 Relevant suggestions Establish relevant units or carry out structural grafting in relevant rural departments, and carry out publicity and promotion work by authoritative (4) Promote the economic development of the central and western regions and narrow the regional development gap. Because the economic level of the central and western regions is weaker than that of the eastern regions, it is necessary to further improve the economic level and lay a good economic foundation for 8 E3S Web of Conferences 275, 03015 (2021) EILCD 2021 E3S Web of Conferences 275, 03015 (2021) https://doi.org/10.1051/e3sconf/202127503015 the expansion and development of Inclusive Financing. Pay attention to the rational distribution of educational resources and fiscal expenditure, pay attention to the infrastructure construction and the distribution of educational resources in the central and western regions, and ensure the development of the central and western regions. Through policy implementation and publicity and guidance, we will provide more employment opportunities for the central and western regions and create more possibilities for capital inflow. For the central and western regions, foreign funds can be introduced to improve the development level of regional enterprises, so that they have certain policy support in the development process. At the same time, actively promote and implement digital Inclusive Financing, do a good job in the early stage of promotion, and improve the degree of digital development in the central and western regions. the expansion and development of Inclusive Financing. Pay attention to the rational distribution of educational resources and fiscal expenditure, pay attention to the infrastructure construction and the distribution of educational resources in the central and western regions, and ensure the development of the central and western regions. Through policy implementation and publicity and guidance, we will provide more employment opportunities for the central and western regions and create more possibilities for capital inflow. For the central and western regions, foreign funds can be introduced to improve the development level of regional enterprises, so that they have certain policy support in the development process. At the same time, actively promote and implement digital Inclusive Financing, do a good job in the early stage of promotion, and improve the degree of digital development in the central and western regions. (5) Strengthen the supervision system to prevent financial risks. The development of Internet finance is accompanied by corresponding problems and risks, which to a great extent hinders and affects the further development of digital Inclusive Financing. 6.2 Relevant suggestions The government should improve the information disclosure mechanism and relevant laws and regulations to provide legal guarantee for the development of Inclusive Financing. Improve the credit information system, enrich the information and situation of financial participants, reduce the probability of financial risks, improve the security of inclusive finance, enhance residents' resistance to risks, maintain financial market stability, promote economic growth, and narrow the income gap between urban and rural areas. References: 1. Allen F,Demirguc-Kunt A,Klapper L,et al. The foundations of financial inclusion:Understanding ownership and use of formal accounts[J]. Journal of Financial Intermediation,2016,27: 2. Zhang Xiaoyan. The impact of Inclusive Financing's development on urban-rural income gap under the background of Internet finance [J]. Accounting Monthly, 2016(17):94-97. 3. Zhang Zihao, Tan Yanzhi. Digital Inclusive Financing and Urban-Rural Income Gap in China: An Empirical Analysis Based on Spatial Econometric Model [J]. Financial Theory and Practice, 2018(06):1-7. 4. Zhao Bingqi. Digital Inclusive Financing and Urban-Rural Income Gap in China: An Empirical Study Based on Panel Threshold Model [J]. Journal of Social Sciences, 2020(01):196-205. 5. Xiong Deping, Chen Yuran. The impact of the development of digital Inclusive Financing on the income gap between urban and rural areas —— An empirical analysis based on unbalanced effect and threshold effect [J]. Changbai Journal, 2020(05): 99-106. 9 9
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First records of Viviparus acerosus (Bourguignat, 1862) (Gastropoda: Viviparidae) from the Czech Republic outside its native range
Folia Malacologica
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Folia Malacol. 27(3): 223–229 https://doi.org/10.12657/folmal.027.021 FIRST RECORDS OF VIVIPARUS ACEROSUS (BOURGUIGNAT, 1862) (GASTROPODA: VIVIPARIDAE) FROM THE CZECH REPUBLIC OUTSIDE ITS NATIVE RANGE Luboš Beran1*, Michal Horsák2, Sebastian Hofman3 Nature Conservation Agency of the Czech Republic, Regional Office Kokořínsko – Máchův kraj Protected Landscape Area Administration, Česká 149, CZ-27601 Mělnik, Czech Republic (e-mail: lubos.beran@nature.cz); https://orcid.org/0000-0002-5851-6048 2 Department of Botany and Zoology, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic; https://orcid.org/0000-0003-2742-2740 3 Department of Comparative Anatomy, Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Cracow, Poland; https://orcid.org/0000-0001-6044-3055 *corresponding author 1 Abstract: Three native species of the Viviparidae are known in the Czech Republic, namely Viviparus acerosus, V. contectus and V. viviparus. The native range of V. acerosus includes the Danube and probably Dnieper and Dvina river basins. In the Czech Republic it inhabits slowly flowing rivers and canals in the floodplain of the Dyje and Morava rivers in South Moravia (Danube River basin). Recently, this species was found outside its native range in four sites in Bohemia (Labe River basin) and in one site in Silesia (Odra River basin). The species identity of the abundant population from the Švihov dam reservoir (Labe River basin) was verified using COI sequences. These finds represent the first records of the species in the Labe and Odra river basins. The possible origin of these populations is discussed. Key words: Viviparus acerosus, Czech Republic, non-native occurrence, new records, recent spread INTRODUCTION There are three native species of the Viviparidae reported from the Czech Republic (Beran 2002, Horsák et al. 2013). Viviparus contectus (Millet, 1813) inhabits stagnant waters in the floodplains of large lowland rivers in the Labe, Odra and Danube river basins, while V. viviparus (Linnaeus, 1758) was known in the Czech Republic only from rivers of the Labe River basin (Beran 2002). Recently, probably native populations of the latter species have been discovered also in the upper section of the Dyje River (Danube River basin) at the boundary between Austria and the Czech Republic (Beran 2013). The third spe- cies, V. acerosus Bourguignat, 1862, typically inhabits slow-flowing rivers and canals in the floodplain of the Dyje and Morava rivers in South Moravia, also parts of the Danube River basin (Beran 2002, 2013). The species is considered endemic to the Danube and probably also Dnieper and Dvina river basins (Vavrova & Van Damme 2011, Welter-Schultes 2012, Vinarski & Kantor 2016) (Fig. 1). However, it has been recently found outside its native range, for example in the Netherlands (Soes et al. 2009). In this paper, we present and discuss the first records of this species in the Czech Republic outside its native range. Folia Malacologica is funded by the Ministry of Science and Higher Education, Republic of Poland, under agreement no 534/P-DUN/2018 of April 4th, 2018 allocated to the activities for disseminating science: Task 1: Preparation of English versions of publications (sum funded by DUN 12,000 PLN) and Task 2: Digitalisation of publications and scientific monographs to enable their open access in the Internet (sum funded by DUN 11,070 PLN). 224 Luboš Beran, Michal Horsák, Sebastian Hofman Fig. 1. Map of the Czech Republic with the known occurrence of Viviparus acerosus. Red dots – native occurrence, black triangles – records outside native range. Drawn by J. Vrba MATERIAL AND METHODS The material used in this study was collected by hand with searching of sediments, vegetation and surfaces of stones, wood and artificial materials. The snails for DNA analysis were fixed in 80% pure ethanol and DNA was extracted from foot tissue. The tissue was hydrated in TE buffer (3 × 10 min.); then total genomic DNA was extracted with the SHERLOCK extracting kit (A&A Biotechnology) and the final product was dissolved in 20 μl TE buffer. Details of PCR conditions, primers used and sequencing as well as of phylogenetic analysis were given in Szarowska et al. (2016) and Rysiewska et al. (2019). The following COI sequences from GenBank were used as references: KY781182 (deposited by David et al. 2017) and MK517417, MK517418, MK517420, MK517421, MK517423–MK5174427 (Rysiewska et al. 2019). Two new sequences MN272426–MN272427 obtained from specimens of the site Tvrdonice were also added to GenBank. We used COI sequences only, because other loci were monomorphic for Viviparus. Selected material of shells and/or specimens fixed in 80% ethanol is deposited in the authors’ collections. RESULTS AND DISCUSSION The first locality of V. acerosus outside the Danube River basin was found in the Švihov (Želivka) dam reservoir in 2015 (Figs 2, 4, 6). An abundant population was recorded in one site (49°37'25.2"N, 15°14'52.5"E, 2.8.2015, 15.9.2018, L. Beran leg.). The collected material was originally identified as V. viviparus (Beran 2017). Both species are morphologically similar and their populations are very variable in size. The pointed apex of V. acerosus, which in the field is the most useful feature distinguishing it from V. viviparus with blunt apex, is in the case of adult individuals very often difficult to use due to corrosion. Later revision and also COI sequences showed that this population actually belonged to V. acero­sus. Our analysis indicated that snails from Švihov and Tvrdonice (V. acerosus from the native range in the Danube River basin) were distinct from other the Viviparus species (Fig. 3), including V. contectus (p-distance 0.115) and V. viviparus (p-distance also 0.115). The sequences from Švihov and Tvrdonice were almost identical (1% differences) and represented one species. Another two populations of this species were found also in two ponds (Sýkora pond, 49°29'32.4"N, 15°23'12.7"E, 4.6.2016, L. Beran leg., Tuksa pond, 49°29'15.9"N, 15°23'52.3"E, 4.6.2016, 15.9.2018, L. Beran leg., Fig. 7), only about 18 km away from Records of Viviparus acerosus outside of its native range 225 Fig 2. Viviparus acerosus from the Švihov dam reservoir. Photo: Aleksandra Rysiewska Fig. 3. Cytochrome oxidase subunit I (COI) maximum likelihood tree; bootstrap supports and Bayesian probabilities and GenBank numbers were given Fig. 4. Live specimens of V. acerosus collected at the Švihov dam reservoir. Photo: Luboš Beran 226 Luboš Beran, Michal Horsák, Sebastian Hofman Fig. 5. Accumulation of fresh empty shells of V. acerosus at the Horní Bartošovický rybník pond (Silesia). Photo: Iva Pospíšilová Fig. 6. Švihov dam reservoir. Photo: Luboš Beran Records of Viviparus acerosus outside of its native range Fig. 7. Tuksa Pond. Photo: Luboš Beran Fig. 8. Horní Bartošovický rybník Pond. Photo: Iva Pospíšilová 227 228 Luboš Beran, Michal Horsák, Sebastian Hofman the first mentioned locality. In the case of the extensive Švihov dam reservoir (1,602 ha) other six sites were surveyed between 2015 and 2019 and V. acero­ sus was found in one other site only (49°36'51.5"N, 15°15'10"E, 25.7.2019, L. Beran leg.). Suprisingly, an abundant population of this species was also found in the pond of Horní Bartošovický rybník in the Poodří Protected Landscape Area in the Odra River basin in Silesia (49°40'38.6"N, 18°01'44.9"E, 5.9.2018, M. Horsák leg., Figs 5 and 8). These new records outside the native range represent the first records of this species from the Labe and Odra river basins (Fig. 1). The origin of these populations can be only deduced. This species (and other species of the Viviparidae) are often sold in garden centres and pet shops, so the first possible way might be an accidental escape from garden pools or a deliberate release. This way is mentioned as the most likely explanation for the occurrence of V. acerosus in Dordrecht in the Netherlands (Soes et al. 2009). As gastropods are capable to passive dispersal by birds over long distances, natural dispersal cannot be ruled out either. The nearest sites with the native populations of this gastropod are ca. 123 km and 105 km away from the sites in Bohemia in the Labe River basin and ca. 110 km away from the Horní Bartošovický rybník pond in Silesia. This site is near the boundary with Poland, but V. acerosus has not been reported yet from Poland (Piechocki & WawrzyniakWydrowska 2016). Thus, the nearest populations are known also only from South Moravia. The future population dynamic and even the species presence at these newly colonised sites are hard to predict. In the case of ponds, populations are affected adversely by water level manipulation. This is probably the case of a large extinction recorded in the Tuksa Pond. In 2016, a very abundant population was found there, while during the visit in 2018 only empty shells were recorded. Eutrophication may also have a negative effect on pond populations. However the situation in the Švihov dam reservoir is probably different. The reservoir serves as a source of drinking water and thus the quality of the environment is relatively well secured and stable. The reservoir is also inhabited by an abundant population of the invasive Dreissena poly­ morpha (Pallas, 1771), however no effect on the population of V. acerosus was observed (sessile specimens of D. polymorpha were found only on empty shells of V. acerosus) and the changes in the water level have caused extinction of only a part of the population. The question is whether the species will spread because it has been found in the reservoir only in two sites out of the seven sites surveyed between 2015 and 2019. From another point of view, a possible effect of these non-native populations should be considered. The impact of this species outside its native range is, however, difficult to predict; the most probable is only its potential impact on the two native species of Viviparus. All three species differ somewhat in their habitat preferences in the Czech Republic, but V. acerosus ecologically overlaps with the other two species. V. viviparus is known mostly from rivers, while V. contectus prefers stagnant and overgrown habitats. However, V. acerosus was found mostly in transitional habitats between stagnant and flowing waters and often occupied muddy and slow-flowing canals and rivers in floodplains. ACKNOWLEDGEMENTS The authors would like to thank Jan Vrba for drawing maps, Aleksandra Rysiewska, Iva Pospíšilová for photos and two anonymous reviewers for their valuable comments. REFERENCES Beran L. 2002. Vodní měkkýši České republiky – rozšíření a jeho změny, stanoviště, šíření, ohrožení a ochrana, červený seznam. Sborník přírodovědného klubu v Uh. Hradišti, Supplementum 10. Přírodovědný klub v Uherském Hradišti, Uherské Hradiště. Beran L. 2013. Freshwater molluscs of the Dyje (Thaya) river and its tributaries – the role of these water bodies in expansion of alien species and as a refuge for endangered gastropods and bivalves. Folia Malacologica 21: 143–160. http://doi.org/10.12657/folmal.021.018 Beran L. 2017. Vodní měkkýši Vysočiny. Malacologica Bohemoslovaca 16: 44–76. David A. A., Zhou H., Lewis A., Yhann A., Verra S. 2017. DNA Barcoding of the Banded Mystery Snail, Viviparus georgianus in the Adirondacks with quantifi- cation of parasitic infection in the species. American Malacological Bulletin 35: 175–180. https://doi. org/10.4003/006.035.0211 Horsák M., Juřičková L., Picka J. 2013. Měkkýši České a Slovenské republiky. Molluscs of the Czech and Slovak Republics. Kabourek, Zlín. Piechocki A., Wawrzyniak-Wydrowska B. 2016. Guide to freshwater and marine Mollusca of Poland. Bogucki Wydawnictwo Naukove, Poznań. Rysiewska A., Hofman S., Osikowski A., Beran L., Pešić. V., Falniowski A. 2019. Viviparus mamilla­ tus (Küster, 1852), and partial congruence between the morphology- allozyme- and DNA-based phylogeny in European Viviparidae (Caenogastropoda: Records of Viviparus acerosus outside of its native range 229 Architaenioglossa). Folia Malacologica 27: 43–51, https://doi.org/10.12657/folmal.027.004 e.T23040A9408473. http://doi.org/10.2305/IUCN. UK.2011-2.RLTS.T23040A9408473.en Soes D. M., Glöer P., Winter A. J. de 2009. Viviparus ace­ rosus (Bourguignat, 1862) (Gastropoda: Viviparidae), a new exotic snail species for the Dutch fauna. Aquatic Invasions 4: 373–375. Vinarski M. V., Kantor Y. I. 2016. Analytical catalogue of fresh and brackish water molluscs of Russia and adjacent countries. A. N. Severtsov Institute of Ecology and Evolution of RAS, Moscow. Szarowska M., Osikowski A., Hofman S., Falniowski A. 2016. Pseudamnicola Paulucci, 1878 (Caenogastropoda: Truncatelloidea) from the Aegean Islands: a long or short story? Organisms Diversity & Evolution 16: 121– 139. https://doi.org/10.1007/s13127-015-0235-5 Welter-Schultes F. W. 2012. European non-marine molluscs, a guide for species identification. Planet Poster Editions, Göttingen Vavrova L., Van Damme D. 2011. Viviparus aceros­ us. The IUCN Red List of Threatened Species 2011: Received: July 31st, 2019 Revised: August 6th, 2019 Accepted: August 7th, 2019 Published on-line: September 14th, 2019
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Electrodeposition of Nickel onto 12K Carbon Fibre Tow in a Continuous Manner
Croatica chemica acta
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INTRODUCTION homogeneous throughout the tow, it is therefore very likely that numerous (present) active sites might require different levels of activation energy for this process. The described voltage levels are between 10 and 50 V, and electric charge in the amount of 600 C/1 000 fibre strands, at 30 V. The single plating cell of the Cyanamid system consists of a cylindrical electrolyte tank, a series of contact and non-contact rollers, and several anode baskets, containing electrolytic nickel pellets. The report- ed operational line speed is 1.5 m min–1 and thickness of suchproduced Ni deposit (for 7 µm in diameter 12K fibre tow) about 0.5 µm. It has to be stated, how- ever, that the description of the Cyanamid process (given in Refs. 14 through 19) is quite superficial and does concern a large- scale (80 tow), commercial plating process. Carbon fibre (CF) and nickel-coated carbon fibre (NiCCF) materials are of primary interest to a number of industry sectors. One of the most important applica- tions for such materials is their utilization in electrostat- ic dissipation (ESD) and electromagnetic shielding interference (EMI) technologies. These technologies are especially important for automotive, cell-phone, laptop computer and military industry markets.1–5 Other report- ed applications for carbon fibre-based materials involve electrochemical corrosion processes and lithium-ion battery technologies.6–8 Nickel-coated carbon fibre composites can be pro- duced by means of chemical vapour deposition (CVD) method,9 electroless10–13 or by electrochemical deposi- tion of nickel layer onto the surface of carbon fibre tow material. Originally, the commercial process of Ni electrodeposition on 12K carbon fibre tow was exten- sively studied by L. Morin of American Cyanamid company. The Cyanamid technology (protected by the US patent law)14–19 is a several-step, Watts bath-based Ni deposition process. According to this invention,14 an initial stage of metal electrodeposition (nucleation phase) is realized through application of high external voltages, in order to provide uniform nucleation with the fibrils. As the surface of CF filaments is not very On the other hand, current study reports a small (single tow) laboratory-size CF electroplating system, which in principal was set to study the effect of basic CF pretreatments, working parameters of the plating setup and the plating bath composition on the quality of such-produced NiCCF composite material. CROATICA CHEMICA ACTA CCACAA, ISSN 0011-1643, e-ISSN 1334-417X Croat. Chem. Acta 85 (1) (2012) 1–8. http://dx.doi.org/10.5562/cca1743 Original Scientific Article RECEIVED AUGUST 9, 2010; ACCEPTED DECEMBER 17, 2010 Abstract. Nickel-coated carbon fibre (NiCCF) is an important composite material, which finds nu- merous engineering applications, primarily within: computer, telecommunication, automotive and mili- tary industries. In general, NiCCF can be produced by one of the three methods, namely: chemical va- pour deposition (CVD), electroless, and electrochemical (galvanic) deposition of Ni on a carbon fibre tow material. The present paper reports a study of the process of nickel electrodeposition (at ultrathin layers of ≈ 0.3–0.5 μm) onto the surface of 12K (12 000 single filaments) carbon fibre (CF) tow, carried-out in a continuous way. In addition, the effect of selected pre-treatments applied to carbon fibre, as well as that of operational parameters of the process, on the quality of the NiCCF product were investigated. (doi: 10.5562/cca1743) Keywords: nickel-coated carbon fibre, NiCCF, nickel electrodeposition, CF pre-treatments Electrodeposition of Nickel onto 12K Carbon Fibre Tow in a Continuous Manner Boguslaw Pierozynski Department of Chemistry, Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn, Plac Lodzki 4, 10-957 Olsztyn, Poland (E-mail: bogpierozynski@yahoo.ca) EXPERIMENTAL Figure 2. A PVC-made carbon fibre electroplating unit, where the following components are: spring-loaded, adjustable pinch type cathodic CR assembly (1); electrolyte sprinkler (2); elec- trolyte overflow compartment (3); electrolyte circulation (in) (4); electrolyte circulation (out) (5); DSA anode assembly (6); adjustable wall (7): adjustable, wrap type cathodic CR assem- bly, mounted on electroplater’s top cover (8); electrolyte drain (9); CF tow’s passage (10); distance between an initial cathod- ic contact point and entrance of the nucleation plating com- partment (d). d A laboratory system for electrodeposition of Ni on a single tow of 12K carbon fibre material (see Figure 1) consists of the following elements: d d  fibre pay-out assembly,  PVC-made fibre electroplating unit (including two dc 10 A / 20 V power supplies, with current resolu- tion of 10 mA),  fibre rinse station (run on distilled H2O),  fibre drying station (hair dryer-based chamber) and Figure 2. A PVC-made carbon fibre electroplating unit, where the following components are: spring-loaded, adjustable pinch type cathodic CR assembly (1); electrolyte sprinkler (2); elec- trolyte overflow compartment (3); electrolyte circulation (in) (4); electrolyte circulation (out) (5); DSA anode assembly (6); adjustable wall (7): adjustable, wrap type cathodic CR assem- bly, mounted on electroplater’s top cover (8); electrolyte drain (9); CF tow’s passage (10); distance between an initial cathod- ic contact point and entrance of the nucleation plating com- partment (d).  motorized spool take-up assembly. The PVC electroplating unit (see Figure 2) was built for the purpose of this work. The plating unit al- lowed to study Ni electrodeposition process in single and double-cell configurations, with adjustable effective cell lengths of 2.5, 5 and 8 cm (first cell), and 5 and 10 cm for the second cell. The electroplater was designed to operate at current-densities ranging from 5 to 50 mA cm–2, resulting voltages ≈ 2–10 V (for the first cell) and the plating line speed between 10 and 100 cm min–1. Two DSA (Nb clad Pt) anodes per cell (20×20 and 45×20 mm strip type, with adjustable separation gap of 10 to 20 mm) were used. Two sets of highly-polished (20 mm in diam.), stainless-steel (SS) and/or Cu cathod- ic contact rollers (pinch or wrap configuration, see Fig- ure 3) per each deposition cell were employed, with flexible adjustment of the distance d between the ca- thodic contact points and an entrance (or exit) of the plating chamber. INTRODUCTION Some of the most important parameters that have been addressed in this work are the following:  quality and initial condition of carbon fibre precursor,  fibre passage system: fibre unwinding, cell passage and take-up assemblies, 2 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow  fibre spreading (optimization of the CF tow spreading within an electroplater),  fibre spreading (optimization of the CF tow spreading within an electroplater),  electrochemical and other parameters, and cell design (current-density and voltage, design of fibre contact and non-contact roller assemblies, internal cathode-to-anode distances, bath composition, op- erational pH and temperature parameters, electro- lyte circulation, etc.). Figure 1. Schematic diagram of a laboratory-size carbon fibre electroplating line, where the following elements are: fibre pay-out and spreading assembly (1); electroplating cell assem- bly (2); fibre rinse station (3); fibre drying station (hair dryer- based) (4); and fibre take-up assembly (5). EXPERIMENTAL In addition, the electroplater was equipped with four sets of electrolyte sprinklers, cou- pled with an original design of electrolyte overflow compartments, aimed both at minimizing the d parame- ter, as well as the effect of electrolyte splashing outside the plating cells. The fibre pay-out assembly consisted of a regular CF 12K spool (Hexcel 12K AS4 product), several 10 cm in diam. PTFE-made fibre lead rollers and three, 5 cm in diam. Cu (or SS)-made fibre spread- ing contact rollers. The tension for the CF tow was arbitrarily set through adjustment of the clutch on the pay-out spool assembly. Similar design was employed on the motorized, cross-winding take-up end of the fibre plating system. Figure 3. Cathodic contact rollers, shown in pinch (a) and wrap (b) configurations. Figure 3. Cathodic contact rollers, shown in pinch (a) and wrap (b) configurations. A typical Watts20,21 nickel plating bath was select- ed for the purpose of this work. Base electrolyte con- tained 300 g L–1 NiSO4 · 6H2O, 50 g L–1 NiCl2 · 6H2O and 40 g L–1 H3BO3. In addition, two bath additives (wetting agent: Na lauryl sulphate and Ni grain refiner: Na saccharine, in the amount of 0.1–0.5 g L–1 and 0.5–1.0 g L–1, correspondingly) were also tested. Plating tempera- ture varied between 45 and 60 °C, and pH was kept be- tween 3.0 and 5.0. As pH of the plating bath continued to decline upon Ni electrodeposition, this parameter was constantly monitored during the process and readjusted Croat. Chem. Acta 85 (2012) 1. B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow 3 verified through tow side viewing, as well as by cross- section examination of the composites, at magnifica- tions between 25 and 3 000. For the cross-sectional examination, tow samples were secured in a holder by epoxy resin. Then, they were ground flat on fine-grade silicon carbide abrasive paper of 2 500 grade (Metalogis, Poland). The above procedure was followed by polishing the surface, using 3 and 1 micron polycrys- talline diamond suspensions (also from Metalogis). Finally, powder XRD (x-ray diffraction) technique was employed to determine average sizes of crystallite do- mains27 for chosen NiCCF specimens. (when necessary) through addition of small quantities of NaOH solution (w = 10 %). Based on the deposited mass of Ni, the electrolyte was also replenished (at regular intervals) with nickel and chloride ions. EXPERIMENTAL ) Hexcel 12K AS4 carbon fibre22 is polyacrylonitrile (PAN) based product that is delivered to the market as a continuous 12K ribbon, in ≈ 3.6 kg spools. The tow is sized by the manufacturer with an epoxy resin (0.21 % on average), in order to ease fibre handling and thus to re- duce damage to the material. This organic sizing was removed from the fibre (prior to Ni deposition) by heat- treatment, carried-out in a muffle (tube) furnace at low oxygen atmosphere (with fibre residence time of 4 hours at 350 °C). Another, alternative CF pre-treatment in- volved subjection of "as received" Hexcel CF tow to desizing in acetone, which was followed by electrochem- ical oxidation in 0.5 mol dm–3 H2SO4 (performed at an anodic current-density, ja = 0.5 mA cm–2 for 5 min, at room temperature with a Ti/Pt auxiliary electrode). The latter (anodic oxidation) treatment leads to functionaliza- tion23–26 of the CF surface, which process could potential- ly have a strong impact on both mechanical and electro- chemical properties of such-treated carbon fibre tow. In addition, a simple, SEM-supported knot test was used to qualitatively evaluate (and compare) the strength of interfacial adhesion of Ni (potentially, also its ductility) to the surface of CF filaments. The knot test28,29 is a mechanical bending experiment, in which a piece of NiCCF tow is bent sharply enough (a constant load of 500 g for 5 s was applied to tie the NiCCF knots in this work) to break the metal coating on the tension side of the bend. Then, the knots are qualitatively exam- ined and compared by the SEM method. Examination of the NiCCF product started with randomly carried-out selection of NiCCF samples (20 cm long, collected every 10 m of the product). Firstly, these fibre specimens were (again) carefully washed in distilled water. Then, they were placed in an excicator for 48 hours, followed by weighing on a precision bal- ance (Sartorius CP224-OCE) to 0.1 mg. The above procedure allowed precise evaluation of characteristic nickel loading levels by means of a typical "weighing method". In addition, resistance measurements were performed on selected samples of the 12K NiCCF tow, by measuring the voltage drop at constant applied cur- rent (100 mA), over a distance of 20 cm. Both electrical contact points on the NiCCF samples were silver- painted in order to reduce the electrical contact re- sistance. RESULTS AND DISCUSSION Table 1 presents an optimized configuration of the car- bon fibre electroplating system, which includes electro- chemical and all operational parameters that happened to play a key role in the plating process. EXPERIMENTAL Fibre resistance was then calculated from equation 1: Croat. Chem. Acta 85 (2012) 1. Operating Parameters and the Plating Cells' Configuration Tow position Vertical Electrolyte Watts (SO4 2– + Cl–) based Bath temperature/°C 48–50 Bath pH/adjustment 4.0–5.0 (10 % NaOH solution) Bath additives Sodium lauryl sulphate: 0.2 g L–1 Sodium saccharine: 0.5 g L–1 12K50 NiCCF product wNi ≈ 50 %; Ni coating ≈ 0.4 µm; Ni grain size ≈ 25 nm (PXRD- calculated) Table 1. An optimized configuration for a laboratory-size carbon fibre electroplating system, employed during deposi- tion of nickel onto 12K carbon fibre tow Carbon Fibre Electroplating Cell Assembly Plating cells 2 cells; 5 cm long each Contact rollers (CR) Highly-polished SS, 20 mm diam. Anodes (A) 4.5 × 2.0 cm DSA strips (two A per each cell) Distances First cell: 1st CR-A: 2 cm, pinch contact; 2nd CR-A: 3.5 cm, wrap contact Second cell: 2nd CR-A: 2 cm, pinch contact Both cells: CF-A: 0.5 cm Voltage / V First cell: 7.0–10.5 Second cell: 3.0–5.5 Current / A (current ranges de- pendent on the set Ni loading level) First cell: 4.5–6.0 Second cell: 1.0–3.0 Line speed / m min–1 0.5 Tow spread / cm 0.8 Spreading method Mechanical; Cu rollers, 50 mm diam. Carbon fibre Hexcel 12K AS4 tow CF pre-treatment Heat-treated at 350 °C for 4 hours. Tow position Vertical Electrolyte Watts (SO4 2– + Cl–) based Bath temperature/°C 48–50 Bath pH/adjustment 4.0–5.0 (10 % NaOH solution) Bath additives Sodium lauryl sulphate: 0.2 g L–1 Sodium saccharine: 0.5 g L–1 12K50 NiCCF product wNi ≈ 50 %; Ni coating ≈ 0.4 µm; Ni grain size ≈ 25 nm (PXRD- calculated) With respect to configuration of the plating system, superior quality NiCCF (see later Figure 9a) was pro- duced with the two-cell (5 cm each) plating arrangement, equipped with three (or four) sets of highly-polished, SS- made contact rollers (CR). The foremost, spring-loaded pinch CR set, was positioned just over 2 cm (distance d in Figure 2) away from the DSA anode of the first cell. The above was only possible due to the unique design of the electrolyte overflow compartment, which allowed to minimize an effective, cathode-to-anode voltage (and thus prevented fibre from severe overheating and burn- ing) of the first cell. Then, two (or a single) wrap-type fibre contact roller assemblies were installed, both were mounted on the plater's cover (see also Figure 2 for more details). Another pinchtype CR assembly was fastened at the exit of the second plating cell. Operating Parameters and the Plating Cells' Configuration Thus, pH control of the electrolyte was found to be a crucial element of the plating process. As acidity of the bath kept continuously rising during the course of metal deposition, frequent adjustment of pH was necessary, where optimum pH range was found to be around 4.5– 5.0 (for most favorable plating temperature range of 48– 50 °C). For more acidic solution, hydrogen evolution reaction (HER) becomes more competitive with reduc- tion of Ni2+ ions at low overvoltages (see the two possi- ble cathodic reactions in equations 2 and 3, and the corresponding anodic reaction in equation 4). As a con- sequence, the product (especially at pH around 3.0) appears to have much less homogeneous distribution of nickel throughout the tow than that produced at higher pH values (see Figures 4a and 4b, correspondingly). At pH = 3.0, it is the outer tow sections which primarily become metal-coated (Figure 4a), in relation to the fact that diffu- sion of evolved (and likely surface-covering) hydrogen bubbles from the centre part of the CF tow is much more difficult than the corresponding process, but proceeding at the outer tow parts. V R I l  (1) (1) where: R / Ω cm–1 is calculated resistance of the NiCCF tow sample, V / mV is measured voltage, V / mV is measured voltage, V / mV is measured voltage, I / mA is applied current, l / cm is sample's length. Subsequently, selected NiCCF samples were sent for SEM analysis, where quality of nickel deposits was Croat. Chem. Acta 85 (2012) 1. 4 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Ni2+(aq) + 2e– Ni0(s) (2) 2H+(aq) + 2e– H2(g) (3) 2Cl–(aq) + 2e– Cl2(g) (4) (insoluble DSA anodes) Ni2+(aq) + 2e– Ni0(s) (2) 2H+(aq) + 2e– H2(g) (3) 2Cl–(aq) + 2e– Cl2(g) (4) (insoluble DSA anodes) With respect to configuration of the plating system, superior quality NiCCF (see later Figure 9a) was pro- duced with the two-cell (5 cm each) plating arrangement, equipped with three (or four) sets of highly-polished, SS- made contact rollers (CR). The foremost, spring-loaded pinch CR set, was positioned just over 2 cm (distance d in Figure 2) away from the DSA anode of the first cell. Operating Parameters and the Plating Cells' Configuration The above was only possible due to the unique design of the electrolyte overflow compartment, which allowed to minimize an effective, cathode-to-anode voltage (and thus prevented fibre from severe overheating and burn- ing) of the first cell. Then, two (or a single) wrap-type fibre contact roller assemblies were installed, both were mounted on the plater's cover (see also Figure 2 for more details). Another pinchtype CR assembly was fastened at the exit of the second plating cell. The carbon fibre tow was vertically positioned within the plater, giving on optimum cathode-to-anode distance of ≈ 0.5 cm in both cells. The above configuration of the electroplater al- lowed to maintain (for the CF line speed of 50 cm min–1) voltage ranges between 7.0–10.5 V within the first (nu- cleation) chamber and ≈ 3.0–5.5 V within the second nickel deposition cell. The resulting current-densities came to 32.0–42.4 mA cm–2 (for the nucleation cell) and 7.1–21.3 mA cm–2 for the second plating cell (current ranges varied along with the set loading level of Ni). In addition, it should be stressed here that employment of much lower current densities (on the order of 20 mA cm–2) for the primary deposition cell resulted in inferior Ni deposit homogeneity. Table 1. An optimized configuration for a laboratory-size carbon fibre electroplating system, employed during deposi- tion of nickel onto 12K carbon fibre tow Carbon Fibre Electroplating Cell Assembly Plating cells 2 cells; 5 cm long each Contact rollers (CR) Highly-polished SS, 20 mm diam. Anodes (A) 4.5 × 2.0 cm DSA strips (two A per each cell) Distances First cell: 1st CR-A: 2 cm, pinch contact; 2nd CR-A: 3.5 cm, wrap contact Second cell: 2nd CR-A: 2 cm, pinch contact Both cells: CF-A: 0.5 cm Voltage / V First cell: 7.0–10.5 Second cell: 3.0–5.5 Current / A (current ranges de- pendent on the set Ni loading level) First cell: 4.5–6.0 Second cell: 1.0–3.0 Line speed / m min–1 0.5 Tow spread / cm 0.8 Spreading method Mechanical; Cu rollers, 50 mm diam. Carbon fibre Hexcel 12K AS4 tow CF pre-treatment Heat-treated at 350 °C for 4 hours. Operating Parameters and the Plating Cells' Configuration (a) (a) In this work, experimentally optimized CF tow spreading (investigated for ribbon widths between 0.2 and 1.2 cm) came to about 0.8 cm (see the effect of the CF tow spreading on the quality of the NiCCF product in Figures 5a and 5b), which for the springloaded pinch CR assembly (two cathode rollers) means the presence of ≈ 11 carbon fibre layers between the two cathode rollers (see Figure 6). Thus, for the tow's spread of only 0.2 cm, most of the CF filaments were not in direct contact with the contact roller(s) and as such they did experience dramatically reduced current-densities, as compared to those which were concentrated at the out- side of the CF tow. As a result, the tow was primarily plated with nickel at its external regions (see Figure 5a again and contrast this behaviour with that observed in Figure 5b). The above behaviour is in principal similar (although for different reasons) to that observed in Fig- ure 4a for the plating carried-out at pH = 3.0. On the other hand, for the tow's spread over 0.8 cm, a consider- able increase of fibre nesting within the electroplating unit was observed over time of metal deposition. The above resulted in gradual deterioration of mechanical properties, as well as the plating quality for the NiCCF composite. (b) (b) Figure 5. The effect of CF tow’s spread on the quality of la- boratory-electrodeposited NiCCF tow samples, shown in SEM cross-sectional micrograph pictures for the tow’s spread of 0.2 cm at wNi ≈ 36 % (a) and 0.8 cm at wNi ≈ 40 % (b), taken at a given magnification. On the other hand, the quality of electrodeposited NiCCF significantly depended on the extent of CF tow spreading within the electroplater. As the fibre tow is spread, it forms a ribbon comprised of layers of individ- ual carbon filaments. Then, as the ribbon moves over the contact rollers, each individual carbon fibre changes position and its distance from the cathode roller, leading to variation of contact resistance. For the 12K fibre tow and ideal spreading, each carbon filament should be in intimate contact with the cathode roller. Thus, assuming the 12K tow of 7 µm in diam. Operating Parameters and the Plating Cells' Configuration The carbon fibre tow was vertically positioned within the plater, giving on optimum cathode-to-anode distance of ≈ 0.5 cm in both cells. The above configuration of the electroplater al- lowed to maintain (for the CF line speed of 50 cm min–1) voltage ranges between 7.0–10.5 V within the first (nu- cleation) chamber and ≈ 3.0–5.5 V within the second nickel deposition cell. The resulting current-densities came to 32.0–42.4 mA cm–2 (for the nucleation cell) and 7.1–21.3 mA cm–2 for the second plating cell (current ranges varied along with the set loading level of Ni). In addition, it should be stressed here that employment of much lower current densities (on the order of 20 mA cm–2) for the primary deposition cell resulted in inferior Ni deposit homogeneity. Figure 4. The effect of pH on the quality of laboratory-electrodeposited NiCCF tow samples (at wNi ≈ 30 %), shown in SEM cross-sectional micrograph pictures for pH = 3.0 (a) and pH = 5.0 (b), taken at a given magnification. (a) (b) (a) (b) (b) (a) Figure 4. The effect of pH on the quality of laboratory-electrodeposited NiCCF tow samples (at wNi ≈ 30 %), shown in SEM cross-sectional micrograph pictures for pH = 3.0 (a) and pH = 5.0 (b), taken at a given magnification. Croat. Chem. Acta 85 (2012) 1. 5 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Figure 5. The effect of CF tow’s spread on the quality of la- boratory-electrodeposited NiCCF tow samples, shown in SEM cross-sectional micrograph pictures for the tow’s spread of 0.2 cm at wNi ≈ 36 % (a) and 0.8 cm at wNi ≈ 40 % (b), taken at a given magnification. (a) (b) minimize the electrical contact resistance between the fibres, in order to provide more uniform current distri- bution within the plating CF tow. However, an idea behind a practical configuration of the CF tow spreading within the electroplater is always to compromise be- tween the contact resistance parameter and the extent of fibre damage (the latter effect being directly proportion- al to the width of the fibre ribbon, in relation to the fibre tension parameter). Significant increase of fibre tension leads to extensive formation of fibre nests inside the plating cells and as a consequence to further increase of fibre tension, and its damage. Operating Parameters and the Plating Cells' Configuration fibre (and a single cath- ode contact roller), for perfect tow spreading (each filament is in contact with the CR) the tow spreading should provide about 8.4 cm (12 000 × 7 × 10–4 cm) wide ribbon. In addition, it should be mentioned that any fibre that is not in direct contact with the cathode roller will have to overcome the radial resistivity of the fibre (or fibres) that shield it from the roller. These non- contact fibres will exhibit significantly reduced current- densities upon plating, as compared to those being in direct contact with the cathode roller(s). Therefore, the cathode roller configuration should primarily attempt to Croat. Chem. Acta 85 (2012) 1. CF Pre-treatments and Bath Additives An optimized, pinch-type cathodic CR assembly, with residing 11 layers of carbon fibre filaments. Figure 7. SEM micrograph pictures of knot-tied NiCCF sam- ples (at wNi ≈ 40 %) for NiCCF obtained with surface electrooxidized CF (a) and heat-treated CF tow (b), taken at 300 magnification. (b) (b) Figure 6. An optimized, pinch-type cathodic CR assembly, with residing 11 layers of carbon fibre filaments. as compared to that produced with simply heat-treated CF tow (see Figure 7b). These effects are likely to re- flect improved adhesion between the Ni coating and the CF substrate, in relation to surface functionalization and roughening of electrooxidized CF tow filaments. It has to be stated here that surface-electrooxidized carbon fibre (see an earlier work from this laboratory in Ref. 30) exhibited significant increase of the electrochemi- cally accessible surface area. Based on the calculated double-layer capacitance (Cdl) parameter in Ref. 30, the surface area of H2SO4-electrooxidized Hexcel 12K CF tow was ≈ 6.6 times greater than that of the heat- pretreated carbon fibre tow. as compared to that produced with simply heat-treated CF tow (see Figure 7b). These effects are likely to re- flect improved adhesion between the Ni coating and the CF substrate, in relation to surface functionalization and roughening of electrooxidized CF tow filaments. It has to be stated here that surface-electrooxidized carbon fibre (see an earlier work from this laboratory in Ref. 30) exhibited significant increase of the electrochemi- cally accessible surface area. Based on the calculated double-layer capacitance (Cdl) parameter in Ref. 30, the surface area of H2SO4-electrooxidized Hexcel 12K CF tow was ≈ 6.6 times greater than that of the heat- pretreated carbon fibre tow. Figure 7. SEM micrograph pictures of knot-tied NiCCF sam- ples (at wNi ≈ 40 %) for NiCCF obtained with surface electrooxidized CF (a) and heat-treated CF tow (b), taken at 300 magnification. Figure 8. A resistance vs. nickel loading plot for laboratory- electrodeposited (and optimized) NiCCF tow samples. Nevertheless, it has to be stressed that the knot test is itself somewhat ambiguous in character, as tension and shape of the knot may vary from one sample to another. Thus, the knot method enables only qualitative evaluations and comparisons, and its results have to be discussed with caution. CF Pre-treatments and Bath Additives Interestingly, neither selected bath additives nor an electrooxidation pre-treatment applied to the CF tow (see Experimental part above for details) had any signif- icant effect on the quality of such-produced NiCCF composite (including Ni coverage on the CF tow). However, it is important to maintain a relatively low level of sodium lauryl sulphate (e.g. at 0.1–0.2 g L–1) in the plating bath. Excessive amounts of the wetting agent tend to induce severe bath foaming (accelerated by the presence of hydrogen bubbles), which leads to nonuniform Ni electrodeposits. On the other hand, SEM-supported knot tests showed some improvement in the composite mechanical performance for the NiCCF produced with surface- electrooxidized 12K carbon fibre tow. Here (see Figure 7a), somewhat less extensive delamination and Ni frac- turing effects are observed for a knot-tied tow sample, Croat. Chem. Acta 85 (2012) 1. 6 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Figure 7. SEM micrograph pictures of knot-tied NiCCF sam- ples (at wNi ≈ 40 %) for NiCCF obtained with surface electrooxidized CF (a) and heat-treated CF tow (b), taken at 300 magnification. (a) (b) as compared to that produced with simply heat-treated CF tow (see Figure 7b). These effects are likely to re- flect improved adhesion between the Ni coating and the CF substrate, in relation to surface functionalization and roughening of electrooxidized CF tow filaments. It has to be stated here that surface-electrooxidized carbon fibre (see an earlier work from this laboratory in Ref. 30) exhibited significant increase of the electrochemi- cally accessible surface area. Based on the calculated double-layer capacitance (Cdl) parameter in Ref. 30, the surface area of H2SO4-electrooxidized Hexcel 12K CF tow was ≈ 6.6 times greater than that of the heat- pretreated carbon fibre tow. Nevertheless, it has to be stressed that the knot test is itself somewhat ambiguous in character, as tension and shape of the knot may vary from one sample to Figure 7. SEM micrograph pictures of knot-tied NiCCF sam- ples (at wNi ≈ 40 %) for NiCCF obtained with surface electrooxidized CF (a) and heat-treated CF tow (b), taken at 300 magnification. Figure 6. An optimized, pinch-type cathodic CR assembly, with residing 11 layers of carbon fibre filaments. (a) (b) Figure 6. An optimized, pinch-type cathodic CR assembly, with residing 11 layers of carbon fibre filaments. (a) Figure 6. CONCLUSION Fairly uniform, ultra-thin deposits of Ni on 12 000- filament CF tow can suitably be produced through a continuous metal electrodeposition process. However, technological aspects of this process are quite complex, in addition to the required advanced knowledge of fibre handling. The most important conclusions that can be drawn from this work are the following:  in-line resistance measurement could be utilized for direct monitoring of electrodeposited lev- el/thickness of Ni on the CF tow  in-line resistance measurement could be utilized for direct monitoring of electrodeposited lev- el/thickness of Ni on the CF tow  applied bath additives and basic CF tow pre- treatments had rather insignificant effect on the quality (homogeneity) of such-produced NiCCF composites; however surface electrooxidation treatment applied to CF led to somewhat improved (qualitatively) adhesion between Ni coating and the carbon fibre surface. The thickness of the Ni coating was estimated by measurements conducted on high magnification, cross- sectional SEM images. Thus, 100 sample filaments were representatively selected in 10 different areas of the tow (10 × 10 samples). Such estimated thickness of the Ni coating for wNi ≈ 50 % composite came to 0.39 ± 0.02 μm. Acknowledgements. Grateful acknowledgement is made by the author of this work to the Commission of the European Com- munities for financial support of the research described in this paper. This work was financed by the European Union, Marie Curie International Reintegration Grant, acronym "Fibres", contract No.: 031073. In-line Monitoring of Ni Loading Levels Figure 8 shows the influence of Ni loading on the NiCCF's electrical resistance parameter. The fibre re- sistance parameter was derived (see again the Exper- imental part above for details) in reference to the Ni loading levels, estimated by means of the weighing method. The resistance measurements were performed for selected run tests, all carried-out with optimized plating setup and bath parameters. It is strongly believed that the abovediscussed correlation could be found suit- able for continuous, in-line monitoring of the nickel loading level. Thus, any unwanted change in the re- Figure 8. A resistance vs. nickel loading plot for laboratory- electrodeposited (and optimized) NiCCF tow samples. sistance parameter (caused by variation in the mass fraction, w/% of the metal deposit) could immediately be compensated through an appropriate re-adjustment of preset electrical parameters, or the plating line speed. Croat. Chem. Acta 85 (2012) 1. 7 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Figure 9. The effect of Ni loading on the quality of laborato- ry-electrodeposited NiCCF tow samples, shown in SEM mi- crograph pictures for the Ni loadings of wNi ≈ 40 % (a) and wNi ≈ 10 % (b), taken at 500 and 3000 magnifications. (a) (b) (a) (b) the grain growth phase takes place in the following plating chamber (both cells together constitute a CF tow electroplating unit) (a) (a) (a) (a) superior/homogeneous Ni deposits require: superior/homogeneous Ni deposits require: superior/homogeneous Ni deposits require: (i) electrolyte pH within 4.0 to 5.0 (with proper bath circulation and agitation) (i) electrolyte pH within 4.0 to 5.0 (with proper bath circulation and agitation) (ii) electrolyte temperature about 50 °C (b) (b) (b) (b) (iii) CF tow's spread (optimized in this work at 0.8 cm for 12K CF tow) should be set to compromise between the electrical contact resistance parameter and the extent of actual fibre damage within the plater (more advanced laboratory designs could capitalize on the ap- plication of a professional fibre tension con- trolling device, e.g. Helman tensioner unit) Figure 9. The effect of Ni loading on the quality of laborato- ry-electrodeposited NiCCF tow samples, shown in SEM mi- crograph pictures for the Ni loadings of wNi ≈ 40 % (a) and wNi ≈ 10 % (b), taken at 500 and 3000 magnifications. (iv) minimized distances between cathodic CRs and DSA anode assemblies (to reduce opera- tional voltages and thus to limit fibre over- heating) (v) electroplater's line speeds between 20 and 100 cm min–1 [with cathodic current-densities of ≈ 30–40 and 10–20 mA cm–2 for the nu- cleation and the grain growth cells, corre- spondingly (for the line speed of 50 cm min–1)] However, only NiCCF composites with Ni depos- its on the order of wNi = 30 % (or higher) exhibited fairly continuous (but non-uniform) metal coverage of individual carbon fibre filaments (see Figure 9a) within the 12K CF tow. Conversely, all NiCCF samples with Ni loadings below wNi ≈ 20 % were completely non- homogeneous, with respect to the surface distribution of the metal deposit (Figure 9b). The above-observed phe- nomenon strongly supports the idea of severe incon- sistency of the applied "electrode potential" (and as a consequence also the Gibbs free energy of activation for the process) within the complex, 12 000-filament CF tow entity. REFERENCES 1. D. D. L. Chung, J. Mater. Sci. 39 (2004) 2645–2661. 2. D. Markham, Mater. Design 21 (2000) 45–50. 3. S. S. Tzeng and F. Y. Chang, Mater. Sci. Eng. A302 (2001) 258–267. 4. D. D. L. Chung, Carbon 39 (2001) 279–285. 5. S. Y. Fu, B. Lauke, E. Mader, C. Y. Yue, and X. Hu, Composites: Part A 31 (2000) 1117–1125.  Ni deposition should be realized in two steps; initial, nucleation phase proceeds within the first (high current-density is applied, which allows for more uniform nucleation with the fibrils) cell and 6. L. Bertolini, F. Bolzoni, T. Pastore, and P. Pedeferri, Cement and Concrete Research 34 (2004) 681–694. 6. L. Bertolini, F. Bolzoni, T. Pastore, and P. Pedeferri, Ceme Concrete Research 34 (2004) 681–694. 7. P. J. 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Conservation laws, solitary wave solutions, and lie analysis for the nonlinear chains of atoms
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Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See the published version at https://doi.org/10.1038/s41598-023-38658-w. Conservation Laws, Solitary Wave Solutions, and Lie Analysis for the Nonlinear Chains of Atoms Muhammad Junaid U Rehman  (  muhammad-junaid.u-rehman@dokt.p.lodz.pl ) Lodz University of Technology Jan Awrejcewicz  Lodz University of Technology Grzegorz Kudra  Lodz University of Technology Article Keywords: Nonlinear chains of atoms, Lie symmetry approach, Multiplier scheme, Conserved vectors, New auxiliary method Posted Date: June 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3026335/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See the published version at https://doi.org/10.1038/s41598-023-38658-w. Conservation Laws, Solitary Wave Solutions, and Lie Analysis for the Nonlinear Chains of Atoms Muhammad Junaid U Rehman  (  muhammad-junaid.u-rehman@dokt.p.lodz.pl ) Lodz University of Technology Jan Awrejcewicz  Lodz University of Technology Grzegorz Kudra  Lodz University of Technology Article Keywords: Nonlinear chains of atoms, Lie symmetry approach, Multiplier scheme, Conserved vectors, New auxiliary method Posted Date: June 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3026335/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See the published version at https://doi.org/10.1038/s41598-023-38658-w. Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. d ll License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See the published version at https://doi.org/10.1038/s41598-023-38658-w. Abstract Nonlinear chains of atoms(NCA) are complex systems with rich dynamics, influ- encing various scientific disciplines. Lie symmetry approach is considered to analyze the NCA. The Lie symmetry method is a powerful mathematical tool for analyz- ing and solving differential equations with symmetries, facilitating the reduction of complexity and obtaining solutions. After getting the entire vector field by using the Lie scheme, we find the optimal system of symmetries. Using the optimal system we have converted assumed PDE into nonlinear ODE. The new auxiliary scheme in- troduces novel approaches to complement existing techniques, enhancing accuracy and simplifying computations. Travelling wave solutions describe wave-like prop- agation in systems, while graphical behavior visually represents relationships and patterns in data or mathematical models. The multiplier method enables the iden- tification of conservation laws, fundamental principles in physics that assert certain quantities remain constant over time. Understanding these concepts contributes to a deeper comprehension of nonlinear chains of atoms and their dynamics, fostering advancements in related fields. Keywords: Nonlinear chains of atoms, Lie symmetry approach, Multiplier scheme, Conserved vectors, New auxiliary method. 1Department of Automation, Biomechanics, and Mechatronics, Lodz University of Technology, 1/15 Stefanowski st. (Building A22), Lodz, 90-924, Poland 1Department of Automation, Biomechanics, and Mechatronics, Lodz University o Technology, 1/15 Stefanowski st. (Building A22), Lodz, 90-924, Poland E-mail address: ∗muhammad-junaid.u-rehman@dokt.p.lodz.pl (Corresponding author), grzgorz.kudra@p.lodz.pl, jan.awrjcewicz@p.lodz.pl E-mail address: ∗muhammad-junaid.u-rehman@dokt.p.lodz.pl (Corresponding author), grzgorz.kudra@p.lodz.pl, jan.awrjcewicz@p.lodz.pl Conservation Laws, Solitary Wave Solutions, and Lie Analysis for the Nonlinear Chains of Atoms June 11, 2023 June 11, 2023 Muhammad Junaid-U-Rehman1,∗, Grzegorz Kudra1, Jan Awrejcewicz1 1 Introduction The Lie symmetry analysis approach [1–8] has many applications in different fields, in- cluding physics, engineering, and mathematical modeling. It can be used to study a wide range of nonlinear PDEs, including those that are difficult to solve using other meth- ods. Additionally, this approach provides a powerful tool for developing new theories and models that improve our understanding of complex physical systems. Overall, the 1 Lie symmetry analysis approach is a valuable tool for studying nonlinear PDEs and has many significant applications in different branches of science and engineering. The Lie symmetry analysis approach [12–22] is a powerful method used in the study of nonlinear PDEs. It is based on the concept of Lie groups and Lie algebras, which are mathematical structures that describe the symmetries of a system. The Lie symme- try analysis approach involves transforming a given PDE into an equivalent system of ODEs using a Lie group transformation. This transformation is constructed from a set of symmetry generators that preserve the form of the original PDE. Once the PDE is transformed into an equivalent system of ODEs, it is possible to use various analytical and numerical methods to solve the system and obtain the solution to the original PDE. Additionally, the Lie symmetry analysis approach can be used to identify the conservation laws that govern the physical behavior of the system under study. These conservation laws provide important insights into the underlying physical mechanisms responsible for the observed behavior of the system. Nonlinear PDEs [23–26] play a critical role in mechanical engineering by modeling complex phenomena such as stress and deformation in materials, fluid flow, and heat transfer. Unlike linear PDEs, which can be solved analytically in many cases, nonlinear PDEs require numerical or approximate methods to solve due to their complex nature. The use of nonlinear PDEs is essential in the design and optimization of mechanical systems such as turbines, engines, and aircraft. They also provide a framework for pre- dicting the behavior of materials under different conditions, such as high temperatures, high pressure, and rapid deformation. By incorporating nonlinear PDEs into mechanical engineering models, engineers can improve the accuracy of their designs and ensure that their systems are safe, reliable, and efficient. Nonlinear chains of atoms [27–33] have a wide range of applications in mechanical engineering, particularly in the study of materials science and solid mechanics. 1 Introduction These models provide valuable insights into the behavior of materials at the atomic level, en- abling the design of high-performance materials for various applications. Applications of nonlinear chains of atoms include the study of thermal conductivity in materials, in- vestigating the deformation and fracture mechanisms of materials under various loading conditions, and studying the dynamics of crystals, such as the propagation of waves and the formation of defects. Nonlinear chains of atoms models are crucial in understanding the behavior of materials under extreme conditions and can inform the design of materi- als for high-performance applications, leading to the development of new materials with enhanced mechanical and thermal properties. The new auxiliary method [10,11] is a recently proposed method for solving challenging nonlinear PDEs. This method involves introducing an auxiliary variable and constructing a system of coupled equations involving both the original variables and the auxiliary variable. The resulting system of equations can be solved using numerical methods to obtain the solution to the original PDE. The new auxiliary method can handle highly nonlinear PDEs that are difficult to solve using other numerical methods, such as the finite difference approach or the finite element scheme. Additionally, this method can be used to obtain exact solutions to certain types of nonlinear PDEs, reducing the computational cost required to solve some types of nonlinear PDEs. Overall, the new auxiliary method is a promising tool for solving challenging nonlinear PDEs in various fields, including physics, engineering, and mathematical modeling. Conservation laws of nonlinear PDEs [36–39] are essential concepts that relate to the principle of conservation of physical quantities like mass, energy, and momentum. These 2 laws are expressed in terms of PDEs and have crucial importance in various fields, in- cluding engineering, physics, and mathematical modeling. They provide a mathematical framework to predict the behavior of complex physical systems accurately and develop new theories and models to improve our understanding of the underlying physical mech- anisms. Furthermore, conservation laws play a vital role in the design and analysis of physical systems and the development of numerical methods for solving challenging non- linear PDEs, making them fundamental concepts in the study of nonlinear PDEs. 2 Formation of model The Hamiltonian of the system is Foroutan et al. [34], The Hamiltonian of the system is Foroutan et al. [34], H = X n ( X l̸=n V(|Un −Ul|) + 1 2mU .2 n ) , (1) (1) where m is the mass of the atom, V(|Un −Ul|) stands for nonlinear potential and dot indicates for derivative w.r.t time. We consider l = 1 ± n and the subsequent potential: where m is the mass of the atom, V(|Un −Ul|) stands for nonlinear potential and dot indicates for derivative w.r.t time. We consider l = 1 ± n and the subsequent potential: V(hnl) = 1 4βih4 nl + 1 3αih3 nl + 1 2γih2 nl, (2) (2) where hnl is relative displacement among l −th atom and n −th. The index i shows the distinct interactions via the particles. We omit our focus on the first and second neighbors. From Eqs. (1) and (2) through Hamiltonian equations which are where hnl is relative displacement among l −th atom and n −th. The index i shows the distinct interactions via the particles. We omit our focus on the first and second neighbors. From Eqs. (1) and (2) through Hamiltonian equations which are ∂H ∂Un = −P . n, ∂H ∂P . n = ∂Un ∂τ = U . n, ∂H ∂Un = −P . n, ∂H ∂P . n = ∂Un ∂τ = U . n, which gives us the equation of motion; d2Un dτ 2 =γ1 Un+1 −2Un + Un−1  + γ2 Un+2 −2Un + Un−2  + α1 Un+1 −Un 2 − Un −Un−1 2  + α2 Un+2 −Un 2 − Un −Un−2 2  + β1 Un+1 −Un 2 − Un −Un−1 2  + β2 Un+2 −Un 2 − Un −Un−2 2  . (3) (3) In Hamiltonian’s equations, P . n stands for generalized momentum. Assuming that the δ(inter-atom spacing) is small enough so that the continuum limit is reached, we substi- tute δn →χ. Then Un±1 = U ± δUχ + 1 2δ2Uχχ ± 1 6δ3Uχχχ + 1 24δ4Uχχχχ + . . . (4) Un±2 = U ± 2δUχ + 4 2δ2Uχχ ± 8 6δ3Uχχχ + 16 24δ4Uχχχχ + . . . , (5) Un±1 = U ± δUχ + 1 2δ2Uχχ ± 1 6δ3Uχχχ + 1 24δ4Uχχχχ + . . . 2 Formation of model (4) (4) and and Un±2 = U ± 2δUχ + 4 2δ2Uχχ ± 8 6δ3Uχχχ + 16 24δ4Uχχχχ + . . . , (5) (5) 3 hence, Eq.(3) can be supposed as Foroutan et al. [35] hence, Eq.(3) can be supposed as Foroutan et al. [35] ∂2U ∂τ 2 = δ2 o ∂2U ∂χ2 + po ∂U ∂χ ∂2U ∂χ2 + qo ∂U ∂χ 2∂2U ∂χ2 + r∂4U ∂χ4 , (6) (6) with the subsequent constants; δ2 o = δ2 m γ1 + 4γ2  , po = 2δ3 m γ1 + 8α2  , qo = 3δ4 m β1 + 16β2  , r = δ4 12m γ1 + 16γ2  . (7) (7) Here in this paper, we will find out ...... for nonlinear Eq.(6) with the use of appropriate transformation method. 3.1 New auxiliary approach (14) Case 2: When B1 2 −B2B3 > 0 and B3 ̸= 0 Fq(ϱ) = −B1 B3 + p (B1 2 −B2B3) B3 tanh p (B1 2 −B2B3) 2 ϱ  , (15) Fq(ϱ) = −B1 B3 − p (B1 2 −B2B3) B3 coth p (B1 2 −B2B3) 2 ϱ  . (16) Case 3: When B1 2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p (B1 2 + B2 2) B3 tanh p (B1 2 + B2 2) 2 ϱ  , (17) Fq(ϱ) = B1 B3 + p (B1 2 + B2 2) B3 coth p (B1 2 + B2 2) 2 ϱ  . (18) Case 4: When B1 2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (19) Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . (20) Case 5: When B1 2 −B2 2 < 0 and B3 ̸= −B2 Fq(ϱ) = −B1 B3 + p −(B1 2 −B2 2) B3 tan p −(B1 2 −B2 2) 2 ϱ  , (21) Fq(ϱ) = −B1 B3 + p −(B1 2 −B2 2) B3 cot p −(B1 2 −B2 2) 2 ϱ  . (22) Case 6: When B1 2 −B2 2 > 0 and B3 ̸= −B2 Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 tanh p (B1 2 −B2 2) 2 ϱ  , (23) Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 coth p (B1 2 −B2 2) 2 ϱ  . 3.1 New auxiliary approach (16) (15) (16) Fq(ϱ) = B1 B3 + p (B1 2 + B2 2) B3 tanh p (B1 2 + B2 2) 2 ϱ  , (17) Fq(ϱ) = B1 B3 + p (B1 2 + B2 2) B3 coth p (B1 2 + B2 2) 2 ϱ  . (18) 2 (17) (18) B3 B3  2  ( ) Case 4: When B1 2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (19) Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . (20) Case 5: When B1 2 −B2 2 < 0 and B3 ̸= −B2 Fq(ϱ) = −B1 B3 + p −(B1 2 −B2 2) B3 tan p −(B1 2 −B2 2) 2 ϱ  , (21) Fq(ϱ) = −B1 B3 + p −(B1 2 −B2 2) B3 cot p −(B1 2 −B2 2) 2 ϱ  . (22) Case 6: When B1 2 −B2 2 > 0 and B3 ̸= −B2 Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 tanh p (B1 2 −B2 2) 2 ϱ  , (23) Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 coth p (B1 2 −B2 2) 2 ϱ  . (24) Case 7: When B2B3 > 0, B3 ̸= 0 and B1 = 0 Case 4: When B1 2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (19) Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . 3.1 New auxiliary approach Assuming the general form of partial PDE is of the form: Assuming the general form of partial PDE is of the form: (8) F(U, Uτ, Uχ, Uχχ, ...) = 0, (8) where τ is the time part and χ is the spatial part and U = U(χ, τ) is the dependent variable. We will follow the following steps. g Step 1: Suppose the new similarity variables or transformation is of the form U(χ, τ) = H(ϱ), where ϱ = k(χ + cτ), (9) (9) where k and c both are actual parameters for equation (8). Putting the Eq.(9) into Eq. (8) and we get the new ODE below. P(H, H′, H′′, ...) = 0. (10) (10) Step 2: Assume the general solution for Eq.(10) is of the form Step 2: Assume the general solution for Eq.(10) is of the form H(ϱ) = N X i=0 CiFiq(ϱ), (11) (11) in the above solution, the Ci’s are constants and we will fine later and the 1st ODE satisfied q(ϱ). in the above solution, the Ci’s are constants and we will fine later and the 1st ODE satisfied q(ϱ). q′(ϱ) = 1 ln(F){B2F−q(ϱ) + B1 + B3Fq(ϱ)}, F > 0, F ̸= 1. (12) (12) Step 3: In this step, we will use the balancing scheme to execute the value of N. For this, we have to compare the highest order linear and nonlinear terms to find the value of N. Step 4: Getting the coefficients of the powers of Fq(ϱ) (i = 0, 1, 2, 3..) by Eqs.(11), (12), and (8). Then collecting the same powers terms and put it equal to zero which gives us system of algebraic equations. After solving these system of euations by Maple. 4 Step 5: Finally we will get the different family of solutions for Eq.(12) of the form: Case 1: When B1 2 −B2B3 < 0 and B3 ̸= 0 Fq(ϱ) = −B1 B3 + p −(B1 2 −B2B3) B3 tan p −(B1 2 −B2B3) 2 ϱ  , (13) Fq(ϱ) = −B1 B3 + p −(B1 2 −B2B3) B3 cot p −(B1 2 −B2B3) 2 ϱ  . 3.1 New auxiliary approach (24) Case 7: When B2B3 > 0, B3 ̸= 0 and B1 = 0 Step 5: Finally we will get the different family of solutions for Eq.(12) of the form: Case 1: When B 2 B B < 0 and B ̸ 0 Step 5: Finally we will get the different family of solutions for Eq.(12) of the form: Case 1: When B1 2 −B2B3 < 0 and B3 ̸= 0 p y g Case 1: When B1 2 −B2B3 < 0 and B3 ̸= 0 Fq(ϱ) = −B1 B3 + p −(B1 2 −B2B3) B3 tan p −(B1 2 −B2B3) 2 ϱ  , (13) Fq(ϱ) = −B1 B3 + p −(B1 2 −B2B3) B3 cot p −(B1 2 −B2B3) 2 ϱ  . (14) (13) (14) Case 2: When B1 2 −B2B3 > 0 and B3 ̸= 0 Fq(ϱ) = −B1 B3 + p (B1 2 −B2B3) B3 tanh p (B1 2 −B2B3) 2 ϱ  , (15) Fq(ϱ) = −B1 B3 − p (B1 2 −B2B3) B3 coth p (B1 2 −B2B3) 2 ϱ  . (16) 3 3 ̸ Fq(ϱ) = −B1 B3 + p (B1 2 −B2B3) B3 tanh p (B1 2 −B2B3) 2 ϱ  , (15) Fq(ϱ) = −B1 B3 − p (B1 2 −B2B3) B3 coth p (B1 2 −B2B3) 2 ϱ  . (16) Case 3: When B1 2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p (B1 2 + B2 2) B3 tanh p (B1 2 + B2 2) 2 ϱ  , (17) Fq(ϱ) = B1 B3 + p (B1 2 + B2 2) B3 coth p (B1 2 + B2 2) 2 ϱ  . (18) Case 4: When B1 2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (19) Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . (20) Fq(ϱ) = −B1 B3 + p (B1 2 −B2B3) B3 tanh p (B1 2 −B2B3) 2 ϱ  , (15) Fq(ϱ) = −B1 B3 − p (B1 2 −B2B3) B3 coth p (B1 2 −B2B3) 2 ϱ  . 3.1 New auxiliary approach (20) Case 5: When B1 2 −B2 2 < 0 and B3 ̸= −B2 Fq(ϱ) = −B1 B3 + p −(B1 2 −B2 2) B3 tan p −(B1 2 −B2 2) 2 ϱ  , (21) Case 4: When B1 2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (19) Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . (20) Case 5: When B1 2 −B2 2 < 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (19) Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . (20) en B1 2 −B2 2 < 0 and B3 ̸= −B2 Fq(ϱ) = B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (19) Fq(ϱ) = B1 + p −(B1 2 + B2 2) cot p −(B1 2 + B2 2)ϱ  . (20) (19) (20) (21) (22) Case 6: When B1 2 −B2 2 > 0 and B3 ̸= −B2 Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 tanh p (B1 2 −B2 2) 2 ϱ  , (23) Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 coth p (B1 2 −B2 2) 2 ϱ  . (24) Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 tanh p (B1 2 −B2 2) 2 ϱ  , (23) Fq(ϱ) = −B1 B3 + p (B1 2 −B2 2) B3 coth p (B1 2 −B2 2) 2 ϱ  . (24) B B 0 B ̸ 0 d B 0 (23) (24) Case 7: When B2B3 > 0, B3 ̸= 0 and B1 = 0 Fq(ϱ) = r −B2 B3 tanh √−B2B3 2 ϱ  , (25) Fq(ϱ) = r −B2 B3 tanh √−B2B3 2 ϱ  , (25) (25) 5 Fq(ϱ) = r −B2 B3 coth √−B2B3 2 ϱ  . Fq(ϱ) = tan B2ϱ + c 2  . 3.1 New auxiliary approach (26) Case 8: When B1 = 0 and B2 = −B3 p Fq(ϱ) = r −B2 B3 coth √−B2B3 2 ϱ  . (26) Case 8: When B1 = 0 and B2 = −B3 Fq(ϱ) = −(1 + e2B2ϱ) ± p 2(1 + e2B2ϱ) e2B2ϱ −1 . (27) (26) Case 8: When B1 = 0 and B2 = −B3 Fq(ϱ) = −(1 + e2B2ϱ) ± p 2(1 + e2B2ϱ) e2B2ϱ −1 . (27) Case 9: When B1 2 = B2B3 Case 9: When B1 2 = B2B3 Fq(ϱ) = −B2(B1ϱ + 2) B1 2ϱ . Case 10: When B1 = k, B2 = 2k and B3 = 0 Fq(ϱ) = eϱ −1. Case 11: When B1 = k, B3 = 2k and B2 = 0 Fq(ϱ) = eϱ 1 −eϱ. (28) Case 10: When B1 = k, B2 = 2k and B3 = 0 (29) (30) Case 12: When 2B1 = B2 + B3 Case 12: When 2B1 = B2 + B3 Case 12: When 2B1 = B2 + B3 Fq(ϱ) = 1 + B2e 1 2 (B2−B3)ϱ 1 + B3e 1 2 (B2−B3)ϱ. (31) Case 13: When −2B1 = B2 + B3 Case 13: When −2B1 = B2 + B3 (32) Case 14: When B2 = 0 Case 14: When B2 = 0 (33) Case 15: When B2 = B1 = B3 ̸= 0 Case 15: When B2 = B1 = B3 ̸= 0 (34) Case 16: When B2 = B3, B1 = 0 Case 16: When B2 = B3, B1 = 0 Fq(ϱ) = tan B2ϱ + c 2  . (35) Case 17: When B3 = 0 Case 17: When B3 = 0 (36) 6 3.2 Multiplier approach Supposing the Eq.(8) and applying the following steps below: 1) D fii h l diff i l Supposing the Eq.(8) and applying the following steps below: 1). Definig the total differential as: 1). Definig the total differential as: Di = ∂ ∂χi + Ui ∂ ∂U + Uij ∂ ∂Uj + ..., i = 1, 2, 3...m, (37) (37) 2). Defining the Euler operator as below: 2). Defining the Euler operator as below: δ δU = ∂ ∂U −Di ∂ ∂Ui + Dij ∂ ∂Uij −Dijk ∂ ∂Uijk + ... , (38) (38) 3). Let us define n-tuple f = (f1, f2, f3, ..., fm), i = 1, 2, ...m, Difi = 0, (39) (39) eq.(39) is said to be the conservation laws and it is fulfils the all results of Eq. (8). 4). The purpose of Λ(χ, τ, U) of the Eq. (8): eq.(39) is said to be the conservation laws and it is fulfils the all results of Eq. (8). 4). The purpose of Λ(χ, τ, U) of the Eq. (8): Difi = Λ(χ, τ, U)H, (40) (40) for some function U(µ1, µ2, ..., µm). for some function U(µ1, µ2, ..., µm). 5). We will obtain the determining equations for Λ(χ, τ, U) after calculating the derivative of Λ(χ, τ, U) in Eq.(40): δ δ δU (Λ(χ, τ, U)H) = 0. (41) (41) Eq. (41) depends for some function U(µ1, µ2, ..., µm). Finally, when we calculate the Λ(X, t, U) with use of Eq. (41), the conservation laws can be acquired by Eq. (40). 4 Lie group analysis of Eq. (6) Here, we are supposing the Lie approach for assumed Eq.(6). Now, suppose the one- parameter Lie group of infinitesimal transformations on (τ, χ, U) given by ¯τ = τ + ε ζ1(τ, χ, U) + O(ε2), ¯χ = χ + ε ζ2(τ, χ, U) + O(ε2), ¯U = u + ε η(τ, χ, U) + O(ε2), and ε ≪1 is a Small parameter. The associated Lie algebra of infinitesimal symmetries is generated by vector fields and ε ≪1 is a Small parameter. The associated Lie algebra of infinitesimal symmetries is generated by vector fields X =ζ1(τ, χ, U)∂τ + ζ2(τ, χ, U)∂χ + η(τ, χ, U)∂U. (42) (42) Eq. (42) creates a symmetry of Eq. (6), and X satisfy the Lie group conditions Pr(4)X ∂2U ∂τ 2 = δ2 o ∂2U ∂χ2 + po ∂U ∂χ ∂2U ∂χ2 + qo ∂U ∂χ 2∂2U ∂χ2 + r∂4U ∂χ4  |Eq.(6)=0 = 0. 7 The Pr(4)X for X can be written as: for X can be written as: Pr(4)X =X + ηχ ∂ ∂Uχ + ηττ ∂ ∂Uττ + ηχχ ∂ ∂Uχχ + ηχχχχ ∂ ∂Uχχχχ , (43) (43) furthermore, we have                ηχ = Dχ(η) −UχDχ(ζ1) −UτDχ(ζ2), ηχχ = Dχ(ηχ) −UχχDχ(ζ1) −UτχDχ(ζ2), ητ = Dτ(η) −UχDt(ζ1) −UτDτ(ζ2), ηττ = Dτ(ητ) −UττDτ(ζ1) −UχτDt(ζ2), ηχχχ = Dχ(ηχχ) −UχχχDχ(ζ1) −UτχχDχ(ζ2). (44) (44) Let (x1, x2) = (χ, τ), where Di can be written as: Let (x1, x2) = (χ, τ), where Di can be written as: Di = ∂ ∂χi + Ui ∂ ∂U + Uij ∂ ∂Uj + ..., i = 1, 2. Substituting the values of ηi which gives us the following vectors: Substituting the values of ηi which gives us the following vectors: X1 = ∂ ∂χ, X2 = ∂ ∂τ , X3 = ∂ ∂U , X4 = τ ∂ ∂U . (45) (45) We see that [Xi, Xj] = 0, where i, j = 1, 2, 3. 5 Optimal system In this section, we observe that from the obtained vector field Eq. (45), the X = {X1, X2} forms an abelian algebra. So we can use the (42) and get: £1 = < X1 >, £2 = < X1 + k1X2 > . (46) (46) 5.2 Application of new auxiliary method Here, our aim is to construct the wave patterns for Eq.(6) from Eq.(50) with use of proposed technique. Using the balancing method and we obtain N = 1. Using the value of N = 1 in (11) and we have H(ϱ) = Co + C1Fq(ϱ). (51) (51) We have to put Eq.(51) into Eq.(50) and we get the system of equation after comparing the coefficients of Fq(ϱ). With the use of Maple, we have solved the obtained system of equations and get the following results. C0 = V1, C1 = −12rB3 po , k1 = ± q rB2 1 −4rB2B3 + δ2 o. (52) (52) Using Eq.(52) into Eq.(51) and which gives us the following set of solutions. U(χ, τ) = V1 −12rB3 po Fq(ϱ), where ϱ = χ ± q rB2 1 −4rB2B3 + δ2 o τ (53) U(χ, τ) = V1 −12rB3 po Fq(ϱ), where ϱ = χ ± q rB2 1 −4rB2B3 + δ2 o τ (53) (53) where V1 is orbitraryconstant. 2 where V1 is orbitraryconstant. where V1 is orbitraryconstant. where V1 is orbitraryconstant. y Case:1 When B1 2 −B2U3 < 0 and B3 ̸= 0 Case:1 When B1 2 −B2U3 < 0 and B3 ̸= 0 U1(χ, τ) = V1 −12rB3 po −B1 B3 + p −(B1 2 −B2B3) B3 tan p −(B1 2 −B2B3) 2 ϱ  , (54) U2(χ, τ) = V1 −12rB3 po −B1 B3 + p −(B1 2 −B2B3) B3 cot p −(B1 2 −B2B3) 2 ϱ  . (55) Case:2 When B1 2 −B2B3 > 0 and B3 ̸= 0 U3(χ, τ) = V1 −12rB3 po −B1 B3 + p (B1 2 −B2B3) B3 tanh p (B1 2 −B2B3) 2 ϱ  , (56) U4(χ, τ) = V1 −12rB3 po −B1 B3 − p (B1 2 −B2B3) B3 coth p (B1 2 −B2B3) 2 ϱ  . (57) Case:3 When B1 2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2 U5(χ, τ) = V1 −12rB3 po B1 B3 + p (B1 2 + B2 2) B3 tanh p (B1 2 + B2 2) 2 ϱ  , (58) U6(χ, τ) = V1 −12rB3 po B1 B3 + p (B1 2 + B2 2) B3 coth p (B1 2 + B2 2) 2 ϱ  . 5.1 Similarity reduction of Eq. (6) Here, we will find the similarity variables and analytical results for Eq.(6). 5.1.1 £1 =< X1 > 5.1.1 £1 =< X1 > 5.1.1 £1 =< X1 > Using the vector £1, we get the new variable u(τ, χ) = H(ϱ), where ϱ = χ, (47) (47) u(τ, χ) = H(ϱ), where ϱ = χ, putting the (47) into Eq. (6), which gives us U(τ, χ) = m1τ + m2 (48) (48) U(τ, χ) = m1τ + m2 where m1 and m2 are integration constants. where m1 and m2 are integration constants. 8 5.1.2 £2 =< X1 + k1X2 Using the vector £2, we get the new variable Using the vector £2, we get the new variable u(τ, χ) = H(ϱ), where ϱ = χ + k1τ, (49) (49) u(τ, χ) = H(ϱ), where ϱ = χ + k1τ, (49) putting the (49) into Eq. (6), which gives us 6(k2 1 −δ2 o)H′ −3po(H′)2 −2qo(H′)3 −3rH′′′ = 0. (50) putting the (49) into Eq. (6), which gives us putting the (49) into Eq. (6), which gives us 6(k2 1 −δ2 o)H′ −3po(H′)2 −2qo(H′)3 −3rH′′′ = 0. (50) (50) 5.2 Application of new auxiliary method (59) 1 2 3 3 ̸ U1(χ, τ) = V1 −12rB3 po −B1 B3 + p −(B1 2 −B2B3) B3 tan p −(B1 2 −B2B3) 2 ϱ  , (54) U2(χ, τ) = V1 −12rB3 po −B1 B3 + p −(B1 2 −B2B3) B3 cot p −(B1 2 −B2B3) 2 ϱ  . (55) U3(χ, τ) = V1 −12rB3 po −B1 B3 + p (B1 2 −B2B3) B3 tanh p (B1 2 −B2B3) 2 ϱ  , (56) U4(χ, τ) = V1 −12rB3 po −B1 B3 − p (B1 2 −B2B3) B3 coth p (B1 2 −B2B3) 2 ϱ  . (57) 2 Case:3 When B1 2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2 Case:3 When B1 2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2 U5(χ, τ) = V1 −12rB3 po B1 B3 + p (B1 2 + B2 2) B3 tanh p (B1 2 + B2 2) 2 ϱ  , (58) U6(χ, τ) = V1 −12rB3 po B1 B3 + p (B1 2 + B2 2) B3 coth p (B1 2 + B2 2) 2 ϱ  . (59) (58) (59) 9 Case: 4 When B1 2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 U7(χ, τ) = V1 −12rB3 po B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (60) U8(χ, τ) = V1 −12rB3 po B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . (61) Case: 5 When B1 2 −B2 2 < 0 and B3 ̸= −B2 U9(χ, τ) = V1 −12rB3 po −B1 B3 + p −(B1 2 −B2 2) B3 tan p −(B1 2 −B2 2) 2 ϱ  , (62) U10(χ, τ) = V1 −12rB3 po −B1 B3 + p −(B1 2 −B2 2) B3 cot p −(B1 2 −B2 2) 2 ϱ  . (63) Case: 6 When B1 2 −B2 2 > 0 and B3 ̸= −B2 U11(χ, τ) = V1 −12rB3 po −B1 B3 + p (B1 2 −B2 2) B3 tanh p (B1 2 −B2 2) 2 ϱ  , (64) U12(χ, τ) = V1 −12rB3 po −B1 B3 + p (B1 2 −B2 2) B3 coth p (B1 2 −B2 2) 2 ϱ  . 5.2 Application of new auxiliary method (65) Case: 7 When B2B3 > 0, B3 ̸= 0 and B1 = 0 Case: 4 When B1 2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 U7(χ, τ) = V1 −12rB3 po B1 B3 + p −(B1 2 + B2 2) B3 tan p −(B1 2 + B2 2) 2 ϱ  , (60) U8(χ, τ) = V1 −12rB3 po B1 B3 + p −(B1 2 + B2 2) B3 cot p −(B1 2 + B2 2) 2 ϱ  . (61) Case: 5 When B1 2 −B2 2 < 0 and B3 ̸= −B2 (60) (61) U9(χ, τ) = V1 −12rB3 po −B1 B3 + p −(B1 2 −B2 2) B3 tan p −(B1 2 −B2 2) 2 ϱ  , (62) Case: 6 When B1 2 −B2 2 > 0 and B3 ̸= −B2 U11(χ, τ) = V1 −12rB3 po −B1 B3 + p (B1 2 −B2 2) B3 tanh p (B1 2 −B2 2) 2 ϱ  , (64) U12(χ, τ) = V1 −12rB3 po −B1 B3 + p (B1 2 −B2 2) B3 coth p (B1 2 −B2 2) 2 ϱ  . (65) Case: 7 When B2B3 > 0, B3 ̸= 0 and B1 = 0 (64) (65) U13(χ, τ) = V1 −12rB3 po r −B2 B3 tanh √−B2B3 2 ϱ  , (66) U14(χ, τ) = V1 −12rB3 po r −B2 B3 coth √−B2B3 2 ϱ  . (67) Case: 8 When B1 = 0 and B2 = −B3 U13(χ, τ) = V1 −12rB3 po r −B2 B3 tanh √−B2B3 2 ϱ  , (66) (66) U14(χ, τ) = V1 −12rB3 po r −B2 B3 coth √−B2B3 2 ϱ  . (67) Case: 8 When B1 = 0 and B2 = −B3 (67) U15(χ, τ) = V1 −12rB3 po −(1 + e2B2ϱ) ± p 2(1 + e2B2ϱ) e2B2ϱ −1  . (68) (68) 15(χ, ) 1 po  e2B2ϱ −1  ( ) Case: 9 When B1 2 = B2B3 U16(χ τ) = V1 −12rB3 −B2(B1ϱ + 2) (69) Case: 9 When B1 2 = B2B3 Case: 9 When B1 2 = B2B3 Case: 9 When B1 2 = B2B3 Case: 9 When B1 2 = B2B3 U16(χ, τ) = V1 −12rB3 po −B2(B1ϱ + 2) B1 2ϱ  . 5.2 Application of new auxiliary method (69) Case: 10 When B1 = k, B2 = 2k and B3 = 0 U17(χ, τ) = V1 −12rB3 po  eϱ −1  . (70) Case: 11 When B1 = k, B3 = 2k and B2 = 0 U18(χ, τ) = V1 −12rB3 po  eϱ 1 −eϱ  . (71) U16(χ, τ) = V1 −12rB3 po −B2(B1ϱ + 2) B1 2ϱ  . (69) U16(χ, τ) = V1 −12rB3 po −B2(B1ϱ + 2) B1 2ϱ  . (69) Case: 10 When B1 = k, B2 = 2k and B3 = 0 (69) U16(χ, τ) = V1 − po  B1 2ϱ  . (69) Case: 10 When B1 = k, B2 = 2k and B3 = 0 U17(χ, τ) = V1 −12rB3 po  eϱ −1  . (70) Case: 11 When B1 = k, B3 = 2k and B2 = 0 U18(χ, τ) = V1 −12rB3 po  eϱ 1 −eϱ  . (71) (70) (71) 10 Case: 12 When 2B1 = B2 + B3 U19(χ, τ) = V1 −12rB3 po 1 + B2e 1 2 (B2−B3)ϱ 1 + B3e 1 2 (B2−B3)ϱ  . (72) (72) Case: 13 When −2B1 = B2 + B3 Case: 13 When −2B1 = B2 + B3 Case: 13 When −2B1 = B2 + B3 Case: 13 When −2B1 = B2 + B3 U20(χ, τ) = V1 −12rB3 po B2 + B2e 1 2 (B2−B3)ϱ B3 + B3e 1 2 (B2−B3)ϱ  . (73) (73) Case: 14 When B2 = 0 Case: 14 When B2 = 0 Case: 14 When B2 = 0 U21(χ, τ) = V1 −12rB3 po  B1eB1ϱ 1 + B3 2 eB1ϱ  . (74) Case: 15 When B2 = B1 = B3 ̸= 0 U22(χ, τ) = V1 −12rB3 −(B2ϱ + 2) . (75) U21(χ, τ) = V1 −12rB3 po  B1eB1ϱ 1 + B3 2 eB1ϱ  . (74) U21(χ, τ) = V1 −12rB3 po  B1eB1ϱ 1 + B3 2 eB1ϱ  . U21(χ, τ) = V1 −12rB3 po  B1eB1ϱ 1 + B3 2 eB1ϱ  . (74) Case: 15 When B2 = B1 = B3 ̸= 0 U22(χ, τ) = V1 −12rB3 po −(B2ϱ + 2) B2ϱ  . (75) Case: 16 When B2 = B3, B1 = 0 U23(χ, τ) = V1 −12rB3  tan B2ϱ + c . 5.2 Application of new auxiliary method (76) (74)  2  Case: 15 When B2 = B1 = B3 ̸= 0 U22(χ, τ) = V1 −12rB3 po −(B2ϱ + 2) B2ϱ  . (75) C 16 Wh B B B 0 Case: 15 When B2 = B1 = B3 ̸= 0 U22(χ, τ) = V1 −12rB3 po −(B2ϱ + 2) B2ϱ  . (75) U22(χ, τ) = V1 −12rB3 po −(B2ϱ + 2) B2ϱ  . (75) Case: 16 When B2 = B3, B1 = 0 U22(χ, τ) = V1 −12rB3 po −(B2ϱ + 2) B2ϱ  . (75) (75) po  B2ϱ  Case: 16 When B2 = B3, B1 = 0 U23(χ, τ) = V1 −12rB3 po  tan B2ϱ + c 2  . (76) Case: 16 When B2 = B3, B1 = 0 U (χ τ) V 12rB3  tan B2ϱ + c (76) U23(χ, τ) = V1 −12rB3 po  tan B2ϱ + c 2  . (76) (76) Case: 17 When B3 = 0 Case: 17 When B3 = 0 Case: 17 When B3 = 0 Case: 17 When B3 = 0 U24(χ, τ) = V1 −12rB3 po  eB1ϱ −B2 2B1  . (77) (77) Where ϱ = χ ± p rB2 1 −4rB2B3 + δ2 o τ is given according to. (a) 3D (b) 2D (a) 3D (b) 2D Figure 1: Graphics of u1(χ, τ) for the choice of parameters δo = 1, r = 2, po = 3, B1 = 2, B2 = 1,B3 = 2, τ = 1. (a) 3D Figure 1: Graphics of u1(χ, τ) for the choice of parameters δo = 1, r = 2, po = 3, B1 = 2, B2 = 1,B3 = 2, τ = 1. 11 11 (a) 3D (a) 3D (b) 2D Figure 2: Graphics of u2(χ, τ) for the choice of parameters δo = 2, r = 1, po = 1, B1 = 3, B2 = 2,B3 = 1, τ = 2. (b) 2D (a) 3D Figure 2: Graphics of u2(χ, τ) for the choice of parameters δo = 2, r = 1, po = 1, B1 = 3, B2 = 2,B3 = 1, τ = 2. (a) 3D (b) 2D Figure 3: Graphics of u3(χ, τ) for the choice of parameters δo = 1.5, r = 1, po = 4, B1 = 4, B2 = 3,B3 = 5, τ = 2. 6 Graphics and discussion Graphical representation of obtained solutions is discussed here in this section. By using the new auxiliary method we have constructed the analytical behaviour of considered model in the form of trigonometric functions, hyperbolic trigonometric functions, expo- nential, and algebraic type results. The graph of the tangent function is periodic with a period of Π and has vertical asymptotes at odd multiples of Pi 2 . As χ approaches these vertical asymptotes, the tangent function approaches positive or negative infinity depend- ing on the direction of approach. The graph of the cotangent function is also periodic with a period of Π and has horizontal asymptotes at even multiples of Π. As χ approaches these horizontal asymptotes, the cotangent function approaches zero. We have plotted the behaviour of some obtained results. Fig.(1) shows the graphical behavior of u1(χ, τ) for the choice of parameters δo = 1, r = 2, po = 3, B1 = 2, B2 = 1,B3 = 2, τ = 1. Fig. (2) represent the behaviour of u2(χ, τ) for the choice of parameters δo = 2, r = 1, po = 1, B1 = 3, B2 = 2,B3 = 1, τ = 2. Fig. (3) shows the Graphics of u3(χ, τ) for the choice of parameters δo = 1.5, r = 1, po = 4, B1 = 4, B2 = 3,B3 = 5, τ = 2. Fig. (4) shows the Graphics of u4(χ, τ) for the choice of parameters δo = 5, r = 0.5, po = 3.5, B1 = 1.5, B2 = 3.5,B3 = 2.5, τ = 3. Fig. (5) represent the behaviour of u19(χ, τ) for the choice of parameters δo = 6, r = 3, po = 5, B1 = 1, B2 = 3,B3 = 5, τ = 5. 5.2 Application of new auxiliary method (b) 2D (a) 3D (a) 3D (a) 3D Figure 3: Graphics of u3(χ, τ) for the choice of parameters δo = 1.5, r = 1, po = 4, B1 = 4, B2 = 3,B3 = 5, τ = 2. (a) 3D (b) 2D Figure 4: Graphics of u4(χ, τ) for the choice of parameters δo = 5, r = 0.5, po = 3.5, B1 = 1.5, B2 = 3.5,B3 = 2.5, τ = 3. (b) 2D (a) 3D (a) 3D Figure 4: Graphics of u4(χ, τ) for the choice of parameters δo = 5, r = 0.5, po = 3.5, B1 = 1.5, B2 = 3.5,B3 = 2.5, τ = 3. 12 (a) 3D (b) 2D Figure 5: Graphics of u19(χ, τ) for the choice of parameters δo = 6, r = 3, po = 5, B1 = 1, B2 = 3,B3 = 5, τ = 5. (a) 3D (b) 2D (a) 3D Figure 5: Graphics of u19(χ, τ) for the choice of parameters δo = 6, r = 3, po = 5, B1 = 1, B2 = 3,B3 = 5, τ = 5. 7 Conservation laws (83) (83) Case2 : For C1 = 0, C2 = 1, then Λ1 = 1, we get the following fluxes: T τ 2 = Uτ, T χ 2 = −1 3 U 3 χqo −1 2U 2 χpo −Uχδ2 o −rUχχχ. (84) (84) 7 Conservation laws In this portion, we will construct the conservation laws by multiplier approach for Eq.(6). We obtain the determinant equation for Λ(X, t, u) by Eq. (41). δ δU  Λ ∂2U ∂τ 2 −δ2 o ∂2U ∂χ2 −po ∂U ∂χ ∂2U ∂χ2 −qo ∂U ∂χ 2∂2U ∂χ2 −r∂4U ∂χ4  = 0. (78) (78) Using Eq.(38), we can write the Euler operator is of the form g Eq.(38), we can write the Euler operator is of the form δ δU = ∂ ∂U −Dτ ∂ ∂Uτ −Dχ ∂ ∂Uχ + D2 τ ∂ ∂Uττ + D2 χ ∂ ∂Uχχ + DχDτ ∂ ∂Uτχ −..., (79) δ δU = ∂ ∂U −Dτ ∂ ∂Uτ −Dχ ∂ ∂Uχ + D2 τ ∂ ∂Uττ + D2 χ ∂ ∂Uχχ + DχDτ ∂ ∂Uτχ −..., (79) δ δU = ∂ ∂U −Dτ ∂ ∂Uτ −Dχ ∂ ∂Uχ + D2 τ ∂ ∂Uττ + D2 χ ∂ ∂Uχχ + DχDτ ∂ ∂Uτχ −..., (79) (79) 13 defining the total derivative operators Dτ and Dχ from Eq. (37). Dτ = ∂ ∂τ + Uτ ∂ ∂U + Uττ ∂ ∂Uτ + Uτχ ∂ ∂Uχ ..., Dχ = ∂ ∂χ + Uχ ∂ ∂U + Uχχ ∂ ∂Uχ + Uτχ ∂ ∂Uτ ..., (80) (80) computing Eq. (78) and we obtain the following multipliers and conservation laws as: computing Eq. (78) and we obtain the following multipliers and conservation laws as: Λ = C1τ + C2, (81) (81) by Eqs. (40) and (81), The following conservation laws are found. by Eqs. (40) and (81), The following conservation laws are found. T τ =Uτ(C1τ + C2) −C1U, T χ =(C1τ + C2) −1 3 U 3 χqo −1 2U 2 χpo −Uχδ2 o −rUχχχ  . (82) (82) Using Eq. (82) and we get the following two cases of conservation laws. Case1 : For C1 = 1, C2 = 0, then Λ1 = τ, we get the following fluxes: Using Eq. (82) and we get the following two cases of conservation laws. Case1 : For C1 = 1, C2 = 0, then Λ1 = τ, we get the following fluxes: T τ 1 = τUτ −U, T χ 1 = −1 3 U 3 χτqo −1 2U 2 χτpo −Uχτδ2 o −rUχχχτ. Acknowledgements: This work has been supported by the Polish National Science Centre, Poland under the Grant OPUS 18 No.2019/35/B/ST8/00980. 8 Conclusion In this research, nonlinear chains of atoms(NCA) are studied. NCA are complex sys- tems that exhibit rich dynamics and phenomena, making their study crucial in various scientific fields. The Lie symmetry method provides a powerful mathematical tool for analyzing and solving differential equations with symmetries, reducing complexity and obtaining exact or approximate solutions. The introduction of a new auxiliary scheme enhances existing techniques, offering additional insights, improved accuracy, or simpli- fied computations. Travelling wave solutions describe wave-like behavior propagating through systems, while graphical behavior provides visual representations of relation- ships and patterns in data or mathematical models. The multiplier method allows for the identification of conservation laws, which are fundamental principles in physics that state certain quantities remain constant over time. Understanding conservation laws and utilizing mathematical techniques such as the Lie symmetry method, travelling wave so- lutions, and graphical analysis contributes to a deeper understanding of nonlinear chains of atoms and their dynamics. Acknowledgements: This work has been supported by the Polish National Science Centre, Poland under the Grant OPUS 18 No.2019/35/B/ST8/00980. 14 Credit authorship contribution statement: M. Junaid-U-Rehman: Conceptualiza- tion, Methodology, Investigation, Software, Writing - original draft. Jan Awrejcewicz: Visualization, Investigation. Grzegorz Kudra: Supervision, Software, Validation. Declaration of Competing Interest: The Authors declare that they do not have any Credit authorship contribution statement: M. Junaid-U-Rehman: Conceptualiza- tion, Methodology, Investigation, Software, Writing - original draft. Jan Awrejcewicz: Visualization, Investigation. Grzegorz Kudra: Supervision, Software, Validation. Declaration of Competing Interest: The Authors declare that they do not have any conflicts of interest. tion, Methodology, Investigation, Software, Writing - original draft. Jan Awrejcewicz: Visualization, Investigation. Grzegorz Kudra: Supervision, Software, Validation. Declaration of Competing Interest: The Authors declare that they do not have any conflicts of interest. Declaration of Competing Interest: The Authors declare that they do not have any conflicts of interest. Declaration of Competing Interest: The Authors declare that they do not have any conflicts of interest. Data availability statements: All data that support the findings of this study are included within the article (and any supplementary files). Data availability statements: All data that support the findings of this study are included within the article (and any supplementary files). References [1] Riaz, M.B., Atangana, A., Jhangeer, A. and Junaid-U-Rehman, M., 2021. Some exact explicit solutions and conservation laws of Chaffee-Infante equa- tion by Lie symmetry analysis. Physica Scripta, 96(8), p. 084008. [2] Jhangeer, A., Hussain, A., Junaid-U-Rehman, M., Khan, I., Baleanu, D. and Nisar, K.S., 2020. Lie analysis, conservation laws and travelling wave struc- tures of nonlinear Bogoyavlenskii–Kadomtsev–Petviashvili equation. Results in Physics, 19, p. 103492. 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Introduction includes cleft palate (CP) and cleft lip (CL) in isolation or in combination. Children suffering from OFCs in low income countries have high morbidity throughout their life. They face a range of functional and cosmetic problems with poten- tial long-term adverse impact on health, speech, hearing, psy- chology, learning ability and social skills (Agbenorku, 2013). The treatment often requires a multidisciplinary approach, from surgical correction of physical defect to long-term psy- chological counselling (Mossey and Little, 2009). The burden of orofacial clefts (OFC) is a major concern in India and throughout the world (Mossey et al., 2009). OFC The burden of orofacial clefts (OFC) is a major concern in India and throughout the world (Mossey et al., 2009). OFC Additional Supporting information may be found in the online version of this article. Additional Supporting information may be found in the online version of this article. 1Indian Institute of Public Health- Delhi, Public Health Foundation of India, New Delhi, India Estimates from a meta-analysis including 11 hospital based studies reported a prevalence of OFCs ranging from 0.2 to 2.9 per 1000 total births and a pooled prevalence of 1.3 per 1000 total births in India (Allagh et al., 2015). It is estimated that every year 28,600 infants are born with cleft lip and cleft palate, that is, 78 infants per day in the country (Mossey and Little, 2009). 2Indian Institute of Public Health- Hyderabad, Public Health Foundation of India, Hyderabad, India 3Centre for Applied Research and Education on Neurodevelopmental Impairments and Disability related Health Initiatives (CARENIDHI), New Delhi, India 4Kalawati Saran Hospital and Lady Hardinge Medical College, New Delhi, India 7The UK National Institute for Health Research Bristol Nutrition Biomedical Research Unit, University of Bristol, Bristol, United Kingdom The etiology of OFC is polygenic and multi-factorial. Both genetic and environmental factors have been identified though the precise cause in many cases is unclear (Mossey and Little, 2009). The environmental factors include mater- nal illness, infections, drugs, radiation, alcohol, and contami- nation of food and water with pesticides, nitrates, and mercury (Aylsworth et al., 2015). In vitro fertilization and intracytoplasmic sperm injection, consanguinity, dietary, and indigenous medicines used for sex selection are also implicated as risk factors (Mossey and Little, 2009; Neogi et al., 2015). Risk Factors for Orofacial Clefts in India: A Case–Control Study Sutapa Bandyopadhyay Neogi *1, Samiksha Singh2, Dinesh Raj Pallepogula2, Hira Pant2, Sunanda Reddy Kolli3, Priyanka Bharti1, Vikram Datta4, Srinivas Reddy Gosla5, Krishnamurthy Bonanthaya6, Andy Ness7, Sanjay Kinra8, Pat Doyle8, and Venkata Satyanarayana Murthy Gudlavalleti2 p value 5 0.001), and delayed first conception (AOR, 2.55, 95% CI, 1.25– 5.21, p 5 0.01) were found to be strongly associated with higher risk of OFCs. Supplementation with folic acid during first 3 months of pregnancy was not found to be protective against OFCs (AOR, 1.24; 95% CI, 0.59–2.58; p value 5 0.56). Conclusion: Our study confirmed the importance of family history as a risk factor for OFC. Our study did not show an association with folic acid supplementation but was underpowered to detect small effects. Our finding of higher risk among vegetarians requires replication. Background: Orofacial clefts (OFC) are linked with several genetic and environmental factors. The aim of this study was to explore the association of potential risk factors with OFCs in India. Methods: This was a hospital-based, matched case–control (1:4 ratio; matching done for parity) study conducted in Hyderabad, Bengaluru, and Delhi-National Capital Region. Cases (nonsyndromic clefts) were recruited from treatment centers, while controls (live births) were recruited from maternity centers. Information on exposures was collected during personal interviews. Exposures of interest included folic acid supplementation during the peri-conceptional period, consanguineous marriage, exposure to drugs, infections during pregnancy, family history of OFC, and dietary factors. Results: A total of 785 participants were included in the study: 157 cases and 628 controls. A family history of cleft lip/palate (adjusted odds ratio [AOR], 15.48; 95% confidence interval [CI], 4.36–54.96; p value 5 0.001), exclusive vegetarianism (AOR, 4.47; 95% CI, 1.83–10.98; Birth Defects Research 109:1284–1291, 2017. V C 2017 Wiley Periodicals, Inc. Key words: Orofacial clefts; cleft lip/palate; risk factors; folic acid supplementation Introduction 8London School of Hygiene and Tropical Medicine, London, United Kingdom This work was funded by Wellcome Trust Capacity Strengthening Strategic Award to the Public Health Foundation of India and a consortium of UK uni- versities (Grant number WT084754/Z/08/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript This work was funded by Wellcome Trust Capacity Strengthening Strategic Award to the Public Health Foundation of India and a consortium of UK universities (Grant number WT084754/Z/08/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflicts of Interest: None. manuscript. Conflicts of Interest: None. *Correspondence to: Sutapa B. Neogi, Indian Institute of Public Health- Delhi, Public Health Foundation of India, Delhi, India. E-mail: sutapa.bneogi@iiphd.org Th i ht li f thi ti l h d 15th M h 2018 ft i i l *Correspondence to: Sutapa B. Neogi, Indian Institute of Public Health- Delhi, Public Health Foundation of India, Delhi, India. E-mail: sutapa.bneogi@iiphd.org The copyright line for this article was changed on 26 June 2019 after original online publication. The copyright line for this article was changed on 15th March, 2018 after original online publication. Published online 2 August 2017 in Wiley Online Library (wileyonlinelibrary 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. Folic acid deficiency has been linked to the causation of many chronic and developmental disorders (Smithells com). Doi: 10.1002/bdr2.1073 Published online 2 August 2017 in Wiley Online Library (wileyonlinelibrary. com). Doi: 10.1002/bdr2.1073 V C 2017 Wiley Periodicals, Inc. © 2017 The Authors Birth Defects Research Published by Wiley Periodicals, Inc. V C 2017 Wiley Periodicals, Inc. © 2017 The Authors Birth Defects Research Published by Wiley Periodicals, Inc. BIRTH DEFECTS RESEARCH 109:1284–1291 (2017) 1285 et al., 1976; Botto et al., 1999; Caudill M, 2008; Molloy et al., 2008, 2009). Folate deficiency is prevalent globally, especially in low and middle income countries, ranging between 20 and 30% (Gamble et al., 2005; Pathak et al., 2007; McLean et al., 2008). Various studies have suggested a protective effect of peri-conceptional folic acid consump- tion from cleft lip with or without palate. However, the evidence is inconsistent. Materials and Methods STUDY DESIGN AND SETTINGS This was a hospital-based, matched case–control (1:4 ratio; matching for parity) study conducted in Hyderabad, Bengaluru, and Delhi. At each site, cases were selected from a treatment center specialized for managing OFCs. These treatment centers were part of Smile Train, a pro- gram run by a nongovernmental organization having a nationwide chain of accredited centers that provide free treatment for OFCs. The cases were recruited from the fol- lowing treatment centers: GSR Institute of Craniofacial Surgery, Hyderabad; Bhagvan Mahaveer Jain Hospital, Ban- galore, Karnataka; Kalawati Saran Children’s hospital, Delhi; Sant Paramand Hospital, Delhi. For controls, the fol- lowing centers were selected: Koti Maternity Hospital, Mahbubnagar; District Hospital Mahbubnagar District, Telangana; Kalawati Saran Children’s Hospital, Delhi; Srir- ampura referral Hospital, Bangalore; Bhanshankari District Hospital, Bangalore; Ulsoor referral hospital, Bangalore; Siddipur referral Hospital, Bangalore. Controls were selected from maternity centers near/adjoining the treat- ment centers. Women who delivered in these centers dur- ing the study period were recruited after they gave their consent. SAMPLE SIZE For each case, four controls were selected. The estimated sample size calculated using Open Epi software (version 3.1) (Dean et al., 2013) was 750 (150 cases and 600 con- trols) assuming an alpha error of 10%, a power of 80% to detect a 50% decrease in risk (odds ratio [OR] 5 0.5) associated with folic acid supplementation. The prevalence of folic acid supplementation in the control group was assumed to be around 20% with 5% nonresponse rate. Introduction This is further compounded by heterogeneity resulting from different dosages of folic acid and types (with or without other multivitamins) (De-Regil et al., 2010). from other hospitals or who were not from the catchment area, or those not willing to participate in the study. CASES AND CONTROLS A b b Cases. Any baby with nonsyndromic clefts, that is, cleft lip 6 palate or cleft palate, who visited the treatment cen- ter within 4 months of birth was considered as a case. The treating physician ascertained the outcomes based on the standard case definitions used to detect OFCs. OFCs in this study included an opening in a structure around the mouth and face, including clefts in the lip, the roof of the mouth (hard palate), or the tissue in the back of the mouth (soft palate). We interviewed the mothers of the cases at the time of recruitment. Most of these studies on risk factors and prevention of OFCs by folic acid supplementation were done in Western countries (Figueiredo et al., 2015). Comparatively, India has a predominantly vegetarian population, low maternal vitamin-B12 status, and higher prevalence of consanguine- ous marriages, infections, and maternal illnesses. Thus, the risk profile of OFCs may be different (Agbenorku, 2013). Therefore, the aim of our study was to assess risk factors of OFCs, including lack of folic acid supplementation, dur- ing peri-conceptional period. Controls. The controls were live births without any malfor- mation born in a maternity center close to the same region as the treatment center of the case. We interviewed mothers of the controls within 48 hours of delivery. We verified that both cases and controls were inhabitants of the same geo- graphical regions from their addresses, and close to the sites where the study was conducted. Each control selected pur- posively was individually matched to her control according to parity of mother, using the groups: primiparous, parity 1, 2, and 3 1. The study aimed to match four controls to each case. DATA COLLECTION AND MANAGEMENT Data collection took place between January 2015 and July 2016. Structured data collection questionnaires developed by the research team captured information on basic socio- demographic features, details of reproductive, medical and occupational history and other potential risk factors. We translated the tools into local languages, pretested, piloted, and modified before data collection. Trained female field investigators interviewed mothers to collect information on risk factors. Ascertainment of exposures was based on the history elicited using a struc- tured questionnaire designed for the study. Data on folic acid supplementation were collected based on the history of intake of folic acid tablets during the peri-conceptional period and dietary history. The study population consisted of all pregnant women delivering in these hospitals or visiting treatment centers for their children with cleft lip/ palate who gave their con- sent to participate. When selecting controls, we excluded stillbirths or babies who had any apparent structural or chromosomal malformations other than OFC, if they were part of any other on-going studies, had any maternal com- plications that warranted emergency care, babies referred We collected data on paper forms. Data entry was done by staff trained by the data manager using custom- ized forms designed and validated in MS Access with built in consistency, range, and missing data checks by the data base administrator. RISK FACTORS FOR OROFACIAL CLEFTS 1286 TABLE 1. DATA COLLECTION AND MANAGEMENT Socio-demographic Profile of Cases and Controls Variables Cases (n 5 157) Control (n 5 628) p-Value Age of child in days [mean (SD)] 61.9 (39.1) 14.4 (29.1) 0.000 Age of the child more than or equal to 14 days at time of interview [n (%)] 133 (84.7) 114 (18.2) 0.000 Sex of child - male [n (%)] 64 (40.7) 297 (47.3 0.14 Age of mother (in completed years) [mean (SD)] 24.8 (3.8) 24.0 (3.5) 0.01 Age of father (in completed years) [mean (SD)] 29.7 (4.3) 28.8(4.3) 0.02 Mothers less than 30 years of age 138(87.9) 575 (91.5) 0.15 Fathers less than 30 years of age 80 (50.9) 368 (58.6) 0.08 Education status of mother – illiterate or just literate [n (%)] 18 (11.4) 78 (12.4) 0.74 Education status of father- illiterate or just literate [n (%)] 14 (8.8) 90 (14.3) 0.07 Occupation of mother - homemaker [n (%)] 134 (85.4) 542 (86.3) 0.76 Occupation of father- agriculture/ industry [n (%)] 25 (15.9) 77 (12.2) 0.24 Caste backward caste [n (%)] 74 (47.1) 378 (60.1) 0.003 Family income less than 1 lakh rupees per year [n (%)] 110 (98.2) 391 (99.7) 0.09 Age at marriage [mean (SD)] 20.5 (3.3) 20.1 (3.0) 0.2 Drinking water (ground water) [n (%)] 41 (26.1) 130 (20.7) 0.14 Fuel used in cooking- kerosene/ biomass/ coal [n (%)] 18 (11.5) 89 (14.2) 0.08 Place of cooking inside the house [n (%)] 146 (93) 583 (92.8) 0.6 No. Results A total of 785 participants were included in the study: 260 from north India (Delhi) and the rest from south India (250 from Hyderabad and 275 from Bengaluru). There were no refusals. Nearly equal proportion of males and females were present in the study group, and there was nearly equal distribution (48.4%) of primiparous and mul- tigravida (51.6%) mothers. Most of the mothers were liter- ate. Most households (452; 57.6%) belonged to backward castes, and 63.8% had family income less than one lakh Indian rupees (1500 USD) per year. A large proportion (86%) of women were homemakers. Approximately 7% of fathers worked in agriculture. The mean age at marriage for mothers was 20.2 years (SD, 3.1), ranging from 10 to 37 years; 16.4% of the overall study population reported consanguinity or marriage with relatives, with Delhi (20%) reporting the maximum (Supplementary Table S1, which is available online). ETHICAL CONSIDERATION We obtained informed consent from eligible mothers after giving them detailed information about the study in local language. We sought approvals from the respective hospi- tals and Institutional Ethics committee of Public Health Foundation of India. SOCIO-DEMOGRAPHIC CHARACTERISTICS OF CASES AND CONTROLS The study had 157 matched case–control pairs (1:4). The cases and controls were similar with respect to most of the socio-demographic variables. In both the categories, the majority of mothers did not have employment outside the home and most fathers worked in office jobs, as man- ual laborers or were in business. The majority among cases and controls belonged to backward castes, and most cases and controls had an annual family income less than one lakh Indian rupees (Table 1). EXPOSURES confounders (irrespective of p value) were included in the adjusted conditional logistic regression model. The risk factors included in the study were lack of folic acid supplementation during peri-conceptional period along with other documented risk factors, which were con- ception induced by medications or treatment; exposure to radiation (x-rays); infections; medical illness during preg- nancy (such as epilepsy, hypertension, diabetes mellitus, fever, rash, arthritis, common cold, urinary tract infection, and vomiting); intake of any medicinal drugs; food habits (vegetarian/nonvegetarian); consumption of coffee, alco- hol, tobacco, toddy (local alcoholic drink) or any drugs; exposure to smoking, indoor pollution, pesticides during the peri-conceptional period; consanguineous marriage; family history of any malformations; increased maternal and paternal age (Jha, 2001; Agbenorku, 2013; Neogi et al., 2015). Information was collected by interviewing the women as well as by reviewing medical records wher- ever feasible. The risk factors included in the study were lack of folic acid supplementation during peri-conceptional period along with other documented risk factors, which were con- ception induced by medications or treatment; exposure to radiation (x-rays); infections; medical illness during preg- nancy (such as epilepsy, hypertension, diabetes mellitus, fever, rash, arthritis, common cold, urinary tract infection, and vomiting); intake of any medicinal drugs; food habits (vegetarian/nonvegetarian); consumption of coffee, alco- hol, tobacco, toddy (local alcoholic drink) or any drugs; exposure to smoking, indoor pollution, pesticides during the peri-conceptional period; consanguineous marriage; family history of any malformations; increased maternal and paternal age (Jha, 2001; Agbenorku, 2013; Neogi et al., 2015). Information was collected by interviewing the women as well as by reviewing medical records wher- ever feasible. DATA COLLECTION AND MANAGEMENT of living children [mean (SD)] 1.7 (0.07) 1.6 (0.03) 0.07 H/o of consanguineous marriage 28 (17.8) 101 (16.1) 0.6 H/o of abortion 47 (30.0) 131 (20.8) 0.01 H/o neonatal death 5 (3.2) 18 (2.9) 0.83 H/o stillbirths 0 6 (1.0%) Current multiple pregnancy 5 (3.2) 6 (0.9) 0.03 H/o multiple pregnancy in previous pregnancies 3 (1.9) 1(0.2) 0.00 H/o previous child with cleft lip 3 (1.9) 0 Any family history of cleft lip or palate 27 (17.2) 10 (1.6) 0.000 Any exposure to pesticide/ herbicides 56 (35.7) 235 (37.4) 0.6 More than 1 year for conception 106 (67.5) 324 (51.6) 0.000 H/o any treatment taken for infertility 24 (15.3) 32 (5.1) 0.00 Medical illnesses in the first 3 months of pregnancy – Fever 23 (14.6) 85 (13.5) 0.71 – Rash 11 (7.0) 17 (2.7) 0.01 – Hypertension 6 (3.8) 15 (2.3) 0.32 – Epilepsy 2 (1.3) 3 (0.5) 0.263 – Arthritis/ chikungunya 1 (0.6) 8 (1.3) 0.50 – Urinary tract Infection 25 (15.9) 65 (10.3) 0.05 – Diabetes 1 (0.6) 2 (0.3) 0.56 H/o intake of medicine in first 3 months for any illness 76 (48.4) 276 (43.9) 0.3 H/o consumption folic acid – 3 months before pregnancy 9 (5.7) 4 (0.6) 0.00 – First 3 months of pregnancy 48 (30.6) 136 (21.6) 0.02 BIRTH DEFECTS RESEARCH 109:1284–1291 (2017) 1287 TABLE 1. DATA COLLECTION AND MANAGEMENT Continued Variables Cases (n 5 157) Control (n 5 628) p-Value H/o consumption of iron – 3 months before pregnancy 7 (4.4) 5 (0.8) 0.001 – 3 months of pregnancy 70 (44.6) 181 (28.8) H/o consumption of multivitamin during peri-conceptional period 47 (29.9) 134 (21.3) 0.02 H/o X ray in first 3 months of pregnancy 5 (3.2) 5 (0.8) 0.03 Food habit-vegetarianism 46 (29.3) 72 (11.5) 0.00 Exposure to passive smoking in peri-conceptional period 47 (29.9) 110 (17.5) 0.60 Exposure to active smoking 1 (0.6) 1 (0.2) 0.30 H/o chewing tobacco in peri-conceptional period 1 (0.6) 4 (0.6) 0.31 H/o consumption of any form of alcohol in periconceptional period 5 (3.2) 38 (6.0) 0.10 BIRTH DEFECTS RESEARCH 109:1284 1291 (2017) 1287 Cases (n 5 157) Control (n 5 628) p-Value Cases (n 5 157) p – 3 months before pregnancy 7 (4.4) 5 (0.8) 0.001 – 3 months of pregnancy 70 (44.6) 181 (28.8) H/o consumption of multivitamin during peri-conceptional period 47 (29.9) 134 (21.3) 0.02 H/o X ray in first 3 months of pregnancy 5 (3.2) 5 (0.8) 0.03 Food habit-vegetarianism 46 (29.3) 72 (11.5) 0.00 Exposure to passive smoking in peri-conceptional period 47 (29.9) 110 (17.5) 0.60 Exposure to active smoking 1 (0.6) 1 (0.2) 0.30 H/o chewing tobacco in peri-conceptional period 1 (0.6) 4 (0.6) 0.31 H/o consumption of any form of alcohol in periconceptional period 5 (3.2) 38 (6.0) 0.10 Factorsa that differed significantly between cases and controls (p < 0.05) (and having at least five respondents in each cate- ological plausibility as potential confounders (irrespective of the p value). after conception did not seem to have a protective effect in the final adjusted analysis (AOR, 1.2; 95% CI, 0.6–2.6). Age of child emerged as a strong factor (AOR, 34.3; 95% CI, 16.7–70.4), reflecting the fact that cases were older at the time of interview than the controls. (21.6%) during first 3 months of pregnancy. Almost one- third of the mothers of cases were vegetarians compared to 11% of mothers of controls. Among the exposures examined, history of previous abortion, family history of OFC, more than 1 year taken for conception, history of any treatment taken for infertil- ity, history of rash, urinary tract infection in first 3 months of pregnancy, history of consumption of folic acid or iron and folic acid in first 3 months of pregnancy, multivitamin consumption, vegetarianism, backward caste, and age of the child at the time of interview were found to be asso- ciated (p < 0.05 in unadjusted analysis) with OFCs (Table 2). STATISTICAL ANALYSIS STATA 11.0 was used for analysis. The socioeconomic char- acteristics of cases and controls were compared using the unpaired t test for continuous variables and the Chi- square test for categorical variables. Associations between exposures and outcomes were assessed for case–control pairs using unadjusted and adjusted conditional logistic regression. For the adjusted analysis, exposures that had different distributions in cases and controls (p < 0.05) (and with at least five respondents in each category), and those having a biological plausibility as potential Association between exposures and OFC. A total of 13 women out of 785 reported consuming folic acid during 3 months preceding conception. However, more mothers of cases reported taking folic acid (30.6%) compared with controls RISK FACTORS FOR OROFACIAL CLEFTS 1288 Factorsa Unadjusted model Final model OR P value AOR p-Value H/o consumption folic acid in first 3 months of pregnancy 1.61 (1.07-2.41) 0.02 1.24(0.59-2.58) 0.56 H/o consumption of iron in first 3 months of pregnancy 4.80 (1.46-15.73) 0.01 3.29 (0.45- 23.84) 0.24 Food habit -vegetarianism 3.73 (2.32-5.99) 0.000 4.47 (1.83- 10.98) 0.001 Age of the child more than or equal to 14 days at time of interview 31.06(17.08- 56.52) 0.000 34.29 (16.70- 70.42) 0.000 Maternal age more than or equal to 30 years 0.87 (0.47-1.64) 0.68 0.86 (0.29-2.47) 0.77 Backward caste 2.01 (1.34-3.03) 0.001 1.92 (1.01- 3.68) 0.04 H/o of abortion 1.61 (1.06-2.43) 0.02 1.62 (0.75-3.52) 0.22 Current multiple pregnancy 3.33 (1.02-10.92) 0.05 0.6 3(0.07- 6.07) 0.69 Any family history of cleft lip or palate 12.93 (5.86-28.54) 0.000 15.48 (4.36- 54.96) 0.000 More than 1 year for conception 2.10 (1.42-3.12) 0.0001 2.55 (1.25- 5.21) 0.01 H/o any treatment taken for infertility 3.67 (2.05-6.58) 0.000 1.15 (0.42-3.19) 0.78 Medical illnesses in the first 3 months of pregnancy – Rash 2.58 (1.21-5.52) 0.01 3.37 (0.84-13.49) 0.09 – Urinary Tract Infection 1.58 (0.94-2.65) 0.08 1.22 (0.43-3.47) 0.71 H/o X ray in first 3 months of pregnancy 4.93 (1.32-18.35) 0.02 10.66 (1.03- 110.10) 0.05 aFactors included are those that differed significantly between cases and controls (p < 0.05) (and having at least five respondents in each cate- gory), and those having a biological plausibility as potential confounders (irrespective of the p value). Discussion Vegetarian populations may be more exposed to pesticide residues (except for organochlorine compounds) than the general population (Van Audenhaege et al., 2009). Nonvegetarians may also be exposed to con- centrated forms of pesticides because animals eat plant products, and fat soluble contaminants or those that are resistant to degradation accumulate in the fat tissues that ultimately reach humans (Dorea, 2004). It is also important to note that in India supplementation usually starts only after the first antenatal visit, which usu- ally takes place after the missed period or approximately 3 to 4 weeks after conception. The fetal lip usually closes by 5 to 6 weeks after conception and the palate by 10 weeks. This gives a narrow or no window of opportunity for peri- conceptional folic acid supplementation to be administered and to be effective. The reported intake of supplementation in the pre-conceptional period was low (5.7% among moth- ers of cases and 0.6% among controls). Reported intake dur- ing the first 3 months of pregnancy was higher, 30.6% among cases and 21.6% among controls, but we have no details on when they initiated supplementation. One of the limitations of our study was that the data were primarily based on recall. There is generally no doc- umentation or medical records available on the frequency and duration of folic acid or multivitamin supplementation in our settings. Multivitamin preparations are available in diverse formulations, these may or may not contain folic acid, and people tend to interpret supplements as anything that is given for strength and immunity. These were, there- fore, excluded from the final analysis. On the other hand, folic acid preparations are uniform and it is relatively eas- ier to elicit information from mothers because of its con- sistency in color and size. Moreover, when Indian women visit a doctor or a facility in the first trimester of preg- nancy, any medicine prescribed during the first visit is less likely to be forgotten. Nonetheless, there could be a prob- lem with recall bias in both cases and controls giving rise to nondifferential misclassification of folic acid status. Moreover, in our study, cases were interviewed when they came for surgical intervention while controls were inter- viewed 48 hr after the delivery. This could have intro- duced a selection bias because we selected cases whose parents sought care. Discussion Previous studies on the association of multivitamins with OFCs have reported mixed findings, although multivi- tamin preparations containing folic acid have suggested a protective effect (Czeizel, 1993; Bailey and Berry, 2005). The independent role of folic acid is still unclear (Shaw et al., 1995; Itikala et al., 2001). Lack of preventive effect by folic acid supplementation was reported in some stud- ies with a large sample size of over 3000 (Hayes et al., 1996; Czeizelet al., 1999, 2004). Other studies suggested that folic acid was effective only at higher doses (6–8 mg) (Czeizel et al., 1999). One study suggested efficacy of mul- tivitamin and folic acid supplementation in unilateral cleft with a 83% risk reduction, while no effect was observed for bilateral cleft (Tolarova and Harris, 1995). A Cochrane review conducted in 2010 suggests that there is no evi- dence of any effect of folic acid supplementation on pre- vention of cleft palate and/or cleft lip (De-Regil et al., 2010). Yet another study suggests that fortification with folic acid may create an imbalance between folic acid and vitamin-B12, further aggravating the risk of having a child with OFCs (Godbole et al., 2009). Cases and controls should be chosen from the same base population to reduce selection bias in case–control studies. Evidence shows that for hospital-based case–control stud- ies, community controls are the best choice if high response rates can be attained (Neupane et al., 2010). However, con- sidering the budget constraints for any public health research in resource-scarce settings, researchers may con- sider selecting hospital controls for a case–control study if potential confounders are carefully considered, measured, and adjusted for (Rahman et al., 2012). In our study, al- though we attempted to select controls from the same geo- graphical regions from their addresses, they might not have represented the population that gave rise to cases. There is, therefore, a possibility of some degree of selection bias in our study. Of interest, vegetarianism as compared to nonvegetarian diet emerged to be strongly associated with OFC in our study. Women having vegetarian diets have low folate and vitamin B12 levels (Kirchheimer et al., 2001; Refsum et al., 2001) and a diet deficient in folate and vitamin B12 was shown to be related with OFC (Krapels et al., 2004). Most vegetarians in India adopt traditional cooking methods with overexposure of food to heat. Discussion In this case–control study of OFCs, we found that a family history of cleft lip/palate, vegetarianism, and delayed first conception beyond 1 year were associated with a higher risk of OFCs. We did not find any evidence to support a protective association with folic acid supplementation dur- ing first 3 months of pregnancy. A model of multivariable analysis was developed using conditional logistic regression (Table 2). In the final model, a family history of cleft lip/palate, exclusive vegetarianism and delayed first conception beyond one year were associ- ated with OFCs. Cases had 15 times higher likelihood of having a family history than controls (adjusted odds ratio [AOR], 15.5; 95% confidence interval [CI], 4.4–54.9). Like- wise, cases were around 4.5 times more likely than con- trols to be exclusive vegetarians (AOR, 4.5; 95% CI, 1.8– 10.9). Cases were more likely to be born after a delayed conception compared with controls (AOR, 2.55; 95% CI, 1.25–5.21). Supplementation of folic acid 3 months before conception had to be excluded from the adjusted analysis owing to small cell sizes. Supplementation for 3 months A family history of OFC is a strong risk factor for nonsyndromic OFCs as shown in previous studies (Acuna- Gonzalez et al., 2011; Rahimov et al., 2012; Burg et al., 2016). In our study, the strength of association was as high as 15 but with very wide CIs. Our findings are con- sistent with other studies (effect size varying from 17 to 56) (Sivertsen et al., 2008; Grosen et al., 2010). Delayed first conception beyond 1 year emerged as a risk factor in our analysis. Treatment received for infertility was associ- ated with OFCs in unadjusted analysis but did not show any association in the adjusted analysis. In the analysis, supplementation with multivitamin or folic acid preparations in the peri-conceptional period did not seem to have any protective effect. However, these BIRTH DEFECTS RESEARCH 109:1284–1291 (2017) 1289 findings should be interpreted in the Indian context where any preparations consumed for “strength and vitality” are often termed as “multivitamin preparations” by the people. So, self-reported consumption of multivitamins could in- clude either multivitamins, folic acid, iron preparations, or indigenous medicines as well. (Wardlaw, 2004). A vegetarian diet may also increase expo- sure to pesticides through agricultural use although the evi- dence is equivocal. References Acuna-Gonzalez G, Medina-Solis CE, Maupome G, et al. 2011. Family history and socioeconomic risk factors for non-syndromic cleft lip and palate: a matched case-control study in a less devel- oped country. Biomedica 31:381–391. On the other hand, factors related to medical conditions are less likely to be missed because every subject was enrolled from the hospitals where these details are recorded routinely. Gathering information from medical records leads to less recall bias. Information on diet is highly subject to bias because it is likely to get altered during the course of pregnancy. So, we restricted ourselves only to analyzing whether the person was vegetarian or nonvegetarian. Vege- tarianism is strongly linked with sociocultural customs and generally remains unaltered in Indian culture. The possibil- ity of a vegetarian person getting misclassified as a nonvege- tarian is much less, although the converse may be true. However, triangulation was done to overcome this issue by asking mothers about the frequency of intake of nonvegetar- ian food before and after pregnancy. Given the limitations of recall, we could not categorize participants into those con- suming non vegetarian food daily, weekly, or occasionally, and we combined them in one group. Agbenorku P. 2013. Orofacial clefts: A worldwide review of the problem. Available at https://www.hindawi.com/journals/isrn/ 2013/348465/. Accessed May 8, 2017. Allagh KP, Shamanna BR, Murthy GV, et al. 2015. Birth preva- lence of neural tube defects and orofacial clefts in India: a sys- tematic review and meta-analysis. PLoS One 10:e0118961. Aylsworth AS, Allori AC, Pimenta LA, et al. 2015. Issues involved in the phenotypic classification of orofacial clefts ascertained through a state birth defects registry for the North Carolina Cleft Outcomes Study. Birth Defects Res A Clin Mol Teratol 103:899–903. Bailey LB, Berry RJ. 2005. Folic acid supplementation and the occurrence of congenital heart defects, orofacial clefts, multiple births, and miscarriage. Am J Clin Nutr 81:1213S–1217S. Bianchi F, Cianciulli D, Pierini A, Seniori Costantini A. 1997. Con- genital malformations and maternal occupation: a registry based case-control study. Occup Environ Med 54:223–228. To our knowledge, this is one of only a few case–control studies evaluating risk factors of OFC in a largely vegetarian population. Although, we assumed that there would be a 5% nonresponse rate, in our study there were no refusals which adds to its strength. It was conducted in two geographically and culturally different (north India vs. south India) areas within India. References This increased the external validity of the study. Cases were taken based on physician’s diagnosis only and all the subjects were recruited from big hospitals where the sys- tem of recording is better than many hospitals of the country. Our team reviewed medical records and collected information from them for most of the risk factors studied, minimizing information bias as far as possible. In addition, having four controls for every case improved the power and efficiency of the study. Botto LD, Moore CA, Khoury MJ, Erickson JD. 1999. Neural-tube defects. N Engl J Med 341:1509–1519. Burg ML, Chai Y, Yao CA, et al. 2016. Epidemiology, etiology, and treatment of isolated cleft palate. Front Physiol 7:67. Czeizel AE. 1993. Prevention of congenital abnormalities by periconceptional multivitamin supplementation. BMJ 306:1645–1648. Czeizel AE, Dobo M, Vargha P. 2004. Hungarian cohort-controlled trial of periconceptional multivitamin supplementation shows a reduction in certain congenital abnormalities. Birth Defects Res A Clin Mol Teratol 70:853–861. Czeizel AE, Timar L, Sarkozi A. 1999. Dose-dependent effect of folic acid on the prevention of orofacial clefts. Pediatrics 104:e66. In conclusion, our study confirmed the importance of family history as a risk factor for OFCs. Our study did not show an association with folic acid supplementation but was underpowered to detect small differences. The finding of higher risk among vegetarians requires further explora- tion and research. More studies are needed to establish the role of vitamin B12 alone or in combination with folic acid in prevention of OFCs especially in Indian population where majority are vegetarians. De-Regil LM, Fernandez-Gaxiola AC, Dowswell T, Pena-Rosas JP. 2010. Effects and safety of periconceptional folate supplementa- tion for preventing birth defects. Cochrane Database Syst Rev (10):CD007950. Dean AG, Sullivan KM, Soe MM. 2013. OpenEpi: open source epi- demiologic statistics for public health. www.OpenEpi.com, updated 2013/04/06. Accessed June 8, 2017. Dorea JG. 2004. Vegetarian diets and exposure to organochlorine pollutants, lead, and mercury. Am J Clin Nutr 80:237–238. Discussion Fifty to 90% of folate can be destroyed by these traditional cooking methods because of folate acid’s sensitivity to heat (Wardlaw, 2004). Moreover, most of them are exclusive vegetarians and, therefore, prone to vitamin B12 deficiency, which is essential for increasing the bioavailability of folate. Vitamin B12 deficiency could increase risk of OFCs as vitamin B12 plays a role in many enzymatic reactions linked to folic acid metabolism There were no disaggregated data on the type of OFC; literature suggests that determinants (both environmental and genetic etiologies) of OFC vary with the type (Bianchi et al., 1997; Burg et al., 2016). Combining cleft lip and palate together may have attenuated the strength of association that we would have otherwise obtained. There seems to be a strong association of caste with OFCs. Because these were based only on self-reports in our study and there is a 1290 RISK FACTORS FOR OROFACIAL CLEFTS to the mothers and families who participated in the study and contributed significantly. to the mothers and families who participated in the study and contributed significantly. likelihood of people not revealing the actual caste, we com- bined all backward castes as a single category and com- pared it against nonbackward castes in the final analysis. Sociodemographic factors, however, are less amenable to change during the course of pregnancy and, hence, unlikely to be affected by recall or misclassification bias. Acknowledgments The authors acknowledge the contribution of doctors, nurses, and staff of the hospitals for their support in the exe- cution of the study. The team is grateful to the research staff involved in data collection and data entry. They are thankful Figueiredo RF, Figueiredo N, Feguri A, et al. 2015. The role of the folic acid to the prevention of orofacial cleft: an epidemiolog- ical study. Oral Dis 21:240–247. BIRTH DEFECTS RESEARCH 109:1284–1291 (2017) 1291 Gamble MV, Ahsan H, Liu X, et al. 2005. Folate and cobalamin deficiencies and hyperhomocysteinemia in Bangladesh. Am J Clin Nutr 81:1372–1377. Mossey PA, Little J, Munger RG, et al. 2009. Cleft lip and palate. Lancet 374:1773–1785. Mossey PA, Little J, Munger RG, et al. 2009. Cleft lip and palate. Lancet 374:1773–1785. Neogi SB, Negandhi PH, Sandhu N, et al. 2015. Indigenous medi- cine use for sex selection during pregnancy and risk of congeni- tal malformations: a population-based case-control study in Haryana, India. Drug Saf 38:789–797. Godbole K, Deshmukh U, Yajnik C. 2009. Nutrigenetic determi- nants of neural tube defects in India. Indian Pediatr 46:467–475. Grosen D, Chevrier C, Skytthe A, et al. 2010. A cohort study of recurrence patterns among more than 54,000 relatives of oral cleft cases in Denmark: support for the multifactorial threshold model of inheritance. J Med Genet 47:162–168. Neupane B, Walter SD, Krueger P, Loeb M. 2010. Community con- trols were preferred to hospital controls in a case-control study where the cases are derived from the hospital. J Clin Epidemiol 63:926–931. Hayes C, Werler MM, Willett WC, Mitchell AA. 1996. Case-control study of periconceptional folic acid supplementation and oral clefts. Am J Epidemiol 143:1229–1234. Pathak P, Kapil U, Yajnik CS, et al. 2007. Iron, folate, and vitamin B12 stores among pregnant women in a rural area of Haryana State, India. Food Nutr Bull 28:435–438. Itikala PR, Watkins ML, Mulinare J, et al. 2001. Maternal multivi- tamin use and orofacial clefts in offspring. Teratology 63:79–86. Rahimov F, Jugessur A, Murray JC. 2012. Genetics of nonsyn- dromic orofacial clefts. Cleft Palate Craniofac J 49:73–91. Jha P. 2001. Reliable mortality data: a powerful tool for public health. Natl Med J India 14:129–131. Rahman MA, Spurrier N, Mahmood MA, et al. 2012. Choice of controls for a case-control study in Bangladesh: hospital controls versus community controls. Asian J Epidemiol 5:75–86. Kirchheimer S. 2001. Vegetarian diet and B12 deficiency. Wardlaw H. 2004. Perspective in Nutrition. 6th ed. McGraw-Hill College, Boston, MA. Acknowledgments Avail- able at http://www.webmd.com/food-recipes/news/20030618/ vegetarian-diet-b12-deficiency#1 Accessed September 30, 2016. Refsum H, Yajnik CS, Gadkari M, et al. M. 2001. Hyperhomocys- teinemia and elevated methylmalonic acid indicate a high preva- lence of cobalamin deficiency in Asian Indians. Am J Clin Nutr 74:233–241. Krapels IP, van Rooij IA, Ocke MC, et al. 2004. Maternal dietary B vitamin intake, other than folate, and the association with orofa- cial cleft in the offspring. Eur J Nutr 43:7–14. Shaw GM, Lammer EJ, Wasserman CR, et al. 1995. Risks of orofa- cial clefts in children born to women using multivitamins con- taining folic acid periconceptionally. Lancet 346:393–396. Caudill M. 2008. The role of folate in reducing chronic and develop- mental disease risk: an overview. Available at http://onlinelibrary.wiley. com/doi/10.1111/j.1365-2621.2004.tb17887.x/abstract. Accessed May 8, 2017. Sivertsen A, Wilcox AJ, Skjaerven R, et al. 2008. Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives. BMJ 336:432–434. McLean E, de Benoist B, Allen LH. 2008. Review of the magni- tude of folate and vitamin B12 deficiencies worldwide. Food Nutr Bull 29(Suppl):S38–S51. Smithells RW, Sheppard S, Schorah CJ. 1976. Vitamin deficiencies and neural tube defects. Arch Dis Child 51:944–950. Tolarova M, Harris J. 1995. Reduced recurrence of orofacial clefts after periconceptional supplementation with high-dose folic acid and multivitamins. Teratology 51:71–78. Molloy AM, Kirke PN, Brody LC, et al. 2008. Effects of folate and vita- min B12 deficiencies during pregnancy on fetal, infant, and child devel- opment. Food Nutr Bull 29(Suppl):S101–S111; discussion S112–S105. Van Audenhaege M, Heraud F, Menard C, et al. 2009. Impact of food consumption habits on the pesticide dietary intake: compar- ison between a French vegetarian and the general population. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 26:1372–1388. Molloy AM, Kirke PN, Troendle JF, et al. 2009. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics 123:917–923. Molloy AM, Kirke PN, Troendle JF, et al. 2009. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics 123:917–923. Mossey P, Little J. 2009. Addressing the challenges of cleft lip and palate research in India. Indian J Plast Surg 42(Suppl):S9–S18. Wardlaw H. 2004. Perspective in Nutrition. 6th ed. McGraw-Hill College, Boston, MA.
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Parent–Child Relationship Quality and Internet Use in a Developing Country: Adolescents’ Perspectives
Frontiers in psychology
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ORIGINAL RESEARCH published: 28 February 2022 doi: 10.3389/fpsyg.2022.847278 Parent–Child Relationship Quality and Internet Use in a Developing Country: Adolescents’ Perspectives Thao Thi Phuong Nguyen 1,2*, Tham Thi Nguyen 1,2, Ha Ngoc Do 3, Thao Bich Thi Vu 3, Khanh Long Vu 3, Hoang Minh Do 3, Nga Thu Thi Nguyen 4, Linh Phuong Doan 1,2, Giang Thu Vu 5, Hoa Thi Do 6, Son Hoang Nguyen 5, Carl A. Latkin 7, Cyrus S. H. Ho 8,9 and Roger C. M. Ho 9,10 1 Institute for Global Health Innovations, Duy Tan University, Da Nang, Vietnam, 2 Faculty of Medicine, Duy Tan University, Da Nang, Vietnam, 3 Youth Research Institute, Ho Chi Minh Communist Youth Union, Hanoi, Vietnam, 4 Faculty of Social Sciences and Humanities, Hanoi Metropolitan University, Hanoi, Vietnam, 5 Center of Excellence in Health Services and System Research, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam, 6 Institute of Health Economics and Technology (iHEAT), Hanoi, Vietnam, 7Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States, 8 Department of Psychological Medicine, National University Health System, Singapore, Singapore, 9 Department of Psychological Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 10 Institute for Health Innovation and Technology (iHealthtech), National University of Singapore, Singapore, Singapore Edited by: Quan-Hoang Vuong, Phenikaa University, Vietnam Objective: The goal of the study was to explore the relationship between parent–children relationships related to using the internet among kids and potentially associated factors. Objective: The goal of the study was to explore the relationship between parent–children relationships related to using the internet among kids and potentially associated factors. Reviewed by: Quy Khuc, Vietnam National University, Hanoi, Vietnam Minh-Hoang Nguyen, Phenikaa University, Vietnam Reviewed by: Quy Khuc, Vietnam National University, Hanoi, Vietnam Minh-Hoang Nguyen, Phenikaa University, Vietnam Reviewed by: Quy Khuc, Vietnam National University, Hanoi, Vietnam Minh-Hoang Nguyen, Phenikaa University, Vietnam Materials and Methods: A sample of 1.216 Vietnamese students between the ages of 12 and 18 agreed to participate in the cross-sectional online survey. Data collected included socioeconomic characteristics and internet use status of participants, their perceived changes in relationship and communication between parents and children since using the internet, and parental control toward the child’s internet use. An Ordered Logistic Regression was carried out to determine factors associated with parent–children relationship since using the internet. *Correspondence: Thao Thi Phuong Nguyen nguyentphuongthao73@duytan.edu.vn Results: The characteristics of the relationship between children and their parents since using the Internet were divided into three levels: deterioration (7.0%), stability (78.2%), and improvement (14.8%). The topics that children most often communicate with their parents include learning, housework, and future directions. Two-way interactive activities, such as supporting parents to use the Internet, have a positive impact on the parent–child relationship. Stubborn parental control, such as establishing rules about contact or allowing Internet access and setting up global positioning system (GPS) to track negatively affecting parent–child relationships. Specialty section: This article was submitted to Health Psychology, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Health Psychology, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Health Psychology, a section of the journal Frontiers in Psychology Received: 01 January 2022 Accepted: 02 February 2022 Published: 28 February 2022 Received: 01 January 2022 Accepted: 02 February 2022 Published: 28 February 2022 Keywords: internet addiction, internet use, parent–child relationship, children, Vietnam Edited by: Quan-Hoang Vuong, Phenikaa University, Vietnam INTRODUCTION control might lead to several mental health issues in children such as low self-esteem (Younes et al., 2016; Shi et al., 2017), depression (Younes et  al., 2016; Fayazi and Hasani, 2017), and antisocial behaviors (Barber et al., 2012), which triggered excessive internet use in children as an adapting way for satisfying their psychological needs (Yu et al., 2012). Meanwhile, children who had a high relationship quality with their parents were found to be  more likely to have emotional stability, better social competence, and fewer behavioral issues (Schneider et  al., 2001). Several publications demonstrated a negative association between adolescents’ internet addiction and having an open parent–child relationship (Zhang et  al., 2011; Zhu et  al., 2015).h Using the internet has become one of the most popular leisure- time activities worldwide. In Vietnam, since the internet was officially connected in 1997, internet users sharply increased and reached 68.72  million as of January 2021, equivalent to 70.3% of the population. The average internet use/access time of Vietnamese was 6.5 h every day. Notably, 70.1% of the internet users in Vietnam were reported to be  aged 13–34 (Digital 2021: Vietnam: Datareportal, 2021). A 2016 study showed that 72% of Vietnamese aged 15–24 used the internet, of which 74% were reported having the high-risk danger by cyberbullying (The UNICEF, 2017). The popularity of the internet among adolescents poses challenges to parents in protecting their children from excessive internet use and negative influences from the cyber world (Subrahmanyam et  al., 2000; Greenfield, 2004). Excessive internet use may exhibit similar patterns and have similar negative consequences to the life of youths and adolescents compared to other addictive behaviors (Shapira et al., 2003; Young, 2004). From the parents’ perspective, the attachment of youths and adolescents to the internet may lead to the passivity in the young people’s daily activities, changes in their psychological states and behaviors, and negative effects on the quality of the parent–child relationship (van Den Eijnden et al., 2010; Shek et al., 2018; Restrepo et al., 2020; Li et  al., 2021). The research landscape concerning internet use behaviors of Vietnamese adolescents has expanded significantly in recent years, in light of the accelerating popularity of the internet among this population (Sobowale et al., 2016; Tran et al., 2017; Zhang et  al., 2017; Lan et  al., 2020). INTRODUCTION Negative effects of excessive parental Existing literature argued that parents play a vital role in the management of children’s internet use (Shi et  al., 2017). Previous studies found an inverse association between children’s internet problematic use and parental monitoring and bonding (Siomos et al., 2012; Ding et al., 2017). Meanwhile, the relationship between parental control and children’s behaviors appears to be more complicated. Barber et al. (2005) reckoned that parental control could be  in the forms of behavioral or psychological control. Behavioral control referred to the use of explicit management strategies by parents including children’s activities supervision, establishing rules, and restrictions to regulate the children’s behavior (Shek, 2005), while psychological control manifested through implicitly adjusting children’s behavior based on their emotions and thoughts. Psychological tactics that were used by parents included guilt infusion, love withdrawal, and authority assertion (Smetana and Daddis, 2002; Rogers et  al., 2003). The existing scientific findings have consistently shown that parental psychological control diminished child development and increased parent–child conflicts, while behavioral control positively related children’s developmental outcomes and well- being (Barber et al., 2005; Wang et al., 2007; Bleakley et al., 2016). C i hild ’ i i t t t di Citation: Nguyen TTP, Nguyen TT, Do HN, Vu TBT, Vu KL, Do HM, Nguyen NTT, Doan LP, Vu GT, Do HT, Nguyen SH, Latkin CA, Ho CSH and Ho RCM (2022) Parent–Child Relationship Quality and Internet Use in a Developing Country: Adolescents’ Perspectives. Front. Psychol. 13:847278. doi: 10.3389/fpsyg.2022.847278 Conclusion: Findings indicated that changes in the quality of the parent–child relationship were self-assessed by participants regard to kids’ internet use, especially in the COVID-19 epidemic context. Educational campaigns and programs to raise awareness of parents as to the dangers and negative influences that their children may encounter online, psychology of children’s behaviors and effects of different responding strategies are recommended. February 2022 | Volume 13 | Article 847278 1 Frontiers in Psychology | www.frontiersin.org Parent-Child Relationship and Internet Use Nguyen et al. INTRODUCTION Furthermore, in the context of COVID-19 pandemic outbreaks, the Vietnamese government implemented proactive measures to prevent spreading, including school closures (La et  al., 2020). During the stay-at-home period, spending more time on virtual platforms may influence the parent–children relationship (Cuong et  al., 2021). There have been several studies on internet use among youths and adolescents in Vietnam (Sobowale et  al., 2016; Tran et  al., 2017; Zhang et  al., 2017; Lan et  al., 2020; Cuong et  al., 2021). However, these studies had not offered a comprehensive assessment of the effect of Vietnamese parental management on adolescent Internet use, especially amidst the pandemic. The current study aimed to (1) assess the relationship between parent–children relationships related to using the internet among kids; and (2) identify potentially affected factors. Existing literature argued that parents play a vital role in the management of children’s internet use (Shi et  al., 2017). Previous studies found an inverse association between children’s internet problematic use and parental monitoring and bonding (Siomos et al., 2012; Ding et al., 2017). Meanwhile, the relationship between parental control and children’s behaviors appears to be more complicated. Barber et al. (2005) reckoned that parental control could be  in the forms of behavioral or psychological control. Behavioral control referred to the use of explicit management strategies by parents including children’s activities supervision, establishing rules, and restrictions to regulate the children’s behavior (Shek, 2005), while psychological control manifested through implicitly adjusting children’s behavior based on their emotions and thoughts. Psychological tactics that were used by parents included guilt infusion, love withdrawal, and authority assertion (Smetana and Daddis, 2002; Rogers et  al., 2003). The existing scientific findings have consistently shown that parental psychological control diminished child development and increased parent–child conflicts, while behavioral control positively related children’s developmental outcomes and well- being (Barber et al., 2005; Wang et al., 2007; Bleakley et al., 2016). Concerning children’s excessive internet use, some studies found that children were less likely to get addicted to the internet if their parents managed firmly in proximity and consistent manner (e.g., establishing the rules for using the internet, finding how to share internet information; Harakeh et  al., 2004; Van der Vorst et  al., 2005). On the other hand, some empirical evidence showed that the psychological control of parents was positively associated with children’s internet addiction (Giles and Price, 2008; Xiuqin et  al., 2010; Cheung et  al., 2015). Frontiers in Psychology | www.frontiersin.org Socioeconomic Characteristic We asked participants a number of questions about their information, including gender (male/female), grade (secondary school/high school), current location (urban/rural), current living with (parents/grandparents/siblings), academic performance (excellent/good/fair/moderate/below moderate), and the number of family members that participants can confide in. Measurement and Instrument In this study, we  conducted an online questionnaire on SurveyMonkey’s platform1. This approach yields efficiencies, such as saving time, reducing cost, and reaching a large of samples. A structured questionnaire including five major components: (1) General characteristics; (2) Parent–child relationship since participants used the internet; (3) Children’s internet use status; (4) Interactive and supportive activities regarding internet use between parents and children; and (5) Social support. A pilot survey was conducted by 10 people for ensuring logicality and adjusting questions with unclear before collecting data. Participants spend 20 min completing the informed consent and questionnaires. Based on “EU Kids Online 2017, questions for children and young people” and “Children Online in the European Union 2020” to develop five questions to explore about activities of students with their parents on the Internet (EU Kids Online 2017, 2020; The EU kids online network, 2020), including: Children’s Communication With Their Parents Since Using the Internet of sample size was added to prevent incomplete survey responses, thus resulting in a total of 1,460 students being recruited. The total of participants who agreed to involve in and completed the questionnaire was 1,216 at the end of data collection period, with the response rate was 83.2%. To assess the parent–children communication since using the internet, we  asked participants to collect information about children’s attitude with their parents, and the frequency of talking to their parents about daily life. In particular, the question “Do you  ignore advice from your parents about using the internet?” (Yes/No) was used to explore the children’s attitude toward their parents. Furthermore, nine popular topics about children’s daily life, such as future orientation, learning, housework, daily living habits, hobby, privacy at home, relationship with friends, the selection of clothing, and the problem of pocket money were developed to assess the parent–child interaction. For each topic, a Likert-five scale from 1 (Never) to 5 (Always) was used to report the frequency of parent communication in daily life. The Cronbach’s alpha of these questions was 0.753. Relationship Between Children and Their Parents Since Using the Internet Relationship Between Children and Their Parents Since Using the Internet A question “How the relationship between you and your parents has changed since using the internet?” was used to assess the internet affects family relationships. The response options for this question were divided into three levels: deteriorated, constant, and better relationship. 1. Helping parents when they have difficulty using the Internet. 2. Ask for help from parents when the situation is difficult to handle on the Internet. 3. Seek advice from parents on what to do on the Internet. 4. Talk about what you  do on the Internet with parents. 5. Talk to parents about what is bothering them on the Internet. 1. Helping parents when they have difficulty using the Internet.fi 1. Helping parents when they have difficulty using the Internet.fi fi 2. Ask for help from parents when the situation is difficult to handle on the Internet. 2. Ask for help from parents when the situation is difficult to handle on the Internet. 3. Seek advice from parents on what to do on the Internet. 4 T lk b t h t d th I t t ith t y 5. Talk to parents about what is bothering them on the Internet. 5. Talk to parents about what is bothering them on the Internet. Covariates 6. For each question, the response options ranged from 1 (Never) to 5 (Always). The Cronbach’s alpha was 0.825. 6. For each question, the response options ranged from 1 (Never) to 5 (Always). The Cronbach’s alpha was 0.825. Study Design, Sampling Method, and Data Collection From June to July 2020, an online cross-sectional study was conducted in Vietnam. The eligibility criteria for participating in this survey were: (1) studied from grade 6 to grade 12; (2) currently lived in Vietnam, and (3) agreed to join this study by providing online informed consent. We  recruited participants via convenience sampling from four cities/provinces undergoing rapid urbanization, including Can Tho, Ba Ria-Vung Tau, Hung Yen, and Thanh Hoa. Participants who suffered from serious illnesses or could not answer questions were excluded from the recruitment process.h Concerning children’s excessive internet use, some studies found that children were less likely to get addicted to the internet if their parents managed firmly in proximity and consistent manner (e.g., establishing the rules for using the internet, finding how to share internet information; Harakeh et  al., 2004; Van der Vorst et  al., 2005). On the other hand, some empirical evidence showed that the psychological control of parents was positively associated with children’s internet addiction (Giles and Price, 2008; Xiuqin et  al., 2010; Cheung et  al., 2015). Negative effects of excessive parental The formula to estimate a population proportion was used to calculate the sample size with specified relative precision. With confidence level (%) α = 0.05, expected population proportion with better relationship with parent p = 34.0% (according to a previous study in Egypt; Moawad and Ebrahem, 2016), and relative precision ε = 0.15, the sample size estimated to participate by 332 participants per study sites. The total sample size in four research sites in Vietnam was 1,328. After calculating the total sample size of four research sites, 10% February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 2 Parent-Child Relationship and Internet Use Nguyen et al. Children’s Communication With Their Parents Since Using the Internet 1surveymonkey.com The Parental Management and Care With Internet Use in Children Based on the survey “EU Kids Online 2017, Questions for children and young people” (EU Kids Online 2017, 2020), a series of questions about parental care and management were developed, including: The Status of Using the Internet Among Participants Participants were asked to report about time using the internet per day. Additionally, we  based on “EU Kids Online 2017” to build four questions about the frequency of using the internet at four places: Family, school, friend’s house, and public areas. For each question, a Likert-seven scale from 1 (Never) to 7 (Almost at the time) was used to collect information from participants (EU Kids Online 2017, 2020). The total score of four questions was summed to assess the frequency of using the internet among students, with the higher score indicated that the frequency of using the internet among participants was higher. The Cronbach’s alpha of these questions was 0.64. Parental Management With Internet Use in Children. We developed 10 questions to collect information, including: de e oped 0 quest o s to co ect o at o , c ud g: 1. Rules about how long I  am  allowed to go online 2. Rules about when I  am  allowed to go online 3. Knows about what I look at on the internet and the social media 4. Rules about who I  can contact on social networks 5. Checks my messages on the social media 6. Checks my information on the Internet 7. Technology to track where I am [such as global positioning system (GPS)] 8. Check out content what I  look at on the internet 9. Check out adding my friends and friends list 10. Limits content of what I  can see on the internet 1. Rules about how long I  am  allowed to go online g g 2. Rules about when I  am  allowed to go online 3. Knows about what I look at on the internet and the social media 4. Rules about who I  can contact on social networks 5. Checks my messages on the social media 7. Technology to track where I am [such as global positioning system (GPS)] 8. Check out content what I  look at on the int 8. Check out content what I  look at on the internet 9. Check out adding my friends and friends list 9. Check out adding my friends and friends list 10. Statistical Analysis y Both descriptive and analytical statistics were used to address the main aims of the study by STATA version 16. When screening data, we used the Listwise Deletion method to clean data before analyzing it. Listwise Deletion means that any individual in a data set was excluded from the model if they were missing data on any variable in the analysis. Continuous variables were presented as mean and SD, while categorical variables were presented as frequencies with percentages. In this study, to compare differences in the parent–children relationship among participants, the Kruskal-Wallis test for continuous variables and the χ2 test were used for categorical variables. Multivariate Logistic Regression was conducted to determine potential predictors of the parent–child relationship among Vietnamese students (Deteriorates = 1; and Constant/Better = 0). To identify potential predictors related to the relationship between parents and children, firstly, we selected according to univariate analysis, and variables with value of p ≤ 0.25 were included in the multivariate logistic regression model (Hosmer et  al., 2000). Subsequently, these potential covariates could be  used in multivariate logistics regression model, including socio-economic characteristics, parent–children communication, parental management and care, and social support. Furthermore, we  used Hosmer-Lemeshow test and some criteria, such as R-square, AIC, and BIC to assess Relationship Between Parents and Children Since Using Internet Multidimensional Scale of Perceived Social Support Multidimensional Scale of Perceived Social Support (MSPSS) was a brief, and easy to administer self-report questionnaire which consisted of 12 items. This scale was used to the extent to which an individual perceives social support from three sources: significant others (four items), family (four items), and friends (four items; Wongpakaran et  al., 2011). For each item, a seven-point scale from 1 (very strongly disagree) to 7 (very strongly agree) was used (Zimet et  al., 2020). The total score of the four items shows that the higher the score, the higher the degree of support. The Cronbach’s alpha was excellent at 0.905. Table  1 described the characteristics of the relationship between children and their parents since they started using the internet. Participants reported having either deteriorated (7.0%), constant (78.2%), and better (14.8%) relationship with their parents. Participants who reported deteriorating relationships with parents were mainly female (62.4%), high school students (70.6%), living in a nuclear family (92.9%), having an academic performance at fair levels (38.1%). Meanwhile, secondary school students (57.8%), having excellent/good academic performance (68.1%) showed a better relationship with parents. In addition, most people who had a bad relationship with their parents ignored advice about internet use from their parents (91.8%) and had limited internet access (78.8%). People having deteriorated relationship with parents spent significantly more time on the internet compared to those reported having a constant or better relationship. In particular, the internet use frequency of students having deteriorated relationships with their parents was significantly higher than others. The differences were statistically significant with p < 0.05. The Parental Management and Care With Internet Use in Children Limits content of what I  can see on the internet 10. Limits content of what I  can see on the internet February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 3 Parent-Child Relationship and Internet Use Nguyen et al. Parental Care With Internet Use in Children. Eight questions were used to collect data, including: Parental Care With Internet Use in Children. Eight questions were used to collect data, including: Parental Care With Internet Use in Children. Eight questions were used to collect data, including: the goodness of fit for multivariate logistic regression model and select the best models in this study. These models were then combined with the stepwise forward strategies to reduce models with p < 0.2 as the threshold for including variables (Hosmer et  al., 2013). A value of p < 0.05 was considered statistically significant. 1. Explains why some online content is good or bad 1. Explains why some online content is good or bad 1. Explains why some online content is good or bad 2. Encourages me to explore and learn things on the Internet 3. Talks to me about what I  do on the internet 4. Suggests ways to use the internet safely 5. Helps me when something bothers me on the internet Ethical Consideration 6. Talks to me about what to do if something online bothers or upsets me All procedures performed in studies involving human participants were in accordance with the ethical standards of the Youth Research Institute, Vietnam, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Online Informed consent was obtained from all participants. 7. Helps me when something is difficult to do or find on the internet 7. Helps me when something is difficult to do or find on the internet 8. Do sharing activities with me on the internet For all of the above questions, a Likert-five scale was used, with ranging options from 1 (Never) to 5 (Always). Additionally, the Cronbach’s alpha values of the questions about parental management and care were good at 0.857, and 0.859, respectively. Frontiers in Psychology | www.frontiersin.org Communication and Interaction Between Parents and Children Related to Internet Use Children’s communication with their parents since using the internet was shown in Table  2. Learning, housework, and future orientation were the topics that students communicated most frequently with their parents. “Helping parents when they have difficulty using the internet” was the most popular activity that children do with their parents on the Internet. The majority of participants having a good relationship with parents showed the highest frequency of communication about all topics, except for pocket money issues. Similarly, with the mutual support in internet use, participants having a good parent–child relationship were higher frequency of interactive and supportive activities than the other groups. These differences were statistically significant with p < 0.05. February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 4 Parent-Child Relationship and Internet Use Nguyen et al. The Management and Care of Parents With Children’s Internet Use The parent’s management and care with the children’s internet management with their internet use, particularly with rules about how long (2.9 points)/when they were allowed access internet (2.6 points). For the group having a better parent–child TABLE 1  |  Characteristics of relationship between parents and children since using internet. Communication and Interaction Between Parents and Children Related to Internet Use Parent–children relationships since using the internet management with their internet use, particularly with rules about how long (2.9 points)/when they were allowed access internet (2.6 points). For the group having a better parent–child relationship, the highest frequency of parent’s management was controlling what children look at on the internet and social The Management and Care of Parents With Children’s Internet Useh Communication and Interaction Between Parents and Children Related to Internet Use Characteristics Parent–children relationships since using the internet Total Value of p Deteriorates Constant Better n % n % n % n % Total 85 7.0 951 78.2 180 14.8 1,216 100.0 Provinces   Ba Ria—Vung Tau 35 41.2 330 34.7 47 26.1 412 33.9 <0.01   Can Tho 12 14.1 184 19.4 54 30.0 250 20.6   Hung Yen 20 23.5 206 21.7 25 13.9 251 20.6   Thanh Hoa 18 21.2 231 24.3 54 30.0 303 24.9 Education   Secondary school 25 29.4 458 48.3 104 57.8 587 48.4 <0.01   High school 60 70.6 491 51.7 76 42.2 627 51.6 Gender   Male 32 37.6 446 46.9 95 52.8 573 47.1 0.07   Female 53 62.4 505 53.1 85 47.2 643 52.9 Current living   Nuclear family 79 92.9 766 80.5 142 78.9 987 81.2 0.05   Extended family 5 5.9 157 16.5 30 16.7 192 15.8   Others 1 1.2 28 2.9 8 4.4 37 3.0 Location   Rural 43 50.6 626 65.8 122 67.8 791 65.0 0.01   Urban 42 49.4 325 34.2 58 32.2 425 35.0 Academic performance   Excellent (≥ 9/10) 11 13.1 143 15.1 59 33.0 213 17.6 <0.01   Good (8–< 9/10) 22 26.2 399 42.1 69 38.5 490 40.5   Fair (7–< 8/10) 32 38.1 264 27.8 36 20.1 332 27.4   Moderate and below (<7/10) 19 22.6 142 15.0 15 8.4 176 14.5 Number of family members can confide in   None 3 3.5 31 3.3 0 0.0 34 2.8 0.19   One 61 71.8 675 71.0 131 72.8 867 71.3   Two and above 21 24.7 245 25.8 49 27.2 315 25.9 Ignore advice of parents   No/unknown 7 8.2 350 36.8 101 56.1 458 37.7 <0.01   Yes 78 91.8 601 63.2 79 43.9 758 62.3 Parents limit my Internet access   No/unknown 18 21.2 333 35.0 63 35.0 414 34.0 <0.01   Yes, a little 37 43.5 498 52.4 84 46.7 619 50.9   Yes, a lot 30 35.3 120 12.6 33 18.3 183 15.0 Mean SD Mean SD Mean SD Mean SD Value of p Time using the internet per day (hour) 5.6 3.3 3.9 2.5 4.0 2.6 4.1 2.6 <0.01 The frequency of using internet (range: 4–28) 16.5 4.6 14.3 4.4 13.2 4.7 14.3 4.5 <0.01   Family (range: 1–7) 5.5 0.9 5.3 1.1 5.1 1.4 5.3 1.1   0.03   School (range: 1–7) 3.0 1.9 2.3 1.7 2.1 1.4 2.3 1.7 <0.01   Friend’s house (range: 1–7) 3.8 1.8 3.5 1.8 3.0 1.8 3.5 1.8 <0.01   Public area (range: 1–7) 4.2 2.1 3.1 1.7 3.0 1.7 3.1 1.8 <0.01 Multidimensional Scale of Perceived Social Support (Unit: 1 score)   Social support score in friend domain 3.4 1.0 4.0 0.8 4.1 0.9 3.9 0.8 <0.01   Social support score in family domain 3.4 1.0 3.6 0.7 3.8 0.8 3.6 0.8 <0.01   Social support score in other domains 3.7 0.9 3.8 0.8 3.7 0.9 3.7 0.8 <0.01 TABLE 1  |  Characteristics of relationship between parents and children since using internet. The Management and Care of Parents With Children’s Internet Useh Characteristics Parent–child relationships since using the internet Total Value of p Deteriorates Constant Better Mean SD Mean SD Mean SD Mean SD Topics and frequency of parent–child communication (range: 1–5)   Learning 2.9 1.1 3.5 0.9 3.9 0.9 3.5 1.0 <0.01   Housework 3.2 1.1 3.3 1.1 3.4 1.3 3.3 1.1 0.14   Future orientation 2.9 1.3 3.1 1.2 3.6 1.3 3.2 1.3 <0.01   Relationship with Friend 2.8 1.0 2.8 1.1 3.3 1.2 2.9 1.1 <0.01   Daily living habits 3.0 1.4 2.9 1.2 3.1 1.3 2.9 1.3 0.04   Hobby 2.3 1.2 2.7 1.2 3.3 1.3 2.8 1.3 <0.01   The selection of clothing 2.4 1.1 2.7 1.1 3.0 1.2 2.7 1.1 <0.01   The problem of pocket money 2.8 1.3 2.4 1.2 2.3 1.2 2.4 1.2 0.01   Privacy at home 2.6 1.4 2.2 1.3 2.7 1.4 2.3 1.3 <0.01 Interactive and supportive activities regarding internet use between parents and children (range: 1–5)   Helping parents when they have difficulty using Internet 3.2 1.3 3.5 1.2 3.9 1.1 3.5 1.2 <0.01   Ask for help from parents when the situation is difficult to handle on the Internet 1.8 0.9 2.1 1.2 2.6 1.4 2.1 1.2 <0.01   Ask for advice from parents on what to do on the Internet 1.5 0.6 2.0 1.1 2.6 1.4 2.1 1.4 <0.01   Talk about what you do on the Internet with parents 1.8 1.0 2.1 1.1 2.7 1.4 2.1 1.1 <0.01   Talk to parents about what’s bothering them on the Internet 1.5 0.8 2.0 1.1 2.5 1.4 2.0 1.1 <0.01 TABLE 2  |  Topic, frequency communication and interactive activities regarding internet use between parents and children. TABLE 3  |  The parent’s management and care with internet use in their children. TABLE 3  |  The parent’s management and care with internet use in their children. The Management and Care of Parents With Children’s Internet Useh Characteristics Parent-children relationships since using the internet Total Value of p Deteriorates Constant Better Mean SD Mean SD Mean SD Mean SD Parent’s management with internet use in children (range: 1–5)   Rules about how long I am allowed to go online 2.9 1.5 2.5 1.3 2.6 1.3 2.6 1.3 0.14   Rules about when I am allowed to go online 2.6 1.3 2.2 1.2 2.5 1.3 2.3 1.2 <0.01   Knows about what I look at on the internet and social media 2.3 1.1 2.3 1.1 2.6 1.4 2.3 1.2 0.01   Rules about who I can contact on social networks 2.5 1.4 2.0 1.2 2.3 1.4 2.1 1.3 <0.01   Checks my messages on the social media 2.0 1.0 1.6 0.9 1.9 1.2 1.7 1.0 <0.01   Checks my information on the Internet 1.9 1.0 1.7 1.0 2.0 1.3 1.7 1.3 <0.01   Technology to track where I am (such as GPS) 2.4 1.3 1.5 0.9 1.8 1.1 1.6 1.0 <0.01   Check out content what I look at on the internet 1.9 1.1 1.6 0.9 1.8 1.2 1.6 1.0 0.01   Check out adding my friends and friends list 1.3 0.7 1.4 0.8 1.8 1.2 1.5 0.9 <0.01   Limits content of what I can see on the internet 1.6 1.0 1.4 0.8 1.7 1.3 1.4 0.9 <0.01 Parent’s care with internet use in children (range: 1–5)   Explains why some online content is good or bad 3.1 3.2 3.2 1.2 3.4 1.4 3.2 1.4 0.02   Encourages me to explore and learn things on the internet 2.4 1.3 2.8 1.3 3.3 1.4 2.8 1.3 <0.01   Talks to me about what I do on the internet 2.9 1.0 2.6 1.1 2.8 1.3 2.6 1.1 <0.01   Suggests ways to use the internet safely 2.2 1.1 2.5 1.3 2.7 1.4 2.5 1.3 0.03   Helps me when something bothers me on the internet 2.3 1.4 2.4 1.3 3.3 1.4 2.5 1.4 <0.01   Talks to me about what to do if something online bothers or upsets me 2.4 1.2 2.3 1.2 2.8 1.4 2.4 1.3 <0.01   Helps me when something is difficult to do or find on the internet 1.8 0.9 2.2 1.2 2.7 1.6 2.3 1.3 <0.01   Does shared activities with me on the internet 1.9 1.3 2 1.1 2.6 1.2 2.1 1.1 <0.01 Regarding parents’ care with internet use in children, the group having better parent–child relationship gave higher scores about all items than the others, excepted “talks to me about what I do on the internet.” In contrast, the group having deteriorates relationship with parents showed the item of “talks to me Regarding parents’ care with internet use in children, the group having better parent–child relationship gave higher scores about all items than the others, excepted “talks to me about what I do on the internet.” In contrast, the group having deteriorates relationship with parents showed the item of “talks to me media (2.6 points), and setting the rules of how long/when they were allowed access internet. The Management and Care of Parents With Children’s Internet Useh The parent’s management and care with the children’s internet use were shown in Table  3. People having deteriorated and better relationships with parents showed high levels of parental February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 5 Parent-Child Relationship and Internet Use Nguyen et al. media (2.6 points), and setting the rules of how long/when they were allowed access internet. Less managed activities included checking access contents and friends on the internet, and restricting what they can see on the Internet in all three groups. The differences were statistically significant with p < 0.05. Regarding parents’ care with internet use in children, the group having better parent–child relationship gave higher scores about all items than the others, excepted “talks to me about what I do on the internet.” In contrast, the group having deteriorates relationship with parents showed the item of “talks to me TABLE 2  |  Topic, frequency communication and interactive activities regarding internet use between parents and children. The Management and Care of Parents With Children’s Internet Useh Characteristics Parent–child relationships since using the internet Total Value of p Deteriorates Constant Better Mean SD Mean SD Mean SD Mean SD Topics and frequency of parent–child communication (range: 1–5)   Learning 2.9 1.1 3.5 0.9 3.9 0.9 3.5 1.0 <0.01   Housework 3.2 1.1 3.3 1.1 3.4 1.3 3.3 1.1 0.14   Future orientation 2.9 1.3 3.1 1.2 3.6 1.3 3.2 1.3 <0.01   Relationship with Friend 2.8 1.0 2.8 1.1 3.3 1.2 2.9 1.1 <0.01   Daily living habits 3.0 1.4 2.9 1.2 3.1 1.3 2.9 1.3 0.04   Hobby 2.3 1.2 2.7 1.2 3.3 1.3 2.8 1.3 <0.01   The selection of clothing 2.4 1.1 2.7 1.1 3.0 1.2 2.7 1.1 <0.01   The problem of pocket money 2.8 1.3 2.4 1.2 2.3 1.2 2.4 1.2 0.01   Privacy at home 2.6 1.4 2.2 1.3 2.7 1.4 2.3 1.3 <0.01 Interactive and supportive activities regarding internet use between parents and children (range: 1–5)   Helping parents when they have difficulty using Internet 3.2 1.3 3.5 1.2 3.9 1.1 3.5 1.2 <0.01   Ask for help from parents when the situation is difficult to handle on the Internet 1.8 0.9 2.1 1.2 2.6 1.4 2.1 1.2 <0.01   Ask for advice from parents on what to do on the Internet 1.5 0.6 2.0 1.1 2.6 1.4 2.1 1.4 <0.01   Talk about what you do on the Internet with parents 1.8 1.0 2.1 1.1 2.7 1.4 2.1 1.1 <0.01   Talk to parents about what’s bothering them on the Internet 1.5 0.8 2.0 1.1 2.5 1.4 2.0 1.1 <0.01 TABLE 3  |  The parent’s management and care with internet use in their children. The Management and Care of Parents With Children’s Internet Useh Characteristics Parent-children relationships since using the internet Total Value of p Deteriorates Constant Better Mean SD Mean SD Mean SD Mean SD Parent’s management with internet use in children (range: 1–5)   Rules about how long I am allowed to go online 2.9 1.5 2.5 1.3 2.6 1.3 2.6 1.3 0.14   Rules about when I am allowed to go online 2.6 1.3 2.2 1.2 2.5 1.3 2.3 1.2 <0.01   Knows about what I look at on the internet and social media 2.3 1.1 2.3 1.1 2.6 1.4 2.3 1.2 0.01   Rules about who I can contact on social networks 2.5 1.4 2.0 1.2 2.3 1.4 2.1 1.3 <0.01   Checks my messages on the social media 2.0 1.0 1.6 0.9 1.9 1.2 1.7 1.0 <0.01   Checks my information on the Internet 1.9 1.0 1.7 1.0 2.0 1.3 1.7 1.3 <0.01   Technology to track where I am (such as GPS) 2.4 1.3 1.5 0.9 1.8 1.1 1.6 1.0 <0.01   Check out content what I look at on the internet 1.9 1.1 1.6 0.9 1.8 1.2 1.6 1.0 0.01   Check out adding my friends and friends list 1.3 0.7 1.4 0.8 1.8 1.2 1.5 0.9 <0.01   Limits content of what I can see on the internet 1.6 1.0 1.4 0.8 1.7 1.3 1.4 0.9 <0.01 Parent’s care with internet use in children (range: 1–5)   Explains why some online content is good or bad 3.1 3.2 3.2 1.2 3.4 1.4 3.2 1.4 0.02   Encourages me to explore and learn things on the internet 2.4 1.3 2.8 1.3 3.3 1.4 2.8 1.3 <0.01   Talks to me about what I do on the internet 2.9 1.0 2.6 1.1 2.8 1.3 2.6 1.1 <0.01   Suggests ways to use the internet safely 2.2 1.1 2.5 1.3 2.7 1.4 2.5 1.3 0.03   Helps me when something bothers me on the internet 2.3 1.4 2.4 1.3 3.3 1.4 2.5 1.4 <0.01   Talks to me about what to do if something online bothers or upsets me 2.4 1.2 2.3 1.2 2.8 1.4 2.4 1.3 <0.01   Helps me when something is difficult to do or find on the internet 1.8 0.9 2.2 1.2 2.7 1.6 2.3 1.3 <0.01   Does shared activities with me on the internet 1.9 1.3 2 1.1 2.6 1.2 2.1 1.1 <0.01 TABLE 2  |  Topic, frequency communication and interactive activities regarding internet use between parents and children. The Management and Care of Parents With Children’s Internet Useh Less managed activities included checking access contents and friends on the internet, and restricting what they can see on the Internet in all three groups. The differences were statistically significant with p < 0.05. Frontiers in Psychology | www.frontiersin.org February 2022 | Volume 13 | Article 847278 6 Parent-Child Relationship and Internet Use Nguyen et al. about what I  do on the internet” reached highest score than the other groups (2.9 points); the remaining items almost had the lowest scores compared to the remaining groups. Overall, people having a better relationship with parents had significantly higher scores on most parental care items questions, except the “Talks to me about what I  do on the internet” question. These differences were statistically significant with p < 0.05. parents. It also implied that active family communication significantly affected the healthy interaction of Internet access and positive personal behavior.hi This study was conducted during the first year of the COVID-19 epidemic in Vietnam; the government took proactive measures to control its spread which included school closures nationwide (La et  al., 2020). Children and adolescents were promoted to take classes as well as socialize more online. During the stay-at-home period, especially in the condition of parental supervision, spending more time on virtual platforms can make a young person feel upset, uncomfortable, and the parent–child relationship may become more stressed (Tran et  al., 2020; Cuong et  al., 2021). Therefore, our results found that a part of participants reported perceiving relationships with parents deteriorated related to using the internet. This issue echoed the previous findings found that children’s internet usage influences the amount of time spent with family (Jackson et  al., 2003). Moreover, our study also discovered that young people who had a deteriorated parent–child relationship since using the internet were likely to confide less and ignore parents’ advice. Negative parent–child communication was found to have a significant association with the likelihood of Internet addiction (Xu et al., 2014; Venkatesh et al., 2019). Strict parental rules about internet access may promote compulsive tendencies in children, which leads to the children’s opposition status and decreased frequency of parental communication regarding internet use (van Den Eijnden et al., 2010). Good communication regarding children’s internet use has been found to be  a promising tool that parents can use to prevent their teenage children from developing compulsive Internet use (van Den Eijnden et  al., 2010). Identifying Factors Related to the Relationship Between Parents and Children Since Using the Internet Factors related to the relationship between children and their parents since using the internet were shown in Table  4. Individuals in Hung Yen province had a higher likelihood of having deteriorated relationships with their parents (OR = 9.59; 95%CI: 3.25; 28.33), meanwhile, the opposite was true for Thanh Hoa province (OR = 0.18; 95%CI: 0.05; 0.66). Females, high school students, those having ignored advice from using the internet from parents, and those having higher time using the internet per day were more likely to have deteriorated relationships with their parents. In terms of children’s communication with their parents, a higher frequency of talking to parents about learning (OR = 0.29; 95%CI: 0.19; 0.46), selecting of clothing (OR = 0.34; 95%CI: 0.22; 0.51), as well as the higher score of “helping parents when they have difficulty using the internet” (OR = 0.64; 95%CI: 0.45; 0.92), and “ask for advice from parents on what to do on the Internet” (OR = 0.43; 95%CI: 0.23; 0.80) were positively associated with the relationship between children and parents. Regarding parental management and care with internet use in children, higher score of some parent’s activities such as talking to children about what they do on the internet (OR = 1.71; 95%CI: 1.10; 2.66), helps children when something bothers them on the internet (OR = 2.23; 95%CI: 1.46; 3.39), and using technology to track where children are (OR = 6.92; 95%CI: 3.93; 12.16), rules about who children can contact (OR = 1.82; 95%CI: 1.24; 2.68)/when children are allowed to go online (OR = 1.93; 95%CI: 1.28; 2.93), and knows about what I look at on the internet were increased likelihood of having deteriorated relationships between parent and children. The Management and Care of Parents With Children’s Internet Useh Some studies have also suggested that parent–child communication, which can be enhanced by having appropriate parental care and supports (Svetaz et  al., 2014; Jaggers et  al., 2015; Shek et  al., 2019) may be  a protective factor against problematic internet use in adolescents (Kim and Kim, 2003). It is worth noting that, seemingly positive parental control strategies like “talk to children about what they do on the internet” or “help their children when something bothers them on the internet” can be  associated with parent– child conflict, as our study found. This may be  due to the tendency of young people to express their concerns to people rather than their parents, possibly because of existing lack of parent–child communication (Văn, 2018). Thus, having appropriate parental control strategies is essential in regulating health internet usage in children, avoiding the vicious cycle of negative control—impaired parent–child communication— deteriorated parent–child relationship—unmet psychological needs—more excessive internet use (Wang et  al., 2007; Wang and Fredricks, 2014; Lam, 2020). In Vietnamese society, the notion of “thuong cho roi cho vot,” which means “spanking (with a rod) out of love Frontiers in Psychology | www.frontiersin.org DISCUSSION This research has deepened the understanding of the relationship between parent–child relationship and children’s online behavior. Parental controls or supports of children’s internet use not only affect the behaviors of the children but can also have positive or negative consequences in the relationship between family members. Our results confirmed that parental control behaviors of their children’s internet use might affect the children’s excessive internet use, academic performance results, psychological status as well as the openness of communications and sharing between parents and children. On the other hand, our research found that participants who received encouragement, help, and support from their parents when using the Internet were more likely to establish better relationships with their In Vietnamese society, the notion of “thuong cho roi cho vot,” which means “spanking (with a rod) out of love for the child” suggests that strict discipline was considered a cultural norm for generations (Vu, 2016; Vuong et  al., 2018). Therefore, our findings indicated that Vietnamese parents tend to set diehard control strategies such as using GPS tracking on their children’s devices, establishing rules about contacts, or allowing online. The majority of parents February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 7 Parent-Child Relationship and Internet Use Nguyen et al. TABLE 4  |  Logistic Regression for identifying factors related to the relationship between parents and children since using the internet. Characteristics The relationship between parents and children since using internet (Deteriorates = 1 vs. Constant/Better = 0) OR 95%CI INDIVIDUAL CHARACTERISTICS   Province (Vung Tau—Ref)     Can Tho 0.45 0.15; 1.35     Hung Yen 9.59*** 3.25; 28.33     Thanh Hoa 0.18*** 0.05; 0.66   Gender     Female 3.78*** 1.46; 9.77   Grade (vs. secondary school)     High school 7.04*** 2.34; 21.19   Ignored advice about using the internet from parents (Yes vs. ***p < 0.01; **p < 0.05; *p < 0.1. DISCUSSION No) 10.25*** 2.73; 38.54   The frequency of using internet (per score) 1.00 0.90; 1.11   Time using the internet per day (hour) 1.32*** 1.11; 1.57 PARENT-CHILDREN COMMUNICATION SINCE USING THE INTERNET   Frequency of talking to parents (per score)     Learning 0.29*** 0.19; 0.46     The selection of clothing 0.34*** 0.22; 0.51     Future orientation 0.78 0.53; 1.14     The problem of pocket money 2.09*** 1.45; 3.00   Children’ activities with their parents on the internet (per score)     Helping parents when they have difficulty using the Internet 0.64** 0.45; 0.92     Ask for advice from parents on what to do on the Internet 0.43*** 0.23; 0.80 THE PARENT’S MANAGEMENT AND CARE WITH INTERNET USE IN CHILDREN   Parent’s care with Internet Use in Children (per score)     Talks to me about what I do on the internet 1.71** 1.10; 2.66     Helps me when something bothers me on the internet 2.23*** 1.46; 3.39     Helps me when something is difficult to do or find on the internet 0.67* 0.44; 1.03     Parent’s management with Internet Use in Children (per score)     Technology to track where I am (such as GPS) 6.92*** 3.93; 12.16     Checks my messages on the social media 0.42*** 0.23; 0.76     Rules about who I can contact on social networks 1.82*** 1.24; 2.68     Rules about when I am allowed to go online 1.93*** 1.28; 2.93     Knows about what I look at on the internet and the social media 1.87** 1.14; 3.06     Check out adding my friends and friends list 0.13*** 0.05; 0.29 SOCIAL SUPPORT   Multidimensional Scale of Perceived Social Support     Social support from family (per score) 0.64* 0.38; 1.08 ***p<0 01; **p<0 05; *p<0 1 TABLE 4  |  Logistic Regression for identifying factors related to the relationship between parents and children since using the internet. TABLE 4  |  Logistic Regression for identifying factors related to the relationship between parents and children since using the internet. The relationship between parents and children since using internet (Deteriorates = 1 vs. Constant/Better = 0) Frontiers in Psychology | www.frontiersin.org SOCIAL SUPPORT Social support from family (per score) ***p < 0.01; **p < 0.05; *p < 0.1. disciplinary strategies, and setting regulative rules to regulate children’s behavior has to be  associated with positive developmental results in children (Barber et  al., 2005; Bleakley et  al., 2016). regard spending more time on the internet as a distraction from study and a transgression that warrants discipline (Cappa and Dam, 2014). However, the current results proved the diehard parental control strategies negatively influence the parent–child relationship: the higher magnitude and frequency of such control, the worse the parent–child relationship. Negative parental control, which mostly manifests in the form of psychological control through strategies of guilt induction and love withdrawal aiming to make children’ dependent emotion on their parents (Steinberg et  al., 1989; Siomos et  al., 2012), has been found to harm teenagers’ emotional functioning and the sense of self (Wang et  al., 2007), impair the children’ self-esteem and exacerbate developmental difficulties and maladjustment in youths and adolescents (Barber and Harmon, 2002). In contrast, positive parental control in form of behavioral control through monitoring, taking interest in children’s activities, developing Changes in the quality of the parent–child relationship were self-assessed by participants showed improvement or deterioration since youngsters used the internet. The mindsponge mechanism, which was proposed by Vuong and Napier (2015) and Vuong (2016), may be  used to explain how and why parents “accept or not accept” their kids’ behaviors when using the internet. Parents’ awareness and mindset play crucial factors in accepting children’s behaviors of using online platforms including learning, exchanging, and entertaining via the internet. Additionally, belief in their kids is determined as a critical priority card in the mindsponge mechanism because it encouraged parents in promoting support, sharing information, and communicating with their children in participating in social networking activities February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 8 Parent-Child Relationship and Internet Use Nguyen et al. use behaviors, which might result from general family relations such as quality of parent–child attachment. Participants suggested that their family relationship quality had deteriorated since using the internet, which led to decreasing academic outcomes, loneliness, and depression. Stubborn parental control factors, such as establishing rules about contacts or allowing online, setting up GPS tracking can have a negative impact on the quality of parent–child relationships. SOCIAL SUPPORT Meanwhile, parent-adolescent communication quality, frequency (talking about learning, selection of clothing, and future orientation), and bidirectional interactive activities such as supporting their parents for internet use, affected positively the relationship between children and parents. The present research findings are of importance for future research on parent–children relationship quality and internet use in developing countries. Simultaneously, intervention targets the negative impacts of the internet on young Vietnamese people and increases parent-child communication quality also mentioned in this works. (Vuong and Napier, 2015). The positive parent–child relationship might provide a solid emotional foundation that encourages positive living and prevents internet addiction in children (Shek, 2010; Floros and Siomos, 2013).hi The research findings are of importance for future scholarly works on the parent–children relationship related to using the internet in developing countries, especially in the context of prolonged COVID-19 pandemic. Our findings provide insights for developing evidence-based interventions aiming to lessen the negative impacts of the internet on young Vietnamese people and increase the quality of parent–child communication. First, the intervention programs should prioritize improving parental knowledge, awareness, and attitude about the positive and negative influences of using online platforms (e-learning, exchange, and entertainment), and enhancing parent–child communication. Second, there should be psychological training programs for parents so that they can understand the psychology of children’s behavior and have appropriate responding strategies. Third, a sense of self-responsibility among youths and adolescents should be  encouraged through citizen empowerment and emphasizing responsible behavior, so that young people can protect themselves online. Fourth, future studies can benefit from delving deeper into the association between parent–child relationship and children’s internet use by, for instance, exploring the specific roles of father and mother in family relationship quality and their children’s internet use. Last, regardless of the solutions used by parents or educators for reducing the negative effects of the internet on young people and improving the parent–child relationship, it is critical to strictly practice in a disciplined process for an extended period of time until beneficial results are obtained (Vuong et  al., 2022). ETHICS STATEMENT All procedures performed in studies involving human participants were in accordance with the ethical standards of the Youth Research Institute, Vietnam, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The patients/participants provided their written informed consent to participate in this study. This study should be  viewed in light of its limitations. First, the study employed a cross-sectional design, which did not allow for drawing causal conclusions between parent–children relationship quality and associated factors. Second, the use of an online survey platform might lead to lower response rates as well as lower reliability and validity of the data compared to that of the traditional interview method. Even so, in the context of COVID-19 pandemic and resource-scarce setting of Vietnam, the authors believe that online survey is the optimal approach to reach a large sample size in a short period. Furthermore, in order to limit selection bias, we  conducted a pilot survey and tried to present the questionnaire in the most convenient way for participants in the investigation period. Lastly, due to the length constraint on the online platform, we could not measure factors such as individual characteristics of children and parents, or family context; cultural factors inherently influence the internet use (awareness, habits to experiences, and practices). It is recommended that future studies consider evaluating these aspects in order to possibly draw more objective conclusions from the quality of the two-way relationship between parents and children. AUTHOR CONTRIBUTIONS TTPN, HND, TV, HMD, NN, CL, CH, and RH: conceptualization. TTPN, TTN, LD, and GV: data curation. TTPN and TTN: formal analysis. TTPN, TTN, LD, GV, and SN: methodology. HTD, CL, CH, and RH: supervision. TTPN, SN, LD, and TTN: investigation. TTPN, TTN, GV, and HTD: writing— original draft. TTPN, TTN, HND, TV, KV, HMD, NN, LD, GV, HTD, SN, CL, CH, and RH: writing—review and editing. All authors contributed to the article and approved the submitted version. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be  made available by the authors, without undue reservation. REFERENCES A., and Ebrahem, G. G. S. (2016). The relationship between use of technology and parent-adolescents social relationship. J. Educ. Pract. 7, 168–178. 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Introduction OARSI treatment guidelines.22 Viscosupplementa­ tion consists in intra-articular, single or repeated, injections of HA or HA-based derivatives (HAs). Adequate viscoelastic properties are also important to restore the articular homeostasis.23 HA or HAs increase the elastoviscosity of the intercellular ma­ trix creating a mechanical protective socket that surrounds nociceptive nerve endings. This results in a decrease in pain and an increase in joint mobility24 as it has already been demonstrated on animals models.25,26 OARSI treatment guidelines.22 Viscosupplementa­ tion consists in intra-articular, single or repeated, injections of HA or HA-based derivatives (HAs). Adequate viscoelastic properties are also important to restore the articular homeostasis.23 HA or HAs increase the elastoviscosity of the intercellular ma­ trix creating a mechanical protective socket that surrounds nociceptive nerve endings. This results in a decrease in pain and an increase in joint mobility24 as it has already been demonstrated on animals models.25,26 Osteoarthritis (OA) is a chronic, progressive musculoskeletal disorder1–3 that mainly affects the weight-bearing chondro-synovial joints such as knee, hip4 and the upper extremities.5,6 The disease affects the ageing population worldwide7,8 leading to a high socio-economic burden9 with substantial financial consequences.10,11 In a healthy condition, the lubricants present in the synovial fluid, such as Hyaluronic acid (HA), lubricin12 and the surface-active phospholipids (SAPL)13,14 act as shock absorbers protecting the joint against compressive damage.15–17 Hyaluronic acid (HA) is responsible for the viscoelastic proper­ ties.18 One of the most important drawbacks of HA and HAs is the multiple injection therapy due to the enzymatic degradation by hyaluronidases in the body. High molecular weight products and cross- linked hydrogels have been developed, but they are able to retard the hyaluronidases attack without pre­ venting it.27 Furthermore, inflammatory reactions sometimes accompanied by swelling and erythema have also been variably reported.28,29 In OA joint diseases, the concentration and mo­ lecular weight of HA decrease16,19 and the synovial fluid becomes more abundant, less viscous and re­ duced in elastoviscous properties that may culmi­ nate in a painful condition for the patient. Rheological Studies Dedicated to the Development of a Novel Injectable Polymeric Blend for Viscosupplementation Treatment doi: 10.15255/CABEQ.2014.2148 Original scientific paper Received: November 15, 2014 Accepted: November 30, 2015 Viscosupplementation is an intra-articular symptomatic treatment of mild osteoar­ thritis. The treatment involves the injection of high-molecular-weight hyaluronan (HA), and especially of cross-linked HA to restore the lubricating and cushioning properties of the synovial fluid. This work involves the development of a novel viscosupplementation fluid based on amidated carboxymethylcellulose and obtained by blending the soluble polymer with its crosslinked derivative. Rheological analyses carried out under both oscillatory and con­ tinuous shear provided a rationale to assess the viscosupplement formulation and the production process. The hydrogel fraction content and the total polymer concentration can be properly selected in order to ensure an optimal combination of flowability and viscoelastic properties. Key words: amidated carboxymethylcellulose, viscosupplementation, polymeric blend, hydrogel, rheology amidated carboxymethylcellulose, viscosupplementation, polymeric blend, hydrogel, rheology *Corresponding author: romano.lapasin@di3.units.it 511 511 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) R. LAPASIN et al., Rheological Studies Dedicated to the Develo Introduction A new derivative similar to HA, but with a bet­ ter behaviour in terms of hardness and stability to­ wards enzymatic degradation30 has been developed converting 50 % of the carboxylic groups of car­ boxymethylcellulose (CMC) into amidic groups to obtain the amidated carboxymethylcellulose (CMCA).31 In vitro results showed that the amida­ tion significantly ameliorated the biocompatibility of the material and diminished the cell expression Viscosupplementation20,21 was identified as an effective treatment of OA with a small number of adverse effects. Nowadays, it is approved in Eu­ rope/USA for the treatment of pain associated with OA of the knee, but considerable controversy about its efficacy is still ongoing as evidenced in the 512 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) of inflammatory cytokines. CMCA also positively influenced osteoblast activity.30 The vegetal origin of CMCA potentially avoids the risk of allergic re­ actions due to certain protein components present in the HA and Ha-based products injected. molar ratio with the polymer was 0.5:1 to obtain a crosslinking degree of about 50 %. The obtained hydrogel was purified by repeated rinsing in puri­ fied water and freeze dried. The two freeze-dried components, polymer and hydrogel, were milled in order to have a controlled cut off, up to 2 mm, that were subsequently blended and hydrated in saline solution, and eventually filled in syringe and steril­ ized. This work involves the development of a novel viscosupplementation fluid obtained by blending the soluble CMCA polymer (hereinafter indicated as CMCAp) with its crosslinked hydrogel deriva­ tive (CMCAg) by crosslinking the residual 50 % fraction of CMCAp carboxylic groups. Materials The raw material for CMCA preparation is so­ dium carboxymethylcellulose (average molecular weight: 750 kDa, substitution degree 0.8) (CP Kelko). It can be functionalized thanks to the pres­ ence of two carboxyl groups in the polymeric unit through a water-based synthetic pathway. The car­ boxylic groups are first activated with NHS/EDC (N-hydroxysuccinimide purity >97 % (Sigma-Al­ drich); N-(3-dimethylaminopropyl)-N’-ethylcarbo­ diimide hydrochloride purity >99 % (Actim)). Once activated, they quickly react with methylamine (40 % water solution, Sigma-Aldrich), molar ratio of 1:1. The pH of the solution is always kept at ­4.5–5 adjusting hydrochloric acid. The amide-CMCA was then purified in a buffered solution (about pH 8–8.5 in order to increase the solubility of the poly­ mer) through a Tangential Flow Filtration instru­ ment using a membrane with 100 kDa cut off. The obtained solution was separated into two aliquots: the former was freeze-dried, the latter was used for the crosslinking process. Recovery tests were performed in order to evaluate the structural recovery after the application of continuous shear conditions. They were com­ posed of three consecutive segments, alternatively under small amplitude oscillatory conditions (at 1 Hz) and in continuous shear at constant shear rate (5 s–1, 180 s). The steady values of G’ and G” at­ tained in the oscillatory segments before and after shear flow were compared. All the experimental tests were done in dupli­ cate or triplicate. Rheological experiments CMCA-based fluids were investigated to assess the rheological properties of the viscosupplement fluid during the different processing stages. Both continuous and oscillatory shear tests showed that, at equal polymer concentration, aqueous CMCAp and CMCAg systems exhibit quite different rheo­ logical behaviours, typical of polymer solutions and gels, respectively, and hence a wide arc of viscous and viscoelastic responses can be obtained by blending the two polymers in different proportions. The gel fraction (CMCAg/CMCAp ratio) and the total polymer concentration of the blend can be ap­ propriately selected in order to ensure an optimal combination of viscoelastic properties and flow be­ haviour (injectability) together with an acceptable structural recovery after shear. Rheological characterization was carried out with a rotational controlled stress rheometer (Rheostress RS150, Haake GmbH, Germany), equipped with a cone/plate device (d = 60 mm, an­ gle = 1°) or a parallel plate geometry (d = 35 mm, gap = 1 mm) with serrated surfaces in order to avoid wall slippage effects. The apparatus was comput­ er-controlled (RheoWin software program) and the temperature was kept at 25 ± 0.1 °C (Thermostat TC500, Haake GmbH, Germany). The flow proper­ ties were determined applying a logarithmic se­ quence of increasing constant stresses in the range 1-1000 Pa and measuring the corresponding shear rate. Each stress value was kept constant until the corresponding time variation of the shear rate satis­ fied the pre-set constraint for steady state attain­ ment, or at most for 90 s. Oscillatory stress sweep tests were performed at 1 Hz in order to individuate the extension of the linear viscoelasticity regime. All the frequency sweep measurements were performed from 100 to 0.01 Hz within the linear range. Data treatment and modeling The experimental viscosity data can be cor­ related quite satisfactorily with the Carreau-Yasuda model:32,33 η η η η γ = + − + ∞ ∞ 0 1[ ( ) ] a b p  The amide-CMC polymer is crosslinked by a solution of NHS/EDC and a bi-functional group. In particular, 1,3-diaminopropane (purity 99.0 %, Sig­ ma-Aldrich) was used as crosslinking agent. The where h0 and h∞ represent the zero-shear rate vis­ cosity and the infinite-shear rate viscosity, respec­ tively. In this study, the high shear Newtonian pla­ 513 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) LAPASIN et al., Rheological Studies Dedicated to the Developm Fi g . 2 – Mechanical spectra of aqueous CMC and CMCAp 1 % w/w solutions teau is beyond the experimental window and, hence, h∞ was arbitrarily set equal to 0.89 mPa s, i.e. to the water viscosity at 25 °C. The product bp rules the slope of the flow curve in the power law region, while the critical shear rate gc marking the onset of shear thinning is strictly related to the characteristic time a. The mechanical spectra can be described with the generalized Maxwell model through the follow­ ing expressions: ′= + ′′= + = = ∑ ∑ G G G G i i i i N i i i i N ω λ ω λ ωλ ω λ 2 2 2 2 1 2 2 1 1 1 where Gi and li are the modulus and the relaxation time of the ith Maxwell element, respectively. In or­ der to reduce the problem related to parameter cor­ relation, a scaling criterion was adopted for the re­ laxation time sequence (li+1 = 10 li) and not more than 5 Maxwell elements were taken into consider­ ation for data fitting. Fi g . 2 – Mechanical spectra of aqueous CMC and CMCAp 1 % w/w solutions through the variations of two model parameters: the zero shear rate viscosity h0 and the critical shear rate gc. Results and discussion The changes induced by amidation on the lin­ ear viscoelastic behaviour of solutions were coher­ ent with those observed for their flow properties. At equal polymer concentration, the mechanical spec­ trum moved towards lower values of both moduli and the crossover point shifted to higher frequen­ cies because of the faster relaxation modes. The ex­ ample reported in Fig. 2 regards the 1 % solutions. These effects can be evaluated more precisely by comparing the relaxation time spectra obtained from data correlation with the Maxwell generalized model, as illustrated in Fig. 3. The effects produced by amidation on the rheo­ logical behaviour of CMC solutions appeared to be more evident at low shear conditions and higher polymer concentrations. Comparing the flow curves of CMC and CMCAp solutions at equal concentra­ tion, the zero-shear-rate viscosity diminished after amidation and the Newtonian plateau became more extended towards higher shear conditions, as it can be observed in Fig. 1. Similar rheological effects of amidic moieties on carboxymethylcellulose have been observed by Barbucci et al.30 More drastic changes were produced by poly­ mer crosslinking, particularly in linear viscoelastic conditions; this can be seen by comparing the re­ A satisfactory data correlation was provided by the Carreau-Yasuda equation. Accordingly, the above underlined effects can be concisely described Fi g. 1 – Steady viscosity curves of aqueous CMC and CMCAp solutions at different concentrations (expressed in % w/w) Fig. 3 – Relaxation time spectra of aqueous CMC and CMCAp 1 % w/w solutions derived from data correlation with the Maxwell generalized model Fi g. 1 – Steady viscosity curves of aqueous CMC and CMCAp solutions at different concentrations (expressed in % w/w) Fig. 3 – Relaxation time spectra of aqueous CMC and CMCAp 1 % w/w solutions derived from data correlation with the Maxwell generalized model Fi g. 1 – Steady viscosity curves of aqueous CMC and CMCAp solutions at different concentrations (expressed in % w/w) Fig. 3 – Relaxation time spectra of aqueous CMC and CMCAp 1 % w/w solutions derived from data correlation with the Maxwell generalized model R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) 514 514 R. LAPASIN et al., Rheological Studies Dedicated to the D Fi g. Results and discussion 4 – Viscoelastic responses of 2.5 % CMCAp and 2.5 % CMCAg systems; a) storage and loss moduli vs strain (from stress sweep test); b) mechanical spectra (from frequency sweep test) sions). For CMCAg systems, the critical strain marking the borderline of the linear viscoelastic re­ gime became sensibly lower (two orders of magni­ tude or more) than for CMCAp solutions. The quite different profiles of the mechanical spectra of CMCAp and CMCAg systems reflect the neat changes produced by crosslinking in linear vis­ coelastic properties and relaxation modes, thus evi­ dencing the structural transition from solution to physical gel (Fig. 4b). In the experimental window explored, the behaviour of the CMCAp solution was characterized by a prevailing viscous compo­ nent (G”>G’) and short relaxation times, as it can be deduced by the crossover frequency. In the case of the crosslinked polymer, the storage modulus curve is placed clearly above G” values, and reach­ es a plateau condition at low frequencies, where the divergence from the linear viscoelastic responses of CMCAp is quite evident. Studying the steady flow properties, we found that CMCAg systems exhibited much more pro­ nounced shear thinning than CMCAp solutions, ow­ ing to their different structural conditions and shear-induced mechanisms. CMCAg systems be­ have as dispersions of gel microdomains, which may undergo significant deformations and spatial rearrangements with increasing shear rate. Looking at the problems connected to the de­ velopment of a novel polymeric fluid for visco­ supplementation treatment, the first decisive step concerns its formulation in relation to the structural and rheological properties that the fluid must pos­ sess and exhibit. In particular, it should be easily injectable and display appropriate viscoelastic re­ sponses ‘in situ’ in the various kinematic condi­ tions  experienced by articular joints. Furthermore, an adequate resistance to enzymatic degradation and mechanical solicitations for prolonged times should be ensured. Blending CMCAp solutions and CMCAg microgel dispersions it is possible to finely adjust the composition ratio and the total polymer concentration in order to modulate the structural features and rheological properties of both systems. Fi g. 4 – Viscoelastic responses of 2.5 % CMCAp and 2.5 % CMCAg systems; a) storage and loss moduli vs strain (from stress sweep test); b) mechanical spectra (from frequency sweep test) sponses of both of CMCAp and CMCAg systems with the same concentration of soluble and cross­ linked polymer (2.5 % for systems represented in Figs. Results and discussion 4a and 4b, respectively). Stress sweep tests showed that both viscoelas­ tic moduli of the CMCAg system were markedly higher, and in the nonlinear region, their profiles were different from those of the corresponding CM­ CAp solutions (Fig. 4a). For the latter systems, the transition from linear to nonlinear regime is de­ scribed by monotonic decrease of both moduli. This behaviour is usually observed in ordinary polymeric solutions and labelled as type I according to the classification of LAOS responses.34 Conversely, in the case of CMCAg systems, the storage modulus decreased monotonically with increasing strain whereas a weak overshoot appeared in the G’’ pro­ file. Such a pattern (Type III) is typical of weakly structured fluids (physical gels or colloidal disper­ The following figures illustrate how the rheo­ logical properties of CMCAp-CMCAg blends changed with increasing gel fraction at equal total polymer concentration. The steady viscosity strong­ ly increased at low shear rates, while its change was almost negligible at high shear rates. As the gel fraction increased, the shear thinning character of the flow behaviour became more and more marked, being ruled by the level and extension of the New­ tonian plateau (Fig. 5). The experimental data can be correlated quite satisfactorily with the Carreau-Yasuda equation. The effects of increasing gel fraction fg on viscous R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) 515 ti f CMCA CMCA bl d b Fi g. 5 – Steady viscosity curves of 2.5 % CMCAp-CMCAg blends at different gel fractions (1 represents the polymer/gel ratio of 0/10; 0 represents a polymer/gel ratio of 10/0) Fi g . 7 – Mechanical spectra of 2.5 % CMCAp-CMCAg blends at different gel fractions (0 represents the polymer/gel ratio of 10/0; 1 represents a polymer/gel ratio of 0/10) Fi g . 7 – Mechanical spectra of 2.5 % CMCAp-CMCAg blends at different gel fractions (0 represents the polymer/gel ratio of 10/0; 1 represents a polymer/gel ratio of 0/10) Fi g. 5 – Steady viscosity curves of 2.5 % CMCAp-CMCAg blends at different gel fractions (1 represents the polymer/gel ratio of 0/10; 0 represents a polymer/gel ratio of 10/0) properties of CMCAp-CMCAg blends can be rea­ sonably described through the only variations of the zero-shear rate viscosity h0 and the critical shear rate gc, marking the border of the Newtonian plateau (Fig. 6). Results and discussion Thus, it can be noted that the most signifi­ cant changes occurred in the fg interval between 0.2 and 0.3, where gc drops significantly, and the rate of h0 increase became slower. At higher fg values, the increasing concentration of gel microdomains in the whole polymeric phase resulted only in an exponen­ tial h0 increase with no appreciable effect on the critical shear rate. Fi g . 7 – Mechanical spectra of 2.5 % CMCAp-CMCAg blends at different gel fractions (0 represents the polymer/gel ratio of 10/0; 1 represents a polymer/gel ratio of 0/10) served in the shape of the mechanical spectrum, passing from CMCAp solution to CMCAg system. According to the Winter-Chambon criterion35,36, the sol-gel transition could be located near fg = 0.3, since the traces of G’ and G’’ of system 7 were ­almost coincident and obey the same scaling law (G’    G’’    wn, n    0.45) in the experimentally ex­ plored frequency window. Similarly, the linear viscoelastic behaviour of the blend significantly changed with increasing gel fraction. To clarify, the graphical representation re­ ported in Fig. 7 involves only a partial subset of the mechanical spectra experimentally determined. As may be observed, the increasing gel fraction fg led to an increase in the elastic character of the visco­ elastic response, which was more pronounced at lower frequencies where the storage modulus in­ creased more markedly than the viscous compo­ nent. Consequently, a progressive change was ob­ The generalized Maxwell model provides a sat­ isfactory data correlation and, consequently, the re­ sulting relaxation time spectra can be profitably used for analysing the blending effects on the linear viscoelastic responses in the place of the mechani­ cal spectra (Fig. 8). As the CMCAg content of the polymeric phase increased, the relative weight of the slowest relaxation modes became more import­ Fig. 8 – Relaxation time spectra derived from data correlation with the generalized Maxwell model for 2.5 % CMCAp-CMCAg blends (1 represents the polymer/gel ratio of 0/10, 0 represents a polymer/gel ratio of 10/0) Fi g. 6 – Zero-shear rate viscosity and critical shear rate vs gel fraction in 2.5 % CMCAp-CMCAg blends Fig. 8 – Relaxation time spectra derived from data correlation with the generalized Maxwell model for 2.5 % CMCAp-CMCAg blends (1 represents the polymer/gel ratio of 0/10, 0 represents a polymer/gel ratio of 10/0) Fi g. Results and discussion Typical moist-heat sterilization cycles, based on exposition to temperature of 121 °C for 15’ have been applied, and the mechanical spectra obtained for unsterile and sterile blends of equal total poly­ mer concentration and gel fraction have been com­ pared. As highlighted in Fig. 9, it can be seen that steam sterilization induced a loss of both elastic and viscous moduli due to the polymer degradation, and hence a reduction in the main properties of the de­ vice. Consequently, the initial formulation must be readjusted in terms of total polymer concentration and gel fraction in order to compensate the negative effects produced by the manufacturing process on the rheological behaviour of the blend and to re­ store a correct balance among viscoelasticity, stabil­ ity and easy injectability. In fact rheological proper­ ties of numerous viscosupplements available on the market cover a wide spectrum of values and some­ times they are quite different from those peculiar to healthy synovial fluids.20,38–40 Among the several steps of the manufactur­ ing  process, some of them can strongly affect the elastoviscosity of the polymer blend. It has been ­observed that freeze drying, homogenization in ­saline solution and filling in syringes can produce a  drastic decrease in loss and storage moduli in both  soluble and crosslinked CMCA. Moreover, ­because viscosupplements are classified as phar­ maceutical products or medical devices, an essen­ tial  requirement for CMCA blends is represent­ ed  by  their capability to resist the sterilization process. This suggests that the main requirements for such devices are represented by their viscoelastic properties in order to exhibit a proper response to the stress conditions within synovial joints, together with their capability to be easily injected through an 18-20 G needle. It is also acknowledged that the values of the linear viscoelastic moduli measured at 0.5 and 2.5 Hz can represent a suitable way to eval­ uate how synovial fluid and viscosupplements react to walking and running conditions, respectively.20,41 Accordingly, they can serve to discriminate the be­ haviours of synovial fluids in normal and patholog­ ical conditions, as well as to assess the effects of viscosupplementation therapies.39,40,42 The experi­ mental values obtained for a series of commercial viscosupplements are reported in Fig. 10a. The Several techniques based on chemical agents (EtO, plasma) or physical processes (heating, b or γ radiation) are widely used for sterilization in the pharmaceutical and medical devices industries. Results and discussion 6 – Zero-shear rate viscosity and critical shear rate vs gel fraction in 2.5 % CMCAp-CMCAg blends Fig. 8 – Relaxation time spectra derived from data correlation with the generalized Maxwell model for 2.5 % CMCAp-CMCAg blends (1 represents the polymer/gel ratio of 0/10, 0 represents a polymer/gel ratio of 10/0) Fi g. 6 – Zero-shear rate viscosity and critical shear rate vs gel fraction in 2.5 % CMCAp-CMCAg blends 516 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) R. LAPASIN et al., Rheological Studies Dedicated Fi g . 9 – Reduction ratio of viscoelastic moduli between unster­ ile and sterile 2.5 % CMCAp-CMCAg blends vs gel fraction at two reference frequencies (0.5 and 2.5 Hz) ant owing to the increasing contribution of gel mi­ crodomains towards a structural state close to jam­ ming. The analysis of the reversibility of linear visco­ elastic responses (G’, G’’) can be particularly useful in view of the development of a novel viscosupple­ ment, which must be injected, and hence undergoes short but intense shear conditions in the application phase. Recovery tests performed on CMCAp-CMCAg blends showed that the viscoelastic behaviour was completely reversible for CMCAp solution, since the values of G’ and G” before and after shear flow were shown to be equal. On the other hand, only a partial recovery was attained for CMCAg system along the time segment following the application of a constant shear rate. The viscoelastic recovery de­ creased with increasing gel fraction. Fi g . 9 – Reduction ratio of viscoelastic moduli between unster­ ile and sterile 2.5 % CMCAp-CMCAg blends vs gel fraction at two reference frequencies (0.5 and 2.5 Hz) manufacturing process, in particular that caused by steam sterilization. Investigations were also carried out on the de­ velopment of a pilot production process and evalu­ ations were made on the potential effect of each processing step on the blend’s final properties. Standard production implies a set of consecutive steps to obtain both components of the polymeric blend: CMC solubilization, CMC functionalization in order to obtain CMCA, purification of CMCA, concentration and freeze-drying of the polymer, crosslinking of the CMCA, purification of the ob­ tained hydrogel, concentration and freeze-drying of the hydrogel. The final steps cover rehydration in saline solution, polymer and gel blending, filling in syringe and sterilization. Conclusions Blends with tailored viscoelastic properties can be obtained from CMCA in the form of polymer solutions and crosslinked hydrogels. More specifically, the entire range between 10/0 and 0/10 of polymer/hydrogel ratio has been investigated, and the 5/5-6/4-7/3 polymer/hydrogel compositions seem to create a behaviour very close to the main characteristics of an HA-based product for viscosupplementation. Fi g. 10 – a: Viscoelastic moduli (at 0.5 and 2.5 Hz) of the commercial viscosupplements listed below. b: Viscoelastic moduli (at 0.5 and 2.5 Hz) of the 6/4 CMCAp/CMCAg blends at 2.5 %, 3 % and 3.61 % w/w concentrations compared with those of two commercial products (Orthovisc and Monovisc). c: Viscosity values (at 0.1, 10 and 1000 s–1) of the 6/4 CMCAp/CMCAg blends at 2.5 %, 3 % and 3.61 % w/w concentrations compared with those of two commercial products (Orthovisc and Monovisc). Commercial viscosupplement This study has demonstrated the importance of both polymer and hydrogel form in blends, because, if singularly used, (10/0 and 0/10 of polymer/hydro­ gel samples), they are unable to preserve both stor­ age and loss moduli. pp Suplasyn (Mylan N.V., UK), non-heat degraded highly purified sodium hyaluronate (500 to 730 kDa), measured values, ref. 39.; Hyalubrix (Fidia farmaceutici S.p.A., Italy), sodium hyaluronate (1300 to 3600 kDa), from bacteria fermentation, estimated values, ref. 40.; Orthovisc (Anika Ther­ apeutics MA, USA), highly purified sodium hyaluronate in physiologic saline, measured values, ref. 39.; Monovisc (Anika Therapeutics MA, USA) highly purified, partially crosslinked sodium hyaluronate in a phos­ phate buffered saline solution, estimated values, ref. 40.; Noltrex (JSC “Research center “Bioform”, Russia), synthetic polyacrylamide hydrogel with silver ions, measured values in our laboratory; Hymovis (Fidia far­ maceutici S.p.A., Italy), partially hydrophobized derivative of hyaluronic acid, estimated values, ref. 40.; Synvisc (Hylan G-F 20) (Genzyme Bio­ surgery, USA). blend 80/20 of Hylan A (high molecular weight hyaluron­ ic acid) and Hylan B (crosslinked hyaluronic acid), measured values, ref. 39.: Durolane (Bioventus, USA), crosslinked hyaluronic acid, measured values in our laboratory.; Fermathron (Hyaltech Ltd. UK), sodium hyal­ uronate (1300 to 3600 kDa) in a phosphate buffered saline solution, from bacterial fermentation, measured values in our laboratory. Properties, such as lubrication and shock ab­ sorbing, together with an adequate resistance to processing and steam sterilization, can be assured by using a concentration of 3.61 % w/w. Results and discussion Among them, the most suitable and promising method in terms of efficacy and compatibility with solution or emulsions is represented by steam or moist heat. Conversely, it is known that heat induc­ es degradation of CMC gels and polymer solutions37 and at high temperatures CMC degrades and perma­ nently reduces in viscosity. Therefore, a rheological investigation has been dedicated to evaluate the po­ tential degradation of CMCA blends during the R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) 517 Fi g. 10 – a: Viscoelastic moduli (at 0.5 and 2.5 Hz) of the commercial viscosupplements listed below. b: Viscoelastic moduli (at 0.5 and 2.5 Hz) of the 6/4 CMCAp/CMCAg blends at 2.5 %, 3 % and 3.61 % w/w concentrations compared with those of two commercial products (Orthovisc and Monovisc). c: Viscosity values (at 0.1, 10 and 1000 s–1) of the 6/4 CMCAp/CMCAg blends at 2.5 %, 3 % and 3.61 % w/w concentrations compared with those of two commercial products (Orthovisc and Monovisc). Commercial viscosupplement l ( l ) h d d d h hl f d d quite large interval covered by both moduli at both frequencies is strictly related to the different me­ chanical spectra displayed by the systems consid­ ered, spanning from those typical of ordinary poly­ meric solutions to classical gel behaviours. Similar differences can also be found among novel systems proposed as candidates for viscosupplementa­ tion.40,42–45 Tests performed on blends with different gel fractions have shown that an acceptable structural recovery after continuous shear application (more than 80–90 %) is attained when the gel fraction is lower than 50 %. Thus, the 6/4 CMCAp/CMCAg blend has been provisionally selected for the final investigation addressed to individuate the most ­suitable total polymer concentration, apt to compen­ sate the loss in mechanical properties occurring during processing and sterilization. The viscoelastic moduli of three 6/4 CMCAp/CMCAg blends with different total polymer concentrations are compared with those of two commercial products in Fig. 10b, while Fig. 10c reports the viscosity values of the same systems at three different shear rates. 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Polym. 102 (2014) 110. doi: http://dx.doi.org/10.1016/j.carbpol.2013.11.003 21. Stitik, T. P., Levy, J. A., Am. J. Phys. Med. Rehabil. 85 (Suppl 11) (2006) S32. doi: http://dx.doi.org/10.1097/01.phm.0000245677.20294.c2
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The proinflammatory cytokines IL-1beta and TNF-alpha induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway.
Arthritis research & therapy
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Available online http://arthritis-research.com/content/8/6/R172 Research article Vol 8 No 6 Open Access The proinflammatory cytokines IL-1β and TNF-α induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway Beixue Gao1, Karen Calhoun1 and Deyu Fang1,2 1Department 2Department of Otolaryngology-Head and Neck Surgery, University of Missouri School of Medicine, One Hospital Drive, Columbia, MO 65212, USA of Molecular Microbiology and Immunology, University of Missouri School of Medicine, One Hospital Drive, Columbia, MO 65212, USA Corresponding author: Deyu Fang, FangD@missouri.edu Received: 13 Jun 2006 Revisions requested: 13 Jul 2006 Revisions received: 10 Oct 2006 Accepted: 14 Nov 2006 Published: 14 Nov 2006 Arthritis Research & Therapy 2006, 8:R172 (doi:10.1186/ar2081) This article is online at: http://arthritis-research.com/content/8/6/R172 © 2006 Gao et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The overgrowth of synovial tissues is critical in the pathogenesis of rheumatoid arthritis (RA). The expression of Synoviolin (SYN), an E3 ubiquitin ligase, is upregulated in arthritic synovial fibroblasts and is involved in the overgrowth of synovial cells during RA. However, the molecular mechanisms involved in the elevated SYN expression are not known. Here, we found that SYN expression is elevated in the synovial fibroblasts from mice with collagen-induced arthritis (CIA). The proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNFα) induce SYN expression in mouse synovial fibroblasts. Cultivation of mouse synovial fibroblasts with IL-1β activates mitogen-activated protein kinases, including extra-cellular signal-regulated kinase (Erk), JNK (c-Jun N-terminal kinase), and Introduction Rheumatoid arthritis (RA) is a chronic debilitating disease of the joints characterized by leukocyte infiltration, hyperproliferation of synovial cells, and bone destruction. Hyperproliferative synovial fibroblasts play a critical role in the pathogenesis of RA by the following mechanisms: They directly invade bone and cartilage, produce proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β [1], destroy cartilage through the production of metalloproteinase [2], and produce the receptor of nuclear factor-kappa B (NFκB) ligand, which augments osteoclast activity for bone destruction [3-5]. Therefore, inhibition of the proliferative and/ p38, while only Erk-specific inhibitor blocks IL-1β-induced SYN expression. Expression of transcription factor ETS1 further enhances IL-1β-induced SYN expression. The dominant negative ETS1 mutant lacking the transcription activation domain inhibits SYN expression in a dose-dependent manner. The activation of both Erk1/2 and ETS1 is increased in the CIA synovial fibroblasts. Inhibition of Erk activation reduces ETS1 phosphorylation and SYN expression. Our data indicate that the proinflammatory cytokines IL-1β and TNF-α induce the overgrowth of synovial cells by upregulating SYN expression via the Erk1/-ETS1 pathway. These molecules or pathways could therefore be potential targets for the treatment of RA. or invasive capacities of synovial fibroblasts should have protective effects against joint destruction. Synoviolin (SYN), which is also called Hrd1 (3-hydroxy-3methylglutaryl reductase degradation), was identified by Hampton and co-workers [6] as an E3 ubiquitin ligase in yeast. SYN is a multispanning membrane protein with its C-terminal RING (really interesting new gene) finger domain located in the cytoplasm [6,7]. It has been reported that human SYN is involved in the elimination of two endoplasmic reticulum (ER)associated degradation substrates, T-cell receptor-α and CD3-δ, via its E3 ubiquitin ligase activity [8]. Ubiquitination is a process that covalently conjugates ubiquitin to the target protein for degradation. This process requires a cascade of AP-1 = activator protein-1; CFA = complete Freund's Adjuvant; CIA = collagen-induced arthritis; DMEM = Dulbecco's modified Eagle's medium; EBS = ETS binding site; ER = endoplasmic reticulum; Erk = extracellular signal-regulated kinase; ETS1-DN = dominant negative mutant of ETS1; FBS = fetal bovine serum; GST = glutathione S-transferase; IACUC = institutional animal care and use committee; IL = interleukin; JNK = c-Jun Nterminal kinase; MAPK = mitogen-activated protein kinase; NF-κB = nuclear factor-kappa B; NP-40 = Nonidet P-40; PCR = polymerase chain reaction; RA = rheumatoid arthritis; RT-PCR = reverse transcription-polymerase chain reaction; SYN = Synoviolin; TNF-α = tumor necrosis factor-α; Ubc = ubiquitin-conjugation enzyme. Page 1 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Gao et al. three enzymes, E1, E2, and E3. SYN predominantly uses the ubiquitin-conjugation enzyme 7p (Ubc7p) as an E2 but also cooperates with Ubc6p and Ubc1p in ER-associated degradation [9]. SYN is also required for the mouse embryo development because the gene knockout mice die in utero at approximately embryonic day 13.5 [10]. By means of immunoscreening with an anti-rheumatoid synovial cell antibody, SYN was identified and cloned as a rheumatoid regulator. Expression of SYN is highly associated with the development of RA. Mice with overexpressed SYN (SYN transgenic mice) develop spontaneous arthropathy. On the other hand, mice with reduced SYN (SYN+/- mice) are resistant to collagen-induced arthritis (CIA). Further in vitro study revealed that, through its anti-apoptotic activities, SYN triggers the outgrowth of synovial fibroblasts [11,12]. Therefore, inhibition of the expression of SYN has potential therapeutic benefit in the prevention or treatment of RA. However, the molecular mechanisms involved in overexpression of SYN during RA remain unknown. In this study, we found that the proinflammatory cytokines, particularly IL-1β, upregulate SYN expression at the transcriptional level. The extra-cellular signal-regulated kinase (Erk)ETS1 signal pathway is involved in IL-1β-induced SYN expression. Materials and methods Reagents, antibodies, and plasmids All the mitogen-activated protein kinase (MAPK) inhibitors used in this study, including the Erk activation inhibitor, PD98059 [13], the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 [14], p38 inhibitor, SB202190 [15], and the NFκB inhibitor, SN50 [16], were purchased from Calbiochem (San Diego, CA, USA). Antibodies against JNK1, Erk, and p38 were purchased from Promega Corporation (Madison, WI, USA). Murine IL-1β, IL-6, and TNF-α were obtained from BD Pharmingen (San Diego, CA, USA). Anti-actin monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-ETS1 was obtained from EMD Biosciences, Inc. (San Diego, CA, USA). Antibodies against Erk, c-Jun, and ATF2, as well as their phosphorylated forms, were purchased from Promega Corporation. Anti-SYN polyclonal antibody was generated in our laboratory by immunization of mice with glutathione S-transferase (GST)-fusion protein of the C-terminal 152 amino acids of SYN. ETS1 fulllength cDNA was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) (ATCC no. 5844553). The C-terminal DNA binding domain of ETS1 was amplified by polymerase chain reaction (PCR) and subcloned into pEF4 his expression vectors digested by KpnI and Not1. Mice and CIA DBA1 mice (The Jackson Laboratory, Bar Harbor, ME, USA) were bred and maintained in accordance with the guidelines Page 2 of 10 (page number not for citation purposes) of the institutional animal care and use committee (IACUC), and all the experimental procedures were approved by the IACUC of the University of Missouri (Columbia, MO, USA). Native bovine collagen II (Worthington Biochemical Corporation, Lakewood, NJ, USA) was emulsified with an equal volume of complete Freund's Adjuvant (CFA). Disease was induced by intradermal injection of DBA1 mice with 50 μl of emulsion containing 100 μg of collagen in CFA. On day 21, the mice were boosted by intradermal injection with 100 μg of collagen in incomplete Freund's Adjuvant. Clinical arthritis was assessed by the following scoring system: grade 0, no swelling; grade 1, mild but definite redness and swelling of the ankle or wrist or digits; grade 2, moderate redness and swelling of ankle and wrist; grade 3, severe redness and swelling of entire paw, including digits; and grade 4, maximally inflamed limb with involvement of multiple joints. Each limb was graded, giving a maximum possible score of 16 per mouse. Approximately 80% of DBA1 mice developed arthritis 40 days after the first injection with collagen (supplemental Figure 1a in Additional file 1), and most of these mice developed severe arthritis with an average score of 12 (supplemental Figure 1b in Additional file 1). Isolation of synovial fibroblasts Synovial tissues were obtained from DBA1 mice as described previously [17]. These joint tissues were minced and incubated with 1 mg/ml of collagenase (Worthington Biochemical Corporation) in serum-free Dulbecco's modified Eagle's medium (DMEM) for 3 hours at 37°C, filtered through a nylon mesh, extensively washed, and cultured in DMEM supplemented with 10% fetal calf serum (Fisher Scientific Co., Pittsburgh, PA, USA), 100 U penicillin, 100 μg/ml streptomycin, and 50 mg/ml L-glutamine in a humidified atmosphere containing 5% CO2. After overnight culture, we removed the nonadherent cells, trypsinized the adherent cells split at a ratio of 1:3, and cultured them in medium. Synoviocytes were used from passages 3 to 9 in these experiments, during which they consisted of a homogeneous population of synovial fibroblasts monitored by flow cytometry with less than 1% of CD11b, phagocytic, and Fc receptor II-positive cells. SDS-PAGE and Western blotting We analyzed the expression of SYN by SDS-PAGE and Western blotting as described previously [18]. Synovial fibroblasts were collected from culture dishes and lysed with Nonidet P40 (NP-40) lysis buffer (20 mM Tris-HCl with pH 7.5, 150 mM NaCl, 1% NP-40, and protease inhibitor cocktail was added freshly) and boiled in 20 μl of Laemmli's buffer (50 mM TrisHCl, pH 6.8, 30% glycerol, 4% SDS, and 1% β-mercaptoethanol). Samples were subjected to 8% or 10% analysis by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were probed with the indicated primary antibodies (usually 1 μg/ml), followed by horseradishperoxidase-conjugated Available online http://arthritis-research.com/content/8/6/R172 Figure 1 Increased Synoviolin (SYN) expression in the synovial fibroblasts from collagen-induced arthritis (CIA) mice. mice (a) Test of the specificity of anti-SYN antibody. HEK 293 cells were transfected with (lane 1) or without (lane 2) SYN expression plasmids. Lysates from the transfected cells were analyzed by SDS-PAGE and Western blotting using sera from the mice immunized with GST (glutathione S-transferase)-SYN fusion protein (top panel). The same membrane was reprobed with anti-actin (bottom panel). (b) Analysis of the protein expression of SYN in mouse synovial fibroblasts. Mouse synovial fibroblasts were isolated from each of three normal or CIA mice. Cells were lysed and subjected to SDS-PAGE and Western blotting using anti-SYN polyclonal antibodies (top panel). The same membrane was reprobed with anti-actin antibody (bottom panel). (c) Quantification of SYN expression. The densities of each band in (b) were analyzed, and the ratio of SYN/(10-1 actin) was used. Error bars represent three different CIA or normal mice. Statistic analysis indicates that the expression of SYN is significantly increased (p < 0.001). secondary antibodies. Membranes were thenwashed and visualized with an enhanced chemiluminescence detectionsystem (Amersham Pharmacia Biotech, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK). When necessary, membranes were stripped by incubation in stripping buffer (62.5 mM Tris-HCl, pH 5.7,100 mM 2-mercaptoethanol, and 2% SDS) for 30 minutes at 70°C with constant agitation, washed, and then reprobed with other antibodies asindicated. Real-time reverse transcription-PCR Total cellular RNA extraction was performed using the RNA purification kit from Promega Corporation. The oligonucleotide primers used for mouse SYN were forward 5-aggcccatgtacctggccatgagg-3 and reverse 5-caggagcgcaggcagctcgtgtg-3. The QuantiTect SYBR Green PCR kit (Qiagen Inc., Valencia, CA, USA) was used. Reactions were loaded on 96-well thinwall plates and sealed with optical-quality sealing tape. Each reaction was run on an iCycler iQ Multi-Color Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA) under the following conditions: 95°C for 15 minutes (94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds) for 40 cycles and 72°C for 3 minutes. Samples were run in triplicate, and relative copy numbers were determined. Results Elevated expression of SYN during CIA in DBA1 mice Recent studies demonstrated that SYN functions as a rheumatoid regulator for the overgrowth of synovial tissues in patients with RA [11]. To investigate the molecular mechanisms involved in regulating SYN expression during arthritis development, we compared SYN expression in the synovial fibroblasts from CIA mice with that from normal mice. Because the antibodies against SYN are not commercially available, we first generated anti-SYN polyclonal antibody by immunizing a fusion protein of GST with the C-terminus of SYN. We detected both overexpressed and endogenous SYN in HEK 293 cells by using the sera from GST-SYN-immunized mice (Figure 1a). We therefore used these polyclonal anti-SYN antibodies in this study. With Western blotting analysis, we found a significantly higher SYN expression in the synovial fibroblasts from CIA mice than that from normal controls (Figure 1b). The mean relative density values (10 × SYN/actin) were 4.8 ± 0.9 for CIA synovial fibroblasts and 1.1 ± 0.3 for control synovial cells (p < 0.001) (Figure 1c). These results indicate that SYN expression is upregulated during CIA. Page 3 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Gao et al. Figure 2 The expression (CIA) development of Synoviolin (SYN) during collagen-induced arthritis (CIA) development. (a) Synovial fibroblasts were isolated from normal DBA mice (Normal) and DBA mice 20 days after the first collagen immunization (Immunized) or 10 days after the second immunization when CIA is developed (CIA). The expression of SYN in those synovial fibroblasts was analyzed by Western blotting. (b) The densities of each band in (a) were analyzed, and the ratio of SYN/(10-1 actin) was used. Error bars represent three different CIA, immunized, or normal mice. Statistic analysis indicates that the expression of SYN is significantly increased during CIA development. Next, we kinetically analyzed the expression of SYN during the development of CIA. DBA synovial fibroblasts from normal DBA mice, DBA mice 20 days after the first immunization with collagen but without any clinical sign of CIA, and DBA mice 10 days after boosted immunization when these mice developed severe arthritis (score 8 to 12). Surprisingly, the expression of SYN has been significantly upregulated when the DBA mice received only the first immunization and did not develop obvious arthritis. Although statistically not significant (p = 0.06), boosted immunization further enhanced SYN expression (Figure 2). These results suggest that upregulation of SYN expression is involved in the development of CIA in mice. Induction of SYN expression by IL-1β and TNF-α in mouse synovial fibroblasts The production of proinflammatory cytokines by synovial macrophages and fibroblasts is one reason for synovial cell hyperproliferation. We therefore hypothesized that some of these cytokines may also be responsible for the induction of SYN expression. To test this hypothesis, we first examined the Page 4 of 10 (page number not for citation purposes) Figure 3 Induction of Synoviolin (SYN) expression expression by by proinflammatory proinflammatory cytokines. cytokines (a) Mouse synovial fibroblasts were starved for 24 hours and then cultivated for 48 hours with each proinflammatory cytokine, including 10 ng/ml of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, or IL-6. The expression of SYN was tested by Western blotting by using antiSYN antibody (top panel). As a control, the protein level of actin was detected using the same membrane (bottom panel). (b) The total RNA from the cultured synovial fibroblasts was isolated and reverse-transcribed into cDNA. The levels of SYN cDNA were analyzed by real-time polymerase chain reaction by using SYN-specific primers. Error bars represent the results from three independent experiments (mean ± standard deviation). effects of proinflammatory cytokines on the expression of SYN in mouse synovial fibroblasts. Each of 5 × 105 cells was plated and cultivated in the presence of IL-6, TNF-α, or IL-1β. Fortyeight hours after the addition of each cytokine, the protein levels of SYN were analyzed. As shown in Figure 3a, TNF-α slightly induced the protein expression of SYN with a mean density of 3.1 ± 0.6 compared with 1.1 ± 0.2 of controls from three independent experiments (p < 0.05 to control), whereas IL-6 did not affect SYN expression, the mean density of which was 1.3 ± 0.3 (p = 0.28). The cultivation of synovial fibroblasts with IL-1β significantly upregulated SYN expression, the mean density of which was 6.2 ± 1.1 (p < 0.001). These results indicate that both IL-1β and TNF-α induce SYN expression in mouse synovial fibroblasts. Available online http://arthritis-research.com/content/8/6/R172 To determine whether these cytokines induce SYN expression at the transcriptional level, total RNA was isolated from these synovial fibroblasts, reverse-transcribed into cDNA, and examined by reverse transcription (RT)-PCR using SYN-specific primers. Real-time RT-PCR revealed that IL-1β induced SYN expression at the transcriptional level, because the mRNA level of SYN was upregulated by IL-1β. TNF-α had a weaker effect on SYN transcription, whereas IL-6 had no effect (Figure 3b). These results indicate that IL-1β induces SYN expression at the mRNA level in mouse synovial fibroblasts. IL-1β and TNF-α induce SYN expression through activation of Erk IL-1β induces the growth of synovial fibroblasts by activating the MAPK pathways [19-24]. To elucidate the molecular mechanisms of IL-1β-induced SYN expression, we first tested the activation of all three MAPK members, including Erk, JNK, and p38. Because culture of synovial fibroblasts with 10% fetal bovine serum (FBS) highly activates all these MAPKs, we first starved the mouse synovial fibroblasts for 24 hours with media that contain 0.5% FBS, then IL-1β was added. Erk activation was analyzed with anti-phosphorylated Erk. The phosphorylations of c-Jun and ATF2 were used as reporters for the activation of JNK1 and p38, respectively [19]. The activation of these MAPKs was not detectable after starvation (Figure 4b–d, lane 1). Cultivation of mouse synovial fibroblasts with IL1β activated all three MAPK members, including Erk (Figure 4b, top panel), p38 (Figure 4c, top panel), and JNK1 (Figure 4d, top panel), and IL-1β significantly upregulated SYN expression (Figure 4a). These results suggest that MAPK pathways might be involved in IL-1β-induced SYN expression. To further elucidate which MAPK pathway is involved in regulating IL-1β-induced SYN expression, specific inhibitors of MAP kinases were used [13-15,25]. Erk inhibitor, PD98059, specifically inhibited Erk phosphorylation without affecting the activation of either JNK or p38 (Figure 4b, lane 3). In the presence of this inhibitor, SYN expression was reduced to a level that is comparable with non-treated controls (Figure 4a, top panel, lane 3). p38 inhibitor, SB202190, which specifically inhibited ATF2 phosphorylation without affecting Erk and JNK1 activation, had a very mild inhibitory effect on SYN expression (Figure 4c, lane 4). The specific inhibitor of JNK, SP600125, which specifically inhibited c-Jun phosphorylation, had no effects on IL-1β-induced SYN expression (Figure 4d, lane 5). Similarly, the Erk inhibitor also inhibited TNF-αinduced SYN expression (Figure 4e). Both IL-1β and TNF-α are also strong activators of the NF-κB pathway [24]. We therefore tested whether NF-κB activation is also involved in the induction of SYN expression in mouse synovial fibroblasts. The NF-κB-specific inhibitor, SN50, strongly inhibited both IL1β and TNF-α-induced NF-κB reporter activities (Figure 4g) but not the SYN protein expression (Figure 4f). These findings collectively indicate that IL-1β and TNF-α enhance SYN expression through the activation of Erk, but not the p38, JNK, or NF-κB pathways. Transcription factor ETS1 is involved in IL-1β-induced SYN expression ETS1 is a transcription factor, the expression of which is increased in synovial fibroblasts from patients with RA [27]. Recently, the ETS binding site (EBS), termed EBS-1, from position -76 to -69 of the proximal promoter, was identified as being responsible for SYN expression [28]. Interestingly, we found that overexpression of ETS1 further enhanced IL-1βinduced SYN expression in a dose-dependent manner (Figure 5a, lanes 3 and 4; Figure 5b). To further confirm the involvement of ETS1 in SYN expression, we generated a C-terminal truncate mutation of ETS1, which has been shown to be a dominant negative mutant of ETS1 (ETS1-DN) [29]. As shown in Figure 4a (lanes 5 and 6), ETS1-DN dramatically blocked SYN expression induced by IL-1β in mouse synovial fibroblasts. These results indicate that the transcription factor is involved in regulating IL-1β-induced SYN expression in mouse synovial fibroblasts. Activation of Erk and ETS1 is involved in the overgrowth of synovial fibroblasts from CIA mice We next tested whether the activation of Erk is enhanced in the synovial fibroblasts from CIA mice, in which SYN expression is increased. As expected, both Erk1 activation and Erk2 activation are slightly increased in CIA synovial fibroblasts compared with those from normal DBA1 mice (Figure 6a). Interestingly, in treatment with the Erk-specific inhibitor, the expression of SYN was reduced to comparable levels between normal and CIA synovial fibroblasts. These results suggest that Erk activation is involved in upregulating SYN expression in vivo during CIA development. It has been reported that the expression of ETS1 protein is increased in the synovial fibroblasts from patients with RA [27]. We compared the activation and protein expression of ETS1 in the synovial fibroblasts from CIA mice with those from normal DBA1 mice. As shown in Figure 6a, we found that the activation of ETS1 but not its protein expression was increased in CIA synovial fibroblasts. These results suggest that the activation of both Erk and ETS1 is involved in regulating the expression of SYN in mouse synovial fibroblasts during CIA. Based on previous studies that have suggested that Erk may activate ETS1 transcription activity [30] together with our findings that Erk and ETS1 are involved in IL-1β-induced SYN expression, we proposed that IL-1β induces SYN expression in mouse synovial fibroblasts via the Erk-ETS1 pathway. To support this hypothesis, we found that inhibition of Erk activation by Erk-specific inhibitor blocked ETS1 phosphorylation (Figure 6a). Moreover, Erk inhibitor suppressed the cell growth of mouse synovial fibroblasts, and the synovial fibroblasts from CIA mice were more sensitive to Erk inhibition (Figure 6b). Treatment of these cells with the Erk inhibitor did not Page 5 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Gao et al. Figure 4 Interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) induce Synoviolin (SYN) expression via the Erk pathway pathway. (a) Synovial fibroblasts were partially starved for 24 hours by cultivation of these cells with media that contain 0.5% fetal bovine serum and then cultured with 10 ng/ml of IL-1β. Cells were also treated with each of the MAPK inhibitors. The concentrations of each inhibitor used were as follows: Erk inhibitor PD98059, 20 μM; JNK inhibitor SP600125, 10 μM; p38 inhibitor SB202190, 10 μM; and NF-κB inhibitor SN50, 20 μM. The expression of SYN was examined by Western blotting (top panel). The protein level of actin was reprobed as a control (bottom panel). (b) The activation of Erk was analyzed by anti-p-Erk antibody (top panel). The same membrane was reprobed by anti-Erk antibody (bottom panel). (c) p38 activation was analyzed by anti-p-ATF2 antibody (top panel). The same membrane was reprobed by anti-ATF2 antibody (bottom panel). (d) JNK1 activation was analyzed with anti-phospho-Jun antibody (top panels). The total protein levels of c-Jun were examined using anti-Jun (bottom panels). (e) Mouse synovial fibroblasts were starved for 24 hours and then cultured with 10 ng/ml of TNF-α without or with Erk inhibitor. The expression of SYN was analyzed by Western blotting (top panel). The activation of Erk was determined by anti-phosphorylated Erk antibody (middle panel). The protein level of Erk was analyzed by anti-Erk antibody (bottom panel). (f) The effect of NF-κB inhibitor on SYN expression. Mouse synovial fibroblasts were transfected with NF-κB-luc reporter, which contains firefly luciferase gene under control of NF-κB. The control plasmid pRL-TK encoding renillar luciferase was also included to correct transfection efficiency. Transfected cells were starved and then cultivated in the presence of 10 ng/ml of IL-1β or TNF-α without or with SN50 for 24 hours. The expression of SYN in these cells was analyzed by Western blotting (top panel), and the same membrane was reprobed with anti-actin (middle panel). (g) Parallel prepared cell lysates from (f) were used for testing the NF-κB-driven luciferase activity (bottom panel). Error bars represent three different experiments (mean ± standard deviation). Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa B. significantly arrest the cell cycle of these synovial fibroblasts from either normal DBA or CIA mice (Figure 6c). These findings suggest that the activation of Erk and ETS1 is involved in the overgrowth of mouse synovial fibroblasts during CIA. Based on these findings, we collectively concluded that the Erk-ETS1 pathway is involved in SYN expression in mouse synovial fibroblasts induced by IL-1β and TNF-α. Discussion Proinflammatory cytokines, particularly IL-1β and TNF-α, can induce the cell proliferation of synovial fibroblasts both in vitro and in vivo [31,32]. The molecular mechanisms of proinflam- Page 6 of 10 (page number not for citation purposes) matory cytokine-induced cell growth of synovial tissues have been extensively investigated, although there still is room for debate [33]. IL-1β and TNF-α are key activators of the many transcription factors, including activator protein-1 (AP-1), Egr1 (early growth response-1), and NF-κB, in synovial fibroblasts. Activation of the NF-κB/Rel transcription family and AP1 complexes, composed of members of the Jun and Fos families, contributes to the hyperproliferation of fibroblast-like synoviocytes [34]. We found that IL-1β induces the proliferation of synovial fibroblasts by upregulating SYN expression. These findings provide a new mechanism for IL-1β in synovitis during RA development. Available online http://arthritis-research.com/content/8/6/R172 Figure 5 SYN transcription, as indicated by the finding that the specific inhibitors against Erk, but not JNK and p38, block SYN expression. It has been demonstrated that Erk activation is highly increased in the synovial fibroblasts from patients with RA. Consistent with this, we found that the activation of Erk is significantly upregulated in mouse synovial fibroblasts from CIA mice compared with that from normal DBA1. Interestingly, the CIA synovial fibroblasts are more sensitive to Erk inhibitormediated cell growth inhibition. Therefore, Erk inhibitors could be potential candidates for RA treatment. Recently, EBS-1 was identified as a crucial site for the expression of SYN [28]. EBS-1 is a binding site for ETS family transcription factors, including ETS1/2, GABP (growth-associated binding protein)-α/β, and Sp-1 [36]. Our findings that ETS1 expression increases SYN transcription and that ETS1DN blocks SYN expression (Figure 4) confirm that the EBS-1 binding site is the promoter region for IL-1β-induced SYN expression. ETS1 is activated by Erk-mediated phosphorylation [37]. Therefore, inhibition of Erk activation by specific inhibitors blocks SYN transcription (Figure 3). Overexpression of ETS1 family transcription factors has been observed in RA synovial membranes [19,27]. However, we found that the activation of ETS1 but not its protein expression is upregulated in the synovial fibroblasts from CIA mice. The elevated ETS1 activation is possibly a direct consequence of Erk activation, because inhibition of Erk activation blocks ETS1 phosphorylation. expression ETS1 is involved in interleukin-1β(IL-1β)-induced Synoviolin (SYN) expression. (a) ETS1 or ETS1-DN expression plasmids were transfected into mouse synovial fibroblasts. Cells were then cultured in medium containing 10 ng/ml of IL-1β for 48 hours. The expression of SYN was detected by Western blotting (top panel). The expression of ETS1 and its mutant was detected with anti-ETS1 antibody (middle panel). The same membrane was stripped and reblotted with anti-actin antibody (bottom panel).(b) The expression level of SYN was quantified. Error bars represent three independent experiments (mean ± standard deviation). ETS1-DN, dominant negative mutant of ETS1. MAPKs are especially important in synovitis because they control the proliferation of synovial cells in the rheumatoid joint and induce the production of MMPs and cytokines that participate in the rheumatoid process [33,35]. The involvement of all three MAPK family members, JNK, Erk, and p38, in RA has been indicated by the fact that their activation is increased in rheumatoid synovial cells. They have also been implicated in the pathogenesis of RA [33]. The binding of IL-1β to its receptor expressed on the surface of synovial fibroblasts activates all three MAPK members, as demonstrated in our study and previous reports [33]. Intriguingly, only Erk activation is involved in As an E3 ubiquitin ligase on the ER membrane, SYN functions as an ER-associated degradation system in both yeast and mammals [8,9]. The biological functions of SYN were analyzed in SYN transgenic mice and heterozygous knockout mice because the homozygous mice are embryonic-lethal [11]. Interestingly, the expression level of SYN correlates significantly with the onset of arthropathy: Increased SYN expression causes synovium overgrowth and spontaneous arthropathy, whereas reduced SYN expression (heterozygous mutant mice) is associated with resistance to CIA [11]. Our finding that the suppression of SYN inhibits IL-1β-induced synovial fibroblast proliferation provides a direct rationale for SYN as a potential target for the treatment of RA. It will be extremely interesting to investigate the effects of gene therapeutic delivery of dominant-negative SYN or its siRNA (small interfering RNA) on the development of arthritis in animals such as DBA mice with CIA. Conclusion This study demonstrated that the proinflammatory cytokines IL-1β and TNF-α induce synovial fibroblast growth by upregulating the expression of an E3 ubiquitin ligase, SYN. At the molecular level, activation of MAPK Erk and transcription factor ETS1 is required for SYN expression. Based on our findings and the fact that synovial fibroblasts are among the major resources for IL-1β and TNF-α in the rheumatoid joints Page 7 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Gao et al. Figure 6 Erk inhibition suppresses ETS1 activation and hyperproliferation of mouse synovial fibroblasts from collagen-induced arthritis (CIA) mice mice. (a) Synovial fibroblasts isolated from CIA or normal mice were cultured without or with Erk inhibitor. The expression of Synoviolin (SYN) was determined by Western blotting by using anti-SYN antibody (top panel). The activation and protein levels of Erk in the parallel prepared cell lysates were detected by anti-phospho-Erk and anti-Erk, respectively (the second and third panels). Similarly, the activation and protein levels of ETS1 in the parallel prepared cell lysates were detected by anti-phospho-ETS1 and anti-ETS1, respectively (the bottom two panels). (b) Synovial cells were cultured without or with Erk inhibitor in 12-well plates for 24 hours. One microcurie of 3H-tymidine was added to each well of plated cells and further cultured for 16 hours. 3H-tymidine incorporation was analyzed as described previously [18]. Error bars represent three independent experiments (mean ± standard deviation). (c) Cell cycle analysis of Erk inhibitor-treated synovial fibroblasts. Cells treated with or without the Erk-specific inhibitor were collected and fixed in cold methanol and then stained with propidium iodide (PI) in the presence of RNase. PI-stained cells were washed once with phosphatebuffered saline and then analyzed by flow cytometry. The cell death was significantly increased when CIA synovial fibroblasts were treated with Erk inhibitor, PD98059 (PD). CPM, counts per minute; Erk, extracellular signal-regulated kinase; FBS, fetal bovine serum. [31], we proposed a positive feedback model for SYN in RA development (Additional file 2). According to this model, IL-1β induces SYN transcription, SYN enhances IL-1β-induced synovial fibroblast proliferation, and the massive growth of synovial fibroblasts produces more IL-1β for the induction of SYN expression. This positive feedback process may be critical in RA development. Molecules in this signal pathway can therefore be potential targets against RA. Page 8 of 10 (page number not for citation purposes) Competing interests The authors declare that they have no competing interests. Authors' contributions BG conceived and performed most of the experiments, including anti-SYN antibody generation and signal transduction analysis. KC participated in drafting the manuscript. DF designed and organized the study, drafted the manuscript, and Available online http://arthritis-research.com/content/8/6/R172 performed experiments in inducing arthritis and isolating synovial fibroblasts. All authors read and approved the final manuscript. Additional files The following Additional files are available online: 5. 6. 7. Additional file 1 Collagen-induced arthritis in DBA/1 mice. DBA/1 mice at the age of 6 weeks were immunized with 100 μg of collagen in complete Freund's Adjuvant on day 0 and boosted with 100 μg of collagen in incomplete Freund's Adjuvant on day 21. Ten DBA/1 mice were used. Severity of joint inflammation (a) and incidence of arthritis (b) were scored. See http://www.biomedcentral.com/content/ supplementary/ar2081-S1.tiff 9. Additional file 2 11. A proposed model for interleukin-1β(IL-1β)-induced Synoviolin (SYN) expression in rheumatoid arthritis. IL-1β stimulates synovial fibroblasts and activates Erk. Activated Erk drives ETS1 activation for the transcription of SYN mRNA. The upregulated SYN increases the proliferation of synovial cells, which induces arthritis. The increased synovial fibroblasts produce more IL-1β and thereby facilitate the development of arthritis. Erk, extracellular signal-regulated kinase. See http://www.biomedcentral.com/content/ supplementary/ar2081-S2.tiff 8. 10. 12. 13. 14. 15. 16. Acknowledgements We thank Dr. Christopher H. Evans (Center for Molecular Orthopaedics, Harvard Medical School, Boston, MA, USA) for providing protocols for mouse synovial fibroblast isolation. We also thank Dr. Krishna Kannan (Division of Rheumatology, Department of Internal Medicine, University of Missouri-Columbia) for his critical comments on the manuscript. This work was partially supported by a research board grant from the University of Missouri and by an investigator research award from the Arthritis Foundation to DF. 17. 18. 19. References 1. 2. 3. 4. Chen V, Croft D, Purkis P, Kramer IM: Co-culture of synovial fibroblasts and differentiated U937 cells is sufficient for high interleukin-6 but not interleukin-1beta or tumour necrosis factor-alpha release. Br J Rheumatol 1998, 37:148-156. Cunnane G, Warnock M, Fye KH, Daikh DI: Accelerated nodulosis and vasculitis following etanercept therapy for rheumatoid arthritis. Arthritis Rheum 2002, 47:445-449. 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Gortz B, Hayer S, Tuerck B, Zwerina J, Smolen JS, Schett G: Tumour necrosis factor activates the mitogen-activated protein kinases p38alpha and ERK in the synovial membrane in vivo. Arthritis Res Ther 2005, 7:R1140-R1147. Hammaker DR, Boyle DL, Chabaud-Riou M, Firestein GS: Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis. J Immunol 2004, 172:1612-1618. Lu H, Sun T, Yao L, Zhang Y: Role of protein tyrosine kinase in IL-1 beta induced activation of mitogen-activated protein kinase in fibroblast-like synoviocytes of rheumatoid arthritis. Chin Med J (Engl) 2000, 113:872-876. Toh ML, Yang Y, Leech M, Santos L, Morand EF: Expression of mitogen-activated protein kinase phosphatase 1, a negative regulator of the mitogen-activated protein kinases, in rheumaPage 9 of 10 (page number not for citation purposes) Arthritis Research & Therapy 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Vol 8 No 6 Gao et al. toid arthritis: up-regulation by interleukin-1beta and glucocorticoids. Arthritis Rheum 2004, 50:3118-3128. Vergne-Salle P, Leger DY, Bertin P, Treves R, Beneytout JL, Liagre B: Effects of the active metabolite of leflunomide, A77 1716, on cytokine release and the MAPK signalling pathway in human rheumatoid arthritis synoviocytes. Cytokine 2005, 31:335-348. Gao M, Labuda T, Xia Y, Gallagher E, Fang D, Liu YC, Karin M: Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science 2004, 306:271-275. Krasnow SW, Zhang LQ, Leung KY, Osborn L, Kunkel S, Nabel GJ: Tumor necrosis factor-alpha, interleukin 1, and phorbol myristate acetate are independent activators of NF-kappa B which differentially activate T cells. Cytokine 1991, 3:372-379. Redlich K, Kiener HP, Schett G, Tohidast-Akrad M, Selzer E, Radda I, Stummvoll GH, Steiner CW, Groger M, Bitzan P, et al.: Overexpression of transcription factor ETS1 in rheumatoid arthritis synovial membrane: regulation of expression and activation by interleukin-1 and tumor necrosis factor alpha. Arthritis Rheum 2001, 44:266-274. Tsuchimochi K, Yagishita N, Yamasaki S, Amano T, Kato Y, Kawahara K, Aratani S, Fujita H, Ji F, Sugiura A, et al.: Identification of a crucial site for synoviolin expression. Mol Cell Biol 2005, 25:7344-7356. Pourtier-Manzanedo A, Vercamer C, Van Belle E, Mattot V, Mouquet F, Vandenbunder B: Expression of an ETS1 dominant-negative mutant perturbs normal and tumor angiogenesis in a mouse ear model. Oncogene 2003, 22:1795-1806. Jinnin M, Ihn H, Mimura Y, Asano Y, Yamane K, Tamaki K: Matrix metalloproteinase-1 up-regulation by hepatocyte growth factor in human dermal fibroblasts via ERK signaling pathway involves Ets1 and Fli1. 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Ito H, Duxbury M, Benoit E, Clancy TE, Zinner MJ, Ashley SW, Whang EE: Prostaglandin E2 enhances pancreatic cancer invasiveness through an ETS1-dependent induction of matrix metalloproteinase-2. Cancer Res 2004, 64:7439-7446. Page 10 of 10 (page number not for citation purposes)
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Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation
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Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation Vidhu Sharma1, Lisa Young1, Miguel Cavadas2, Kate Owen3, Reproducibility Project: Cancer Biology1* 1Applied Biological Materials, Richmond, Canada; 2University of College Dublin, Dublin, Ireland; 3University of Virginia, Charlottesville, United States 1Applied Biological Materials, Richmond, Canada; 2University of College Dublin, Dublin, Ireland; 3University of Virginia, Charlottesville, United States Abstract The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “COT drives resistance to RAF inhibition through MAPK pathway reactivation” by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D- E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/ TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: 10.7554/eLife.11414.001 Funding: See page 31 Received: 17 September 2015 Accepted: 07 March 2016 Published: 21 March 2016 REGISTERED REPORT Introduction Activation of the canonical mitogen activated protein kinase (MAPK) pathway occurs in response to the binding of growth factors, hormones, or neurotransmitters to receptor tyrosine kinase receptors located at the cell surface (Dhomen and Marais, 2009; Lopez-Bergami et al., 2008). In untrans- formed cells, receptor ligation induces the sequential activation of the small GTPase RAS, followed by RAF, MEK and ERK, which relays proliferative signals generated at the cell periphery into the nucleus to control cellular survival, differentiation and growth (Inamdar et al., 2010; Panka et al., 2006). Not surprisingly, dysregulation of MAPK signaling is common in many human cancers includ- ing melanoma. Mutations in the RAF and RAS genes (Davies et al., 2002; Mercer and Pritchard, 2003) that trigger constitutive activation of the MAPK pathway can result in uncontrolled cell prolif- eration, invasion, metastasis, survival and angiogenesis (Panka et al., 2006; Sharma et al., 2006; Smalley et al., 2006; Smalley and Herlyn, 2006). Received: 17 September 2015 Accepted: 07 March 2016 Published: 21 March 2016 Reviewing editor: Tony Hunter, Salk Institute, United States Copyright Sharma et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. BRAF is one of three members of the RAF family, which includes ARAF, BRAF, and CRAF (or RAF-1) (Dhomen and Marais, 2009). In melanoma, BRAF represents the most commonly mutated Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 1 of 32 Registered report Biochemistry gene in the MAPK signaling cascade where 90% of tumors carry a valine to glutamic acid transition at codon 600 (V600E) that renders BRAF constitutively active and hyperactivates the MAPK cascade (Davies et al., 2002; Dhomen and Marais, 2009; Michaloglou et al., 2008). While preclinical and clinical studies have shown that targeting BRAF (V600E) melanomas with the use of RAF-selective inhibitors results in initial tumor regression (Fedorenko et al., 2015; Flaherty et al., 2010; Shtivelman et al., 2014), responses to RAF inhibitors are transient, with acquired resistance trigger- ing disease progression (Shtivelman et al., 2014). Although progress has been made in the devel- opment of drugs that target RAF, the clinical outcome regarding long-term usage and the mechanisms of acquired resistance warrants further evaluation. In their study, Johannessen and col- leagues sought to identify kinases involved in mediating resistance to the RAF kinase inhibitor PLX4720 (Johannessen et al., 2010). Using a kinase open reading frame (ORF) collection and a high throughput screening methodol- ogy, Johannessen and colleagues identified MAP3K8 (the gene encoding cancer osaka thyroid (COT)/TPL2), as a driver of resistance to BRAF inhibition with PLX4720 (Johannessen et al., 2010). Johannessen and colleagues first examined basal MAP3K8 expression in multiple cell lines harboring the V600E mutation (Johannessen et al., 2010). As shown in Figure 3B and reported by others, RPMI-7951 and OUMS-23 cells were found to express high intrinsic levels of MAP3K8 compared to A375 cells where MAP3K8 was undetectable (Johannessen et al., 2010; Paraiso et al., 2012). RPMI-7951 and OUMS-23 cells also exhibited robust, undiminished ERK and MEK activity concomi- tant with reduced growth sensitivity in the presence of PLX4720 (Figure 3D–E; Johannessen et al., 2010). This is supported by additional findings demonstrating that RPMI-7981 cells treated with the closely related BRAF inhibitor PLX4032/vemurafenib (a successor of PLX4720) also remain refractory to inhibitor treatment as assessed by annexin V staining (Paraiso et al., 2012) and MTS assay (Park et al., 2013). Registered report Materials and methods Materials and methods Protocol 1: MAPK pathway analysis in cells expressing elevated MAP3K8 This experiment assesses the effect the RAF inhibitor, PLX4720, has on the MAPK pathway, in cells expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 and OUMS-23 cells, which express a high level of MAP3K8 and A375 cells, which have undetectable levels. This protocol replicates the experiments reported in Figures 3B and 3E. p g protocol replicates the experiments reported in Figures 3B and 3E. Sampling Experiment to be repeated a total of 4 times for a minimum power of 80%. The original data is qual- itative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells . Cohort 2: RPMI-7951 cells . Cohort 3: OUMS-23 cells Each cohort has four conditions: . Vehicle (DMSO) . 10 mM PLX4720 . 1 mM PLX4720 . 0.1 mM PLX4720 Each condition will be probed with the following antibodies: . pERK1/2 (T202/Y204) . pERK1/2 . pMEK1/2 (S217/221) . MEK1/2 . MAP3K8 . Actin Materials and reagents Reagent Type Manufacturer Catalog # Comments RPMI medium with L-glutamine Cell culture Sigma R8758 Replaces Corning cat no. 10-040-CV. Communicated by authors. MEM with L-glutamine Cell culture Sigma M4655-500ML Replaces Corning cat. No. 10-010-CV. Communicated by authors. Fetal bovine serum (FBS) Cell culture Life Technologies 12483-020 Replaces Corning brand. Communicated by authors. Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. Communicated by authors. A375 cells Cell line ATCC CRL-1619 Original brand not specified. RPMI-7951 cells Cell line ATCC HTB-66 OUMS-23 cells Cell line JCRB JCRB1022 Original brand not specified. 6-well plates Labware Greiner bio-one 657 160 Original brand not specified. Phosphate buffered saline (PBS) Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. Continued on next page Reviewing editor: Tony Hunter, Salk Institute, United States However, others have reported RPMI-7981 cells as exhibiting modest sensitivity to PLX4720 (Schayowitz et al., 2012). In the latter case, ERK activity was reduced by 50% after incu- bation with inhibitor, although these differences in sensitivity may reflect the significantly shorter time course and experimental design used by Park and colleagues. Finally, in Figure 3I, Johannessen and colleagues determined that MAP3K8 kinase activity is required to regulate MEK/ERK activation in RPMI-7951 cells, findings that further confirm MAP3K8 is an essential upstream activator of the MEK-ERK signaling cascade (George and Salmeron, 2009; Johannessen et al., 2010). The key experiments outlined in Figures 3B,D,E, and 3I will be replicated in protocols 1, 2, 3, and 4. p g p p Resistance to targeted agents, such as BRAF inhibitors, is a frequent cause of therapy failure, as noted above. Importantly, chronic BRAF inhibition can lead to cross-resistance to several BRAF- selective inhibitors, indicating that resistance is not likely to be overcome by switching to a new RAF inhibitor (Corcoran et al., 2010; Villanueva et al., 2011). It has been suggested previously that combination treatment with MEK and BRAF inhibitors may be useful in preventing the emergence of resistance or in overcoming resistance to single agent therapies targeting either molecule alone (Corcoran et al., 2010). To examine whether the combined use of RAF and MEK inhibitors bypass MAP3K8-driven resistance, Johannessen and colleagues ectopically expressed MAP3K8 in A375 mel- anoma cells before treatment with BRAF inhibitor (PLX4720) alone or in combination with the MEK inhibitors CI-1040 or AZD6244. As shown in Figures 4E and 4F, both viability and ERK activation was dramatically reduced in MAP3K8-expressing cells treated with either of the combination therapies, similar to cells ectopically expressing MEK1, which remained sensitive to PLX4720 (Johannessen et al., 2010). Similar results were obtained in RPMI-7951 cells expressing high basal levels of MAP3K8 treated with PLX4032 and a second MEK inhibitor AS703026 (Park et al., 2013). Interestingly, overexpression of constitutively active MEK (MEK1DD) resulted in increased sensitivity to BRAF inhibition combined with AZD6244, but not CI-1040 (Johannessen et al., 2010). These find- ings confirm that MAP3K8 is able to reactivate MAPK signaling despite BRAF inhibition and that tar- geting RAF and MEK in combination may be an effective anti-melanoma treatment strategy. These experiments will be replicated in Protocols 5 and 6. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 2 of 32 Registered report Registered report Biochemistry Sampling p g Experiment to be repeated a total of 4 times for a minimum power of 80%. The original data is qual- itative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells . Cohort 2: RPMI-7951 cells . Cohort 3: OUMS-23 cells Each cohort has four conditions: . Vehicle (DMSO) . 10 mM PLX4720 . 1 mM PLX4720 . 0.1 mM PLX4720 Each condition will be probed with the following antibodies: . pERK1/2 (T202/Y204) . pERK1/2 . pMEK1/2 (S217/221) . MEK1/2 . MAP3K8 . Actin Materials and reagents Reagent Type Manufacturer Catalog # Comments RPMI medium with L-glutamine Cell culture Sigma R8758 Replaces Corning cat no. 10-040-CV. Communicated by authors. MEM with L-glutamine Cell culture Sigma M4655-500ML Replaces Corning cat. No. 10-010-CV. Communicated by authors. Fetal bovine serum (FBS) Cell culture Life Technologies 12483-020 Replaces Corning brand. Communicated by authors. Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. Communicated by authors. A375 cells Cell line ATCC CRL-1619 Original brand not specified. RPMI-7951 cells Cell line ATCC HTB-66 OUMS-23 cells Cell line JCRB JCRB1022 Original brand not specified. 6-well plates Labware Greiner bio-one 657 160 Original brand not specified. Phosphate buffered saline (PBS) Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. 3 of 32 Biochemistry Continued Reagent Type Manufacturer Catalog # Comments 10 cm plates Labware CellStar 664 160 Original brand not specified. PLX4720 Inhibitor Selleck Chemicals S1152 Replaces Symansis brand. DMSO Chemical Sigma D4540 Original brand not specified. NP-40 buffer Buffer Life tech FNN0021 Original brand not specified. Protease inhibitors Inhibitor Roche 04693116001 Original catalog # not specified. Phosphatase inhibitor cocktail I Inhibitor Sigma P2850 Replaces CalBioChem brand. Phosphatase inhibitor cocktail II Inhibitor Sigma P5726 Replaces CalBioChem brand. Cell scraper Labware Sarstedt 83.1830 Original brand not specified. BCA kit Reporter assay Pierce 23227 Original catalog # not specified. Communicated by authors. Dithiothreitol (DTT) Chemical Biobasic DB0058 Original brand not specified. Sample buffer Buffer Abm G031 Replaces Invitrogen brand. Protein molecular weight ladder Western materials Abm G252, G494 Original brand not specified. 10% Tris/Glycine gel; 10 well, 1.0 mm thick Western materials Abm Internal Replaces Invitrogen brand. Running buffer Buffer Abm Internal Original brand not specified. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 p p y g . Cells will be sent for mycoplasma testing and STR profiling. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. y g p . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS an 1% penicillin/streptomycin/L glutamine at 37˚C in a humidified atmosphere at 5% CO Sampling Immobilon P Western materials Thermofisher IPVH00010 Original brand not specified. Transfer buffer Buffer Abm Internal Original brand not specified. Mouse anti-pERK1/2 (T202/Y204) (clone E10) antibody (clone E10) Antibodies Cell Signaling 9106 Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-pMEK1/2 (S217/221) (clone 41G9) antibody Antibodies Cell Signaling 9154 Use at 1:11000 dilution. Original catalog # not specified. Mouse anti-p44/42 MAPK (ERK1/2) (clone L34F12) antibody Antibodies Cell Signaling 4696 Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors. Rabbit anti-MEK1/2 (clone D1A5) antibody Antibodies Cell Signaling 8727 Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-MAP3K8 (clone M-20) antibody Antibodies Santa Cruz sc-720 Use at 1:500 dilution. Communicated by authors. Mouse anti-ß-Actin (clone C4) antibody Antibodies Santa Cruz sc-47778 Use at 1:100 – 1:1000 dilution. Original catalog # not specified. Anti-rabbit IgG – HRP conjugated antibody Antibodies Cell Signaling 7074 Use at 1:1000 dilution. Original catalog # not specified. Anti-mouse IgG – HRP conjugated antibody Antibodies Cell Signaling 7076 Use at 1:1000 dilution. Original catalog # not specified. Chemiluminescent reagent Western materials Life Technologies WP20005 Replaces Pierce brand. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 4 of 32 4 of 32 Registered report Registered report Biochemistry 1. Plate 500,000 A375 cells, 750,000 RPMI-7951, and 750,000 OUMS-23 cells in 6-well plates and incubate for 24–36 hr to achieve log phase growth. 1. Plate 500,000 A375 cells, 750,000 RPMI-7951, and 750,000 OUMS-23 cells in 6-well plates and incubate for 24–36 hr to achieve log phase growth. g p g 2. 24–36 hr after seeding treat cells with 0.1, 1, and 10 mM PLX4720 or DMSO. Incubate for 24 hr. a. Add drug directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.1%. 2. 24–36 hr after seeding treat cells with 0.1, 1, and 10 mM PLX4720 or DMSO. Incubate for 24 hr. a Add drug directly to each well using a 1000X stock (in DMSO) a. Add drug directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.1%. a. Add drug directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.1%. 3. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. 3. Sampling Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between 2–3 mg/ml. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between 2–3 mg/ml. mg m b. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifuga- tion at max speed (table-top microfuge) at 4˚C. b. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifuga- b. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifuga- tion at max speed (table-top microfuge) at 4˚C. tion at max speed (table-top microfuge) at 4˚C. 4. Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature at 88˚C. 5. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow- ing replicating lab’s standard protocol. ing replicating lab’s standard protocol g ep cat g ab s sta da d p otoco . a. Samples run per gel: i. Protein molecular weight marker ii. Vehicle (DMSO) treated A375 cells iii. 10 mM PLX4720 treated A375 cells iv. 1 mM PLX4720 treated A375 cells v. 0.1 mM PLX4720 treated A375 cells vi. Vehicle (DMSO) treated RPMI-7951 cells vii. 10 mM PLX4720 treated RPMI-7951 cells viii. 1 mM PLX4720 treated RPMI-7951 cells ix. 0.1 mM PLX4720 treated RPMI-7951 cells x. Vehicle (DMSO) treated OUMS-23 cells xi. 10 mM PLX4720 treated OUMS-23 cells xii. 1 mM PLX4720 treated OUMS-23 cells xiii. 0.1 mM PLX4720 treated OUMS-23 cells a. Samples run per gel: a. Samples run per gel: i. Protein molecular weight marker ii. Vehicle (DMSO) treated A375 cells iii. 10 mM PLX4720 treated A375 cells iv. 1 mM PLX4720 treated A375 cells v. 0.1 mM PLX4720 treated A375 cells vi. Vehicle (DMSO) treated RPMI-7951 cells vii. 10 mM PLX4720 treated RPMI-7951 cells viii. 1 mM PLX4720 treated RPMI-7951 cells ix. 0.1 mM PLX4720 treated RPMI-7951 cells x. Vehicle (DMSO) treated OUMS-23 cells xi. 10 mM PLX4720 treated OUMS-23 cells xii. 1 mM PLX4720 treated OUMS-23 cells xiii. 0.1 mM PLX4720 treated OUMS-23 cells i. Protein molecular weight marker ii. Vehicle (DMSO) treated A375 cells iii. 10 mM PLX4720 treated A375 cells iv. 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, d h d l h l f ll f 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions. Confirmatory analysis plan . Statistical Analysis of the Replication Data:  Two-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of A375, RPMI-7951, and OUMS-23 cells with the following planned comparisons using the Bonferron  Two-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of A375, RPMI-7951, and OUMS-23 cells with the following planned comparisons using the Bonferron correction: & Planned contrast of normalized pERK1/2 levels from A375 cells treated with vehicle compared to cells treated with PLX4720 (all doses). & Planned contrast of normalized pERK1/2 levels from A375 cells treated with vehicle & Planned contrast of normalized pERK1/2 levels from A375 cells tr compared to cells treated with PLX4720 (all doses). p compared to cells treated with PLX4720 (all doses). & Planned contrast of normalized pERK1/2 levels from RPMI-7951 cells treated with vehicle compared to cells treated with PLX4720 (all doses). & Planned contrast of normalized pERK1/2 levels from RPMI- vehicle compared to cells treated with PLX4720 (all doses). p & Planned contrast of normalized pERK1/2 levels from OUMS-23 cells treated with vehicle compared to cells treated with PLX4720 (all doses). p & Planned contrast of normalized pMEK1/2 levels from A375 cells treated with vehicle compared to cells treated with PLX4720 (all doses). p & Planned contrast of normalized pMEK1/2 levels from RPMI-7951 cells treated with vehicle compared to cells treated with PLX4720 (all doses). & Planned contrast of normalized pMEK1/2 levels from OUMS-23 cells treated with vehicle compared to cells treated with PLX4720 (all doses). p . Meta-analysis of original and replication attempt effect sizes:  The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison. Known differences from the original study The replication will not include the other BRAF (V600E) cell lines reported in the original paper. The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and 1% NP-40. The western blots will use Actin, instead of Vinculin, which was reported in Figure 3B. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. Provisions for quality control y The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made pub- lically available. Sampling pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). c. MAP3K8 normalized to Actin. 10. Repeat steps 1–9 independently three additional times. Biochemistry Biochemistry 9. Analyze bands with image analysis software and normalize to loading controls. a pERK1/2 (T202/Y204) normalized to MEK1/2 (total) a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). p pMEK1/2 (S217/221) normalized to ERK1/2 (tota c. MAP3K8 normalized to Actin. 10. Repeat steps 1–9 independently three additional times. Deliverables: . Data to be collected: . Data to be collected:  Full image western blot films of all immunoblots including ladder. (Compare to Figures 3B and 3E)  Raw data of band analysis and normalized bands for each sample.  Full image western blot films of all immunoblots including ladder. (Compare to Figures 3B and 3E)  Full image western blot films of all immunoblots including ladder. (Compare to Figures 3B and 3E) )  Raw data of band analysis and normalized bands for each sample. Confirmatory analysis plan All differences have the same capabilities as the original and are not expected to alter the experimental design. Sampling 1 mM PLX4720 treated A375 cells v. 0.1 mM PLX4720 treated A375 cells m vi. Vehicle (DMSO) treated RPMI-7951 cells vii. 10 mM PLX4720 treated RPMI-7951 cells viii. 1 mM PLX4720 treated RPMI-7951 cells ix. 0.1 mM PLX4720 treated RPMI-7951 cells x. Vehicle (DMSO) treated OUMS-23 cells xi. 10 mM PLX4720 treated OUMS-23 cells xii. 1 mM PLX4720 treated OUMS-23 cells xiii. 0.1 mM PLX4720 treated OUMS-23 cells 6. Wet transfer with supplied wet-transfer cassette apparatus to immobilon P following repli- cating lab’s standard protocol. 6. Wet transfer with supplied wet-transfer cassette apparatus to immobilon P following repli- cating lab’s standard protocol. a. Original transfer protocol was for 120min at 30–35 V at 4˚C. cating lab s standard protocol. a. Original transfer protocol was for 120min at 30–35 V at 4˚C. g a. Original transfer protocol was for 120min at 30–35 V at 4˚C. g p 7. After transfer, block non-specific binding and immunoblot membrane with the following pri- mary antibodies for 18 at 4˚C following manufacturer recommendations: a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa e. rabbit anti- MAP3K8; use at 1:500 dilution; 52, 58 kDa f. mouse anti-ß-Actin; use at 1:100 - 1:1000 dilution; 43 kDa 7. After transfer, block non-specific binding and immunoblot membrane with the following pri- mary antibodies for 18 at 4˚C following manufacturer recommendations: a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa e. rabbit anti- MAP3K8; use at 1:500 dilution; 52, 58 kDa f. mouse anti-ß-Actin; use at 1:100 - 1:1000 dilution; 43 kDa Protocol 1 Western Blot Antibody POI Loading Control Independent Gels Description Working Conc. Description Working Conc. 1 Mouse anti-pERK1/2 (T202/Y204) (42, 44kDa) 1:1000 Rabbit anti-MEK1/2 (45 kDa) 1:1000 2 Rabbit anti-pMEK1/2 (S217/221) (45 kDa) 1:1000 Mouse anti-ERK1/2 (42, 44 kDa) 1:1000 3 Rabbit anti-MAP3K8 (52, 58 kDa) 1:500 Mouse anti-ß-Actin (43 kDa) 1:100 – 1:1000 Protocol 1 Western Blot Antibody Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 5 of 32 Registered report 9. Analyze bands with image analysis software and normalize to loading controls. a. Protocol 2: Determine the range of detection of the replicating lab’s plate reader This is a general protocol that determines the range of detection of the plate reader in order to cal- culate the required number of A375, RPMI-7951, and OUMS-23 cells to yield 90–95% confluency in 5 days for Protocols 3 and 5. 6 of 32 6 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report Biochemistry Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep- tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. Sampling Sampling This experiment is performed a total of once with three cell lines (A375, RPMI-7951, and OUMS-23 cells) p g This experiment is performed a total of once with three cell lines (A375, RPMI-7951, and OUM cells). Each cell line has 5 conditions to be performed with six technical replicates per experiment: . A375 cells:  1600 cells/well  1400 cells/well  1200 cells/well  1000 cells/well  800 cells/well . RPMI-7951 cells  3400 cells/well  3200 cells/well  3000 cells/well  2800 cells/well  2600 cells/well . OUMS-23 cells  3400 cells/well  3200 cells/well  3000 cells/well  2800 cells/well  2600 cells/well Materials and reagents Materials and reagents Reagent Type Manufacturer Catalog # Comments RPMI medium with L-glutamine Cell culture Sigma R8758 Replaces Corning cat no. 10-040-CV. MEM with L-glutamine Cell culture Sigma M4655-500ML Replaces Corning cat. no. 10-010-CV. FBS Cell culture Life Technologies 12483-020 Replaces Corning brand. Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. A375 cells Cell line ATCC CRL-1619 Original brand not specified. RPMI-7951 cells Cell line ATCC HTB-66 OUMS-23 cells Cell line JCRB JCRB1022 Original brand not specified. PBS Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. 10 cm plates Labware CellStar 664 160 Original brand not specified. 96 well clear plates Labware Sarstedt 83.3924 Original brand not specified. WST1 viability assay Reporter assay Roche 11644807001 Original catalog # not specified. Microplate reader (420–480 nm) Instrument Molecular Devices abm Original brand not specified. Registered report Procedure N . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep- tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep- tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 7 of 32 7 of 32 Registered report 1. Plate 800 – 1600 A375 cells, 2600 – 3400 RPMI-7951 cells, and 2600 – 3400 OUMS-23 cells in 96 well plates with 100 ml of medium. Incubate for 5 days. 1. Plate 800 – 1600 A375 cells, 2600 – 3400 RPMI-7951 cells, and 2600 – 3400 OUMS-23 cells in 96 well plates with 100 ml of medium. Incubate for 5 days. a. Plate media alone (no cells) in columns 1 and 12. b Plate cells in remaining wells (columns 2 11) a. Plate media alone (no cells) in columns 1 and 1 a. Plate media alone (no cells) in columns 1 and 12. b. Plate cells in remaining wells (columns 2–11). b. Plate cells in remaining wells (columns 2–11). b. Plate cells in remaining wells (columns 2–11). c. Exclude plating in the first and last row to avoid edge effects and evaporation. 2. 5 days later estimate confluency and determine cell viability with the WST1 viability as c. Exclude plating in the first and last row to avoid edge effects and evaporation. 2. 5 days later estimate confluency and determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described: according to manufacturer’s instructions. Briefly described: according to manufacturer’s instructions. Briefly described: a. Add 11 ml/well reagent WST-1 (1:10 dilution). m g b. Incubate cells for 20–30 min. b. Incubate cells for 20–30 min. c. Shake thoroughly for 1 min on a shaker. d. Measure the absorbance against a background control as blank using a microplate reader at 420–480 nm. Provisions for quality control The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. This All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available. Protocol 3: PLX4720 growth inhibitory analysis in cells expressing elevated MAP3K8 This experiment assesses the effect the RAF inhibitor, PLX4720, has on cellular viability, in cells expressing elevated MAP3K8. It utilizes RPMI-7951 and OUMS-23 cells, which express a high level of MAP3K8 and A375 cells, which have undetectable levels. This protocol replicates the experiment reported in Figure 3D. Deliverables . Data to be collected: . Data to be collected:  Raw data and background subtracted absorbance at 420–480 nm. Confirmatory analysis plan Confirmatory analysis plan Known differences from the original study All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design. Procedure N (If reference wavelength is to be determined, a filter >600 nm is recommended) e. Exclude rows A-H due to edge effects/evaporation, thus making each seeding six techni- cal replicates. f. Calculate viability after background subtraction. f. Calculate viability after background subtraction. g. Use starting cell numbers that give ~ 90–95% confluency in 5 days and is in the linear range of the viability assay. i. Original report used 1500 A375 cells, 3000 RPMI-7951 cells, and 3000 OUMS-23 cells. Deliverables . See Power Calculations section for details. Sampling p g Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is from a single biological replicate, thus to determine an appropriate number of replicates to initially per- form, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has 3 cohorts: Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 8 of 32 Registered report Registered report Biochemistry . Cohort 1: A375 cells . Cohort 2: RPMI-7951 cel . Cohort 3: OUMS-23 cells . Cohort 1: A375 cells . Cohort 2: RPMI-7951 cells . Cohort 3: OUMS-23 cells Each cohort has 9 conditions to be performed with six technical replicates per experiment: . DMSO (vehicle) . 100 mM PLX4720 . 10 mM PLX4720 . 1 mM PLX4720 . 0.1 mM PLX4720 . 0.01 mM PLX4720 . 0.001 mM PLX4720 . 0.0001 mM PLX4720 . 0.00001 mM PLX4720 Materials and reagents Materials and reagents Reagent Type Manufacturer Catalog # Comments RPMI medium with L-glutamine Cell culture Sigma R8758 Replaces Corning cat no. 10-040-CV. MEM with L-glutamine Cell culture Sigma M4655-500ML Replaces Corning cat. no. 10-010-CV. FBS Cell culture Life Technologies 12483-020 Replaces Corning brand. Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. A375 cells Cell line ATCC CRL-1619 Original brand not specified. RPMI-7951 cells Cell line ATCC HTB-66 OUMS-23 cells Cell line JCRB JCRB1022 Original brand not specified. PBS Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. 10 cm plates Labware CellStar 664 160 Original brand not specified. 96 well clear plates Labware Sarstedt 83.3924 Original brand not specified. PLX4720 Inhibitor Selleck Chemicals S1152 Replaces Symansis brand. DMSO Chemical Sigma D4540 Original brand not specified. WST1 viability assay Reporter assay Roche 11644807001 Original catalog # not specified. Microplate reader (420-480 nm) Instrument Molecular Devices abm Original brand not specified. Deliverables . Data to be collected:  Raw data and back . Data to be collected: Data to be collected: Data to be collected:  Raw data and background subtracted absorbance at 420–480 nm. Raw data and background subtracted absorbance  Raw data and background subtracted ab f GI50 values of each biological replicate.  GI50 values of each biological replicate.  Graph of average GI50 values for each condition. (Compare to Figure 3D.) Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Confirmatory analysis plan . Statistical Analysis of the Replication Data: . Statistical Analysis of the Replication Data:  One way ANOVA of GI50 values from A375, RPMI-7951, and OUMS-23 cells with the fol- lowing planned comparisons using Fisher’s LSD test. y p  One way ANOVA of GI50 values from A375, RPMI-7951, and OUMS-23 cells with the fol- lowing planned comparisons using Fisher’s LSD test. . A375 cells compared to RPMI-7951 cells. . A375 cells compared to OUMS-23 cells. . Meta-analysis of original and replication attempt effect sizes:  The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison. Known differences from the original study The replication will not include the other BRAF (V600E) cell lines reported in the original paper. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design. Provisions for quality control Procedure Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO (100 mM to 0.01 mM). b Dil t 1000X i l dil ti t k 1 100 i l t th di t i ld 10X t k a. Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO (100 mM to 0.01 mM). m b. Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock (1 mM to 10-4 mM) that is added directly to the 90 ml of cell/medium. b. Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock (1 mM to 10-4 mM) that is added directly to the 90 ml of cell/medium. i. Final DMSO concentration kept to 0.1%. Final DMSO concentration kept to 0.1%. 3. Determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described: y a. Add 11 ml/well reagent WST-1 (1:10 dilution). a. Add 11 ml/well reagent WST-1 (1:10 dilution). b. Incubate cells for 20–30 min. c. Shake thoroughly for 1 min on a shaker. c. Shake thoroughly for 1 min on a shaker. d. Measure the absorbance against a background control as blank using a microplate reader at 420–480 nm. (If reference wavelength is to be determined, a filter >600 nm is recommended) e. Exclude rows A-H due to edge effects/evaporation, thus making each cohort six techni- cal replicates, except DMSO (vehicle), which has 12. f. Calculate viability as a percentage of control (DMSO (vehicle) cells) after background subtraction. g. Determine GI50 value by fitting data using a nonlinear regression curve fit with a sigmoid dose-response curve (four-parameter log-logistic function). 4. Repeat steps 1–3 independently two additional times. Deliverables Procedure . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep- tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. y g . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. 1. Plate number of A375, RPMI-7951, and OUMS-23 cells as determined in Protocol 2 in a 96 well plate with 90 ml of medium per well. Incubate for 24 hr. a Plate media alone (no cells) in columns 1 and 12 a. Plate media alone (no cells) in columns 1 and 12. b. Plate cells in remaining wells (columns 2–11). g c. Exclude plating in the first and last row to avoid edge effects and evaporation. d. One plate is needed for each cell line. c. Exclude plating in the first and last row to avoid edge effects and evaporation. f p g g p d. One plate is needed for each cell line. d. One plate is needed for each cell line. d. One plate is needed for each cell line. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 9 of 32 Registered report Biochemistry 2. Treat cells with 10 ml of 10X serial dilutions of PLX4720 to yield final dilutions of 100 mM to 10-5 mM (8 dilutions) (columns 3 through 10), or treat with DMSO (vehicle) control (columns 2 and 11). Incubate for 96 hr. a. Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO (100 mM to 0.01 mM). b. Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock (1 mM to 10-4 mM) that is added directly to the 90 ml of cell/medium. i Final DMSO concentration kept to 0 1% 2. Treat cells with 10 ml of 10X serial dilutions of PLX4720 to yield final dilutions of 100 mM to 10-5 mM (8 dilutions) (columns 3 through 10), or treat with DMSO (vehicle) control (columns 2 and 11). Incubate for 96 hr. 10-5 mM (8 dilutions) (columns 3 through 10), or treat with DMSO (vehicle) control (columns 2 and 11). Incubate for 96 hr. a. Provisions for quality control Continued on next page Protocol 4: MAPK pathway analysis after MAP3K8 inhibition in cells expressing elevated MAP3K8 This experiment assesses the effect a MAP3K8 kinase inhibitor has on the MAPK pathway, in cells expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 cells, which express a high level of MAP3K8. This protocol replicates the experiment reported in Figure 3I. Provisions for quality control q y The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. The seeding density of each cell line was determined in Protocol 2. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available. 10 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report Registered report Biochemistry Protocol 4: MAPK pathway analysis after MAP3K8 inhibition in cells expressing elevated MAP3K8 This experiment assesses the effect a MAP3K8 kinase inhibitor has on the MAPK pathway, in cells expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 cells, which express a high level of MAP3K8. This protocol replicates the experiment reported in Figure 3I. Sampling Experiment to be repeated a total of 8 times for a minimum power of 80%. The original data is qual- itative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has five conditions: . Vehicle (DMSO) treated RPMI-7951 cells . 20 mM MAP3K8 inhibitor treated RPMI-7951 cells . 10 mM MAP3K8 inhibitor treated RPMI-7951 cells . 5 mM MAP3K8 inhibitor treated RPMI-7951 cells . 1 mM MAP3K8 inhibitor treated RPMI-7951 cells Each condition will be probed with the following antibodies: . pERK1/2 (T202/Y204) . pERK1/2 . pMEK1/2 (S217/221) . MEK1/2 . Vinculin Materials and reagents Reagent Type Manufacturer Catalog # Comments MEM with L-glutamine Cell culture Sigma M4655-500ML Replaces Corningcat. no. 10-010-CV FBS Cell culture Life Technologies 12483-020 Replaces Corning brand. Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. RPMI-7951 cells Cell line ATCC HTB-66 PBS Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. 10 cm plates Labware CellStar 664 160 Original brand not specified. 6 well plates Labware Greiner bio-one 657 160 Original brand not specified. MAP3K8 kinase inhibitor Inhibitor EMD 616373 DMSO Chemical Sigma D4540 Original brand not specified. NP-40 buffer Buffer Life Technologies FNN0021 Original brand not specified. Protease inhibitors Inhibitor Roche 04693116001 Original catalog # not specified. Phosphatase inhibitor cocktail I Inhibitor Sigma P2850 Replaces CalBioChem brand. Phosphatase inhibitor cocktail II Inhibitor Sigma P5726 Replaces CalBioChem brand. Cell scraper Labware Sasrstedt 83.1830 Original brand not specified. Registered report Sampling p g Experiment to be repeated a total of 8 times for a minimum power of 80%. The original data is qual- itative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has five conditions: . Vehicle (DMSO) treated RPMI-7951 cells . 20 mM MAP3K8 inhibitor treated RPMI-7951 cells . 10 mM MAP3K8 inhibitor treated RPMI-7951 cells . 5 mM MAP3K8 inhibitor treated RPMI-7951 cells . 1 mM MAP3K8 inhibitor treated RPMI-7951 cells Each condition will be probed with the following antibodies: Each condition will be probed with the following antibodies: Materials and reagents Materials and reagents Reagent Type Manufacturer Catalog # Comments MEM with L-glutamine Cell culture Sigma M4655-500ML Replaces Corningcat. no. 10-010-CV FBS Cell culture Life Technologies 12483-020 Replaces Corning brand. Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. RPMI-7951 cells Cell line ATCC HTB-66 PBS Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. 10 cm plates Labware CellStar 664 160 Original brand not specified. 6 well plates Labware Greiner bio-one 657 160 Original brand not specified. MAP3K8 kinase inhibitor Inhibitor EMD 616373 DMSO Chemical Sigma D4540 Original brand not specified. NP-40 buffer Buffer Life Technologies FNN0021 Original brand not specified. Protease inhibitors Inhibitor Roche 04693116001 Original catalog # not specified. Phosphatase inhibitor cocktail I Inhibitor Sigma P2850 Replaces CalBioChem brand. Phosphatase inhibitor cocktail II Inhibitor Sigma P5726 Replaces CalBioChem brand. Cell scraper Labware Sasrstedt 83.1830 Original brand not specified. 11 of 32 11 of 32 Biochemistry Continued Reagent Type Manufacturer Catalog # Comments BCA kit Reporter assay Pierce 23227 Original catalog # not specified. Communicated by authors. DTT Chemical Biobasic DB0058 Original brand not specified. Sample buffer Buffer abm G031 Replaces Invitrogen brand. Protein molecular weight ladder Western materials abm G252, G494 Original brand not specified. 10% Tris/Glycine gel; 10 well, 1.0 mm thick Western materials abm internal Replaces Invitrogen brand. Running buffer Buffer abm internal Original brand not specified. Immobilon P Western materials Thermofisher IPVH00010 Original brand not specified. Transfer buffer Buffer abm internal Original brand not specified. Mouse anti-pERK1/2 (T202/Y204) (clone E10) antibody Antibodies Cell Signaling 9106 Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-pMEK1/2 (S217/221) (clone 41G9) antibody Antibodies Cell Signaling 9154 Use at 1:11000 dilution. Original catalog # not specified. Sampling Mouse anti-p44/42 MAPK (ERK1/2) (clone L34F12) antibody Antibodies Cell Signaling 4696 Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors. Rabbit anti-MEK1/2 (clone D1A5) antibody Antibodies Cell Signaling 8727 Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-Vinculin antibody Antibodies Sigma V4139 Use at 1:20,000 dilution. Original catalog # not specified. Anti-rabbit IgG – HRP conjugated Antibodies Cell Signaling 7074 Use at 1:1000 dilution. Original catalog # not specified. Anti-mouse IgG – HRP conjugated Antibodies Cell Signaling 7076 Use at 1:1000 dilution. Original catalog # not specified. Chemiluminescent reagent Western materials Life Technologies WP20005 Replaces Pierce brand. Registered report Biochemistry Procedure . RPMI-7951 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/ streptomycin at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. 1. Plate 750,000 RPMI-7951 cells in 6-well plates and incubate for 24–36 hr to achieve log phase growth p g 2. Wash twice with 1X PBS and incubate overnight in serum-free growth medium. g g 3. Treat cells with 20, 10, 5, and 1 mM MAP3K8 inhibitor or DMSO for 1 hr. g g 3. Treat cells with 20, 10, 5, and 1 mM MAP3K8 inhibitor or DMSO for 1 hr. a Add drug directly to each well 3. Treat cells with 20, 10, 5, and 1 mM MAP3K8 inhibitor or DMSO for 1 hr. a. Add drug directly to each well. g y b. Make stocks of MAP3K8 inhibitor at 10 mM in DMSO. (500X dilution for 20 mM) c. Dilute stock of MAP3K8 to achieve 1000X final concentration of serial dilution stocks in DMSO (10 mM to 1000 mM). m d. Final DMSO concentration kept to 0.2%. . RPMI-7951 cells maintained in MEM medium supplemented with 10% FBS and 1% penicilli streptomycin at 37˚C in a humidified atmosphere at 5% CO2. m d. Final DMSO concentration kept to 0.2%. (Compare to Figure 3I)  Raw data of band analysis and normalized bands for each sample Full image western blot films of all immunoblots including ladder. (Compare to Figure 3I) Raw data of band analysis and normalized bands for each sample  Full image western blot films of all immunoblots including ladder. (Compare to Figure 3  Raw data of band analysis and normalized bands for each sample.  Raw data of band analysis and normalized bands for each sample. m d. Final DMSO concentration kept to 0.2%. d. Final DMSO concentration kept to 0.2%. 4. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 4. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. y y 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between 2–3 mg/ml. mg m b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation at max speed (table-top microfuge) at 4˚C. p p g 5. Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature at 88˚C. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 12 of 32 Registered report Biochemistry 6. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow- ing replicating lab’s standard protocol. 6. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow- ing replicating lab’s standard protocol. a. Samples run per gel: a. Samples run per gel: i. Protein molecular weight marker ii. Vehicle (DMSO) treated RPMI-7951 cells iii. 20 mM MAP3K8 inhibitor treated RPMI-7951 cells iv. 10 mM MAP3K8 inhibitor treated RPMI-7951 cells v. 5 mM MAP3K8 inhibitor treated RPMI-7951 cells vi. 1 mM MAP3K8 inhibitor treated RPMI-7951 cells Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to g p g p 8. After transfer, block non-specific binding and immunoblot membrane with the following pri- mary antibodies for 18 hr at 4˚C following manufacturer recommendations: a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa e. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa 8. After transfer, block non-specific binding and immunoblot membrane with the following pri- mary antibodies for 18 hr at 4˚C following manufacturer recommendations: a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa b. m d. Final DMSO concentration kept to 0.2%. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa e. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa Protocol 4 Western blot antibody combinations POI Loading Control Independent Gels Description Working Conc. Description Working Conc. 1 Mouse anti-pERK1/2 (T202/Y204) (42, 44 kDa) 1:1000 Rabbit anti-MEK1/2 (45 kDa) 1:1000 2 Rabbit anti-pMEK1/2 (S217/221) (45 kDa) 1:1000 Mouse anti-ERK1/2 (42, 44 kDa) 1:1000 3 Rabbit anti-Vinculin (116 kDa) 1:20000 Protocol 4 Western blot antibody combinations 9. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions. 10. Analyze bands with image analysis software, normalize to loading controls, and normalize each dose of MAP3K8 inhibitor to Vehicle (DMSO). a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). 9. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions. g g g a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions. g g g a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions. g 10. Analyze bands with image analysis software, normalize to loading controls, and normalize each dose of MAP3K8 inhibitor to Vehicle (DMSO). g 0. Analyze bands with image analysis software, normalize to loading controls, and normalize each dose of MAP3K8 inhibitor to Vehicle (DMSO). a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b pMEK1/2 (S217/221) normalized to ERK1/2 (total) a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). p 11. Repeat steps 1–10 independently seven additional times. Deliverables . Data to be collected:  Full image western blot films of all immunoblots including ladder. Provisions for quality control Provisions for quality control The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made pub- lically available. Protocol 5: Viability analysis following combinatorial MAPK pathway inhibition in cells expressing elevated MAP3K8 This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI- 1040 or AZD6244, has on cellular viability, in cells expressing MAP3K8. It utilizes A375 cells express- ing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment reported in Figure 4E. Known differences from the original study The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the fol- lowing composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and 1% NP-40. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design. Confirmatory analysis plan . Statistical Analysis of the Replication Data: . Statistical Analysis of the Replication Data:  One-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor with the following analysis using the Bonferroni correction: & One-way ANOVA of pERK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor. . One-sample t-test of pERK1/2 levels of 20 mM treated cells compared to 1 (vehi- cle treated cells). Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 13 of 32 Registered report Biochemistry & One-way ANOVA of pMEK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor. . One-sample t-test of pMEK1/2 levels of 20 mM treated cells compared to 1 (vehicle treated cells). (vehicle treated cells).  IC50 values of normalized pERK1/2 and pMEK1/2 levels treated with vehicle or MAP3K8 inhibitor. . Meta-analysis of original and replication attempt effect sizes: . Meta-analysis of original and replication attempt effect sizes:  The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison. Known differences from the original study Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sampling p g Generation of A375 cells expressing MEK1, MEK1DD, and MAP3K8 to be performed once. Experiment (steps 4–6) to be repeated a total of 4 times for a minimum power of 80%. The origi- nal data is from a single biological replicate, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. . See Power Calculations section for details Experiment has 3 cohorts: . Cohort 1: A375 cells expressing MEK1 . Cohort 2: A375 cells expressing MEK1DD . Cohort 3: A375 cells expressing MAP3K8 . See Power Calculations section for details. . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells expressing MEK1 h ll DD . Cohort 2: A375 cells expressing MEK1DD . Cohort 3: A375 cells expressing MAP3K8 Each cohort has 6 conditions to be done with six technical repeats per experiment: . Untreated [additional control] . DMSO (vehicle) . 10 mM PLX4720 . 1 mM PLX4720 . 1 mM PLX4720 + 1 mM AZD6244 . 1 mM PLX4720 + 1 mM CI-1040 . Untreated [additional control] . Untreated [additional control] . DMSO (vehicle) . 10 mM PLX4720 . 1 mM PLX4720 . 1 mM PLX4720 + 1 mM AZD6244 . 1 mM PLX4720 + 1 mM CI-1040 . 1 mM PLX4720 + 1 mM CI-1040 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 14 of 32 14 of 32 Materials and reagents Reagent Type Manufacturer Catalog # Comments RPMI medium with L-glutamine Cell culture Sigma R8758 Replaces Corning cat no. 10-040-CV. DMEM medium Cell culture Corning 10-013 Original brand not specified. FBS Cell culture Life Technologies 12483-020 Replaces Corning brand. Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. A375 cells Cell line ATCC CRL-1619 Original brand not specified. 293T cells Cell line ATCC CRL-11268 PBS Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. 10 cm plates Labware CellStar 664 160 Original brand not specified. 6 cm plates Labware Biolite 11825275 Original brand not specified. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sampling Nucleobond Maxiprep Kit Kit Macherey-Nagel 740414 Not originallyspecified pLX-Blast-V5-MEK1 DNA construct Provided from original authors pLX-Blast-V5-MEK1DD DNA construct Provided from original authors pLX-Blast-V5-MAP3K8 DNA construct Provided from original authors D8.9 (gag,pol) DNA construct Provided from original authors VSV-G DNA construct Provided from original authors FuGene HD transfection reagent Transfection reagent Promega E2311 Replaces FuGene6 Roche brand. OptiMEM medium Buffer Life Tech 51985034 Original brand not specified. 6-well plates Labware Greiner bio-one 657 160 Original brand not specified. Polybrene Cell culture Sigma H9268 Original brand not specified. Blasticidin Cell culture Invivogen ant-bl-1 Original brand not specified. 96 well clear plates Labware Sarstedt 83.3924 Original brand not specified. PLX4720 Inhibitor Selleck Chemicals S1152 Replaces Symansis brand. AZD6244 Inhibitor Selleck Chemicals S1008 Original catalog # not specified. CI-1040 Inhibitor Selleck Chemicals S1020 Replaces Shanghai Lechen International Trading Co. brand. DMSO Chemical Sigma D4540 Original brand not specified. WST1 viability assay Reporter assay Roche 11644807001 Original catalog # not specified. Microplate reader (420–480 nm) Instrument Molecular Devices abm Original brand not specified. Registered report Biochemistry Biochemistry y g p . 293T cells maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. 1. Grow and prepare endotoxin-free plasmid constructs according to the manufacturer’s proto- col for an endotoxin-free Plasmid Maxiprep Kit. a. Viral packaging vectors: i. D8.9 (gag,pol) 1. Grow and prepare endotoxin-free plasmid constructs according to the manufacturer’s proto- col for an endotoxin-free Plasmid Maxiprep Kit. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 15 of 32 Registered report Biochemistry ii. VSV-G b. DNA construct expression vectors: i. pLX-Blast-V5-MEK1 ii. pLX-Blast-V5-MEK1DD iii. pLX-Blast-V5-MAP3K8 b. DNA construct expression vectors: b. DNA construct expression vectors: b. DNA construct expression vectors: i. pLX-Blast-V5-MEK1 ii. pLX-Blast-V5-MEK1DD iii. pLX-Blast-V5-MAP3K8 p i. pLX-Blast-V5-MEK1 ii. pLX-Blast-V5-MEK1DD iii. pLX-Blast-V5-MAP3K8 2. Sequence expression plasmids to confirm identity and run on gel to confirm vector integrity. U th f ll i i i t fi th id tif f th LX CMV l id 2. Sequence expression plasmids to confirm identity and run on gel to confirm vector integrity. Use the following sequencing primers to confirm the identify of the pLX_CMV plasmids: a. pLX_CMV-ORF-fwd primer: 5’-CACCAAAATCAACGGGACTT-3’ b. pLX-ORF-rev primer: 5’-AGGAGGAGAAAATGAAAGCC-3’ 2. Sequence expression plasmids to confirm identity and run on gel to confirm vector integrity. Use the following sequencing primers to confirm the identify of the pLX_CMV plasmids: Use the following sequencing primers to confirm the identify of the pLX_CMV plasmids a. pLX_CMV-ORF-fwd primer: 5’-CACCAAAATCAACGGGACTT-3 b LX ORF i 5’ AGGAGGAGAAAATGAAAGCC 3’ 3. Produce MEK1, MEK1DD, and MAP3K8 lentivirus: 3. Produce MEK1, MEK1DD, and MAP3K8 lentivirus: a. Seed 8 x 105 293T cells per 6 cm dish, incubate. 3. Produce MEK1, MEK1DD, and MAP3K8 lentivirus: a. Seed 8 x 105 293T cells per 6 cm dish, incubate. b. 24 hr later, transfect each plate of 293T cells by adding the following: i. 1 mg pLX-Blast-V5-MEK1, pLX-Blast-V5-MEK1DD, or pLX-Blast-V5-MAP3K8 ii. 900 ng D8.9 (gag,pol) iii. 100 ng VSV-G iv. 6 ml FuGene transfection reagent v. 94 ml OptiMem medium (free of FBS and Pen/Strep). Add OptiMem medium to the aliquots of DNA, then add the FuGene to the OptiMem/DNA mix. vi. Incubate 30 min at RT, then add to cells. H t i 72 h t t f ti li t d f t 80˚C f t l t 24 h b f a. Seed 8 x 105 293T cells per 6 cm dish, incubate. b. 24 hr later, transfect each plate of 293T cells by adding the following: DD i. 1 mg pLX-Blast-V5-MEK1, pLX-Blast-V5-MEK1DD, or pLX-Blast-V5-MAP3K8 ii. 900 ng D8.9 (gag,pol) iii. 100 ng VSV-G iv. 6 ml FuGene transfection reagent v. 94 ml OptiMem medium (free of FBS and Pen/Strep). Add OptiMem medium to the aliquots of DNA, then add the FuGene to the OptiMem/DNA mix. c. Harvest virus 72 hr post-transfection, aliquot, and freeze at -80˚C for at least 24 hr before using. ii. VSV-G i Note: You will need to freeze down enough virus for both Protocols 5 and 6 i. Note: You will need to freeze down enough virus for both Protocols 5 and 6. 4. Titrate lentivirus: a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions. a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate fo 24 hr in normal growth conditions. i. Seeding density is such that cells will be near confluent 3 days after removing virus ii. Seed two wells per viral concentration for each virus (total wells = 30). 24 hr in normal growth conditions. i. Seeding density is such that cells will be near confluent 3 days after removing virus. ii. Seed two wells per viral concentration for each virus (total wells = 30). i. Seeding density is such that cells will be near confluent 3 days after removing virus. d ll l f h ( l ll ) b. Add polybrene (4–10 mg/ml final concentration) to plates, swirl to mix, then infect cells with varying concentrations of virus (1:5, 1:10, 1:12, 1:15, and 1:20) in duplicate (i.e. two wells per viral concentration). i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus get above 4˚C. ii. Add virus to the wells and swirl to mix. ii. Add virus to the wells and swirl to mix. iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. iv. Incubate cells overnight with virus, then change medium the following morning. c. Incubate for 24 hr, then remove medium and replace with growth medium with or with- out 10 mg/ml blasticidin. d. After 5–7 days of selection, count the cells in all wells and divide the counts with blastici- din by the non-selected control for each viral dilution. e. Use the lowest viral dilution that yields an 85–95% ratio of selected/non-selected cells (originally observed to be in the 1:8 – 1:15 range). 5. Infect A375 cells with viral supernatant: a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions. i. Seed three wells per virus. (total wells = 9). a. Biochemistry 6. After 48 hr trypsinize the experimental wells (one per virus), count, and plate number of A375 infected cells as determined in Protocol 2 in a 96 well plate in 90 ml. Incubate for 24 hr. a. Plate media alone (no cells) in columns 1,2,11, and 12. b. Plate cells in remaining wells (columns 3–10). 6. After 48 hr trypsinize the experimental wells (one per virus), count, and plate number of A375 infected cells as determined in Protocol 2 in a 96 well plate in 90 ml. Incubate for 24 hr. Pl di l ( ll ) i l 1 2 11 d 12 6. After 48 hr trypsinize the experimental wells (one per virus), count, and plate number of A375 infected cells as determined in Protocol 2 in a 96 well plate in 90 ml. Incubate for 24 hr. Pl di l ( ll ) i l 1 2 11 d 12 a. Plate media alone (no cells) in columns 1,2,11, b. Plate cells in remaining wells (columns 3–10). g c. Exclude plating in the first and last row to avoid edge effects and evaporation. d. One plate is needed for each of the A375 infected cells. 7. Treat cells with 10 ml of 10X dilutions of PLX4720 with or without AZD6244 or CI-1040 to yield appropriate final concentrations (columns 5 through 8), or treat with DMSO (vehicle) control (columns 4 and 9). Add 10 ml medium for untreated control (columns 3 and 10). Incu- bate for 96 hr. a. Dilute stock of PLX4720 at 1000X final concentration in DMSO (10 mM and 1 mM). b. Dilute stock of AZD6244 and CI-1040 at 1000X final concentration in DMSO (1 mM). c. Dilute 1000X dilution stocks 1:100 in complete growth medium to yield a 10X stock of appropriate treatment. pp p i. Final DMSO concentration kept to 0.2%. 8. Determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described: a. Add 11 ml/well reagent WST-1 (1:10 dilution). b. Incubate cells for 20–30 min. c. Shake thoroughly for 1 min on a shaker. i. Measure the absorbance against a background control as blank using a microplate reader at 420–480 nm. (If reference wavelength is to be determined, a filter > 600 nm is recommended) d. Biochemistry Exclude rows A-H due to edge effects/evaporation, thus making each cohort six techni- cal replicates, except DMSO (vehicle) and untreated control, which has 12. p p e. Calculate viability as a percentage of control (DMSO(vehicle) cells) after background subtraction. 9. Repeat steps 5–8 independently three additional times. Deliverables: . Data to be collected: . Data to be collected:  Raw counts and titration percentages (step 2 above).  Raw counts and infection efficiency (step 3 above).  Raw data and background subtracted absorbance at 420–480 nm. g  Relative viability of A375 cells expressing MEK1, MEK1DD, and MAP3K8 as a percentage g  Relative viability of A375 cells expressing MEK1, MEK1DD, and MAP3K8 as a percentage of DMSO treatment for each cell line. (Compare to Figure 4E.)  Relative viability of A375 cells expressing MEK1, MEK1DD, and MA of DMSO treatment for each cell line. (Compare to Figure 4E.) y p g , , of DMSO treatment for each cell line. (Compare to Figure 4E.) . Sample for additional protocol: DD  MEK1, MEK1DD, and MAP3K8 lentivirus for further use (Protocol 6). ii. VSV-G Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions. i. Seed three wells per virus. (total wells = . Add polybrene (4–10 mg/ml final concentration) to plates, swirl to mix, then infect cells with dilution determined by titration protocol (step 4 above). with dilution determined by titration protocol (step 4 above). i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus get above 4˚C. y p p i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus get above 4˚C. g ii. Add virus to the wells and swirl to mix. ii. Add virus to the wells and swirl to mix. 1. Alternatively, make a 3X virus/polybrene mixture, such that the addition of 1 ml of 3X virus to 2 ml of cells/medium yields an appropriate dilution of virus and polybrene. 1. Alternatively, make a 3X virus/polybrene mixture, such that the addition of 1 ml of 3X virus to 2 ml of cells/medium yields an appropriate dilution of virus and polybrene. p y iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. p y iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. iv. Incubate cells overnight with virus, then change medium the following morning. b f h h d d l h h d h h c. Incubate for 24 hr, then remove medium and replace with growth medium with or with- out 10 mg/ml blasticidin. c. Incubate for 24 hr, then remove medium and replace with growth medium with or with- out 10 mg/ml blasticidin. i. Two wells without selection and one well with blasticidin for each virus. out 10 mg/ml blasticidin. i. Two wells without selection and one well with blasticidin for each virus. mg i. Two wells without selection and one well with blasticidin for each virus. d. After 5–7 days of selection, count cells in one well of no-blasticidin and the blasticidin- treated well to calculate infection efficiency. y i. Divide the counts with blasticidin by the non-selected control. y i. Divide the counts with blasticidin by the non-selected control. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 16 of 32 Registered report Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Confirmatory analysis plan . Statistical Analysis of the Replication Data: . Statistical Analysis of the Replication Data:  One-way ANOVA of normalized viability of A375 cells expressing MAP3K8 treated with vehicle, 1 mM PLX4720, 1 mM PLX4720 + CI-1040, 1 mM PLX4720 + AZD6244, or 10 mM PLX4720 with the following planned comparisons using the Bonferroni correction: & 1 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. & 1 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244. & 10 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. 10 M PLX4720 d 1 M PLX4720 AZD6244 y  One-way ANOVA of normalized viability of A375 cells expressing MAP3K8 treated with vehicle, 1 mM PLX4720, 1 mM PLX4720 + CI-1040, 1 mM PLX4720 + AZD6244, or 10 mM PLX4720 with the following planned comparisons using the Bonferroni correction: g g & 1 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. m p m & 1 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244. & 1 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244. & 10 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. m p m & 10 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244.  Two-way ANOVA of normalized viability of A375 cells expressing MEK1 or MEK1DD treated with vehicle, 1 mM PLX4720, 1 mM PLX4720 + CI-1040, 1 mM PLX4720 + AZD6244, or 10 mM PLX4720.  The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 17 of 32 Registered report Biochemistry Known differences from the original study Known differences from the original study All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design. g y All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design. Confirmatory analysis plan Provisions for quality control The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. The seeding density of the A375 cell line was determined in Protocol 2. Infection efficiency will be determined for each replicate. The expression of the kinase of interest will be assessed using antibodies against the V5 tag as well as MAP3K8 and MEK1 as described in Protocol 6. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available. Protocol 6 MAPK pathway analysis following combinatorial MAPK pathway inhibition in cells expressing elevated MAP3K8 p y p g This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI- 1040 or AZD6244, has on the MAPK pathway, as analyzed via Western blot. It utilizes A375 cells expressing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment reported in Figure 4F. Registered report Sampling p g Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is qual- itative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined. based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells expressing MEK1 . Cohort 2: A375 cells expressing MEK1DD . Cohort 3: A375 cells expressing MAP3K8 Each cohort has 4 conditions: . DMSO (vehicle) . 1 mM PLX4720 . 1 mM PLX4720 + 1 mM AZD6244 . 1 mM PLX4720 + 1 mM CI-1040 Each condition will be probed with the following antibodies: . pERK1/2 (T202/Y204) . ERK1/2 . V5 [additional] . Vinculin . MEK1/2 [additional] . MAP3K8 [additional] Materials and reagents Reagent Type Manufacturer Catalog # Comments RPMI medium with L-glutamine Cell culture Sigma R8758 Replaces Corning cat no. 10-040-CV. FBS Cell culture Life Technologies 12483-020 Replaces Corning brand. Continued on next page Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 18 of 32 . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells expressing MEK1 . Cohort 2: A375 cells expressing MEK1DD . Cohort 3: A375 cells expressing MAP3K8 . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells expressing MEK1 D . Cohort 2: A375 cells expressing MEK1DD . Cohort 3: A375 cells expressing MAP3K8 Each cohort has 4 conditions: . DMSO (vehicle) . 1 mM PLX4720 . 1 mM PLX4720 + 1 mM AZD6244 . 1 mM PLX4720 + 1 mM CI-1040 . DMSO (vehicle) . 1 mM PLX4720 . 1 mM PLX4720 + 1 mM AZD6244 . 1 mM PLX4720 + 1 mM CI-1040 Each condition will be probed with the following antibodies: 18 of 32 Biochemistry Continued Reagent Type Manufacturer Catalog # Comments Pen/strep/glutamine Cell culture Abm G255 Replaces Corning brand. A375 cells Cell line ATCC CRL-1619 Original brand not specified. PBS Buffer Sigma D8537-500ML Original brand not specified. Trypsin Cell culture Sigma T4049 Original brand not specified. 10 cm plates Labware CellStar 664 160 Original brand not specified. 6 well plates Labware Greiner bio-one 657 160 Original brand not specified. Polybrene Cell culture Sigma H9268 Original brand not specified. Blasticidin Cell culture Invivogen ant-bl-1 Original brand not specified. Sampling PLX4720 Inhibitor Selleck Chemicals S1152 Replaces Symansis brand. AZD6244 Inhibitor Selleck Chemicals S1008 Original catalog # not specified. CI-1040 Inhibitor Selleck Chemicals S1020 Replaces Shanghai Lechen International Trading Co. brand. DMSO Chemical Sigma D4540 Original brand not specified. NP-40 buffer Buffer Life technologies FNN0021 Original brand not specified. Protease inhibitors Inhibitor Roche 04693116001 Original catalog # not specified. Phosphatase inhibitor cocktail I Inhibitor Sigma P2850 Replaces CalBioChem brand. Phosphatase inhibitor cocktail II Inhibitor Sigma P5726 Replaces CalBioChem brand. Cell scraper Labware Sasrstedt 83.1830 Original brand not specified. BCA kit Reporter assay Pierce 23227 Original catalog # not specified. Communicated by authors. DTT Chemical Biobasic DB0058 Original brand not specified. Sample buffer Buffer abm G031 Replaces Invitrogen brand. Protein molecular weight ladder Western materials abm G252, G494 Original brand not specified. 10% Tris/Glycine gel; 10 well, 1.0 mm thick Western materials abm internal Replaces Invitrogen brand. Running buffer Buffer abm internal Original brand not specified. Immobilon P Western materials Thermofisher IPVH00010 Original brand not specified. Transfer buffer Buffer abm internal Original brand not specified. Mouse anti-pERK1/2 (T202/Y204) (clone E10) antibody Antibodies Cell Signaling 9106 Use at 1:1000 dilution. Original catalog # not specified. Mouse anti-p44/42 MAPK (ERK1/2) (clone L34F12) antibody Antibodies Cell Signaling 4696 Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors. Mouse anti-V5-HRP conjugated antibody Antibodies Invitrogen R961-25 Use at 1:5000 dilution. Original catalog # not specified. Rabbit anti-Vinculin antibody Antibodies Sigma V4139 Use at 1:20,000 dilution. Original catalog # not specified. Rabbit anti-MEK1/2 (clone D1A5) antibody Antibodies Cell Signaling 8727 Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-MAP3K8 (clone M-20) antibody Antibodies Santa Cruz sc-720 Use at 1:500 dilution. Communicated by authors. Continued on next page 19 of 32 Continued Reagent Type Manufacturer Catalog # Comments Anti-rabbit IgG – HRP conjugated antibody Antibodies Cell Signaling 7074 Use at 1:1000 dilution. Original catalog # not specified. Anti-mouse IgG – HRP conjugated antibody Antibodies Cell Signaling 7076 Use at 1:1000 dilution. Original catalog # not specified. Chemiluminescent reagent Western materials Life Technologies WP20005 Replaces Pierce brand. Registered report Biochemistry Biochemistry . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep- tomycin at 37˚C in a humidified atmosphere at 5% CO2. y p . Cells will be sent for mycoplasma testing and STR profiling. . Cells will be sent for mycoplasma testing and STR profiling. 1. Sampling Infect A375 cells with viral supernatant: 1. Infect A375 cells with viral supernatant: a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions. i. Seed six wells per virus. (total wells = 18). a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions. b. Add polybrene (4–10 mg/ml final concentration) to plates, swirl to mix, then infect cells following Protocol 5, step 5 using dilution determined by titration protocol (Protocol 5, step 4). p c. Incubate for 24 hr, then remove medium and replace with growth medium with or with- out 10 mg/ml blasticidin. c. Incubate for 24 hr, then remove medium and replace with growth medium with or with- out 10 mg/ml blasticidin. i. Five wells without selection and one well with blasticidin for each virus. f f f mg i. Five wells without selection and one well with blasticidin for each virus. d. After 5–7 days of selection, count cells in one well of no-blasticidin and the blasticidin- treated well to calculate infection efficiency. i. Divide the counts with blasticidin by the non-selected control. 2. 72 hr after infection (96 hr after seeding) treat cells with DMSO or 1 mM PLX4720 with or without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 hr. a. Add drugs directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.2%. 2. 72 hr after infection (96 hr after seeding) treat cells with DMSO or 1 mM PLX4720 with or without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 hr. a Add drugs directly to each well using a 1000X stock (in DMSO) 2. 72 hr after infection (96 hr after seeding) treat cells with DMSO or without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 h a. Add drugs directly to each well using a 1000X stock (in DMSO). ( g) m without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 hr. without 1 mM AZD6244, 1 mM CI 1040, or DMSO. Incubate for 24 a. Add drugs directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.2% 3. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sampling Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 3. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. Add 100 200 l 1% NP 40 l i b ff h i i i b 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between 2–3 mg/ml. mg m b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation at max speed (table-top microfuge) at 4˚C. 4. Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature at 88˚C. 5. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow- l l b d d l 5. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow- ing replicating lab’s standard protocol. ing replicating lab’s standard protocol. a. Samples run per gel(s): i. Protein molecular weight marker ii. Vehicle (DMSO) treated A375 cells expressing MEK1 iii. 1 mM PLX4720 treated A375 cells expressing MEK1 iv. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1 v. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1 vi. Vehicle (DMSO) treated A375 cells expressing MEK1DD vii. 1 mM PLX4720 treated A375 cells expressing MEK1DD viii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1DD ix. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1DD x. Vehicle (DMSO) treated A375 cells expressing MAP3K8 xi. 1 mM PLX4720 treated A375 cells expressing MAP3K8 xii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MAP3K8 xiii. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MAP3K8 a. Samples run per gel(s): i. Protein molecular weight marker ii. Vehicle (DMSO) treated A375 cells expressing MEK1 ii. Vehicle (DMSO) treated A375 cells expressing MEK1 iii. 1 mM PLX4720 treated A375 cells expressing MEK1 iv. Sampling 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1 v. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1 vi. Vehicle (DMSO) treated A375 cells expressing MEK1DD iii. 1 mM PLX4720 treated A375 cells expressing MEK1 iv. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1 v. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1 vi. Vehicle (DMSO) treated A375 cells expressing MEK1DD DD vii. 1 mM PLX4720 treated A375 cells expressing MEK1DD viii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1DD x. Vehicle (DMSO) treated A375 cells expressing MAP3K8 xi. 1 mM PLX4720 treated A375 cells expressing MAP3K8 g xii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MAP3K8 g xiii. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MAP3K8 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 20 of 32 Registered report 6. Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to immobilon P following replicating lab’s standard protocol. 7. After transfer, block non-specific binding and immunoblot membrane with the following pri- mary antibodies for 18 hr at 4˚C following manufacturer recommendations: a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa c. mouse anti-V5-HRP; use at 1:5000 dilution; 45 kDa for MEK1, 52, 58 kDa for MAP3K8 d. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa e. rabbit anti-MAP3K8; use at 1:500 dilution; 52, 58 kDa f. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa Biochemistry Biochemistry 6. Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to immobilon P following replicating lab’s standard protocol. g p g p 7. After transfer, block non-specific binding and immunoblot membrane with the following pri- mary antibodies for 18 hr at 4˚C following manufacturer recommendations: a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa c. mouse anti-V5-HRP; use at 1:5000 dilution; 45 kDa for MEK1, 52, 58 kDa for MAP3K8 d. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa e. rabbit anti-MAP3K8; use at 1:500 dilution; 52, 58 kDa f. Sampling rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa Protocol 6 Western blot antibody combinations Protocol 6 Western blot antibody combinations POI Loading control Independent Gels Description Working Conc. Description Working Conc. 1 Mouse anti-pERK1/2 (T202/Y204) (42, 44 kDa) 1:1000 Rabbit anti-MEK1/2 (45 kDa) 1:1000 2 Mouse anti-V5-HRP (45, 52, 58 kDa) 1:5000 Rabbit anti-Vinculin (116 kDa) 1:20000 3 Rabbit anti-MAP3K8 (52, 58 kDa) 1:500 Mouse anti-ERK1/2 (42, 44 kDa) 1:1000 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions. 9. Analyze bands with image analysis software and normalize to loading controls. a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. V5 (tag on exogenous proteins) normalized to Vinculin. 10. Repeat steps 1–9 independently two additional times. 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation and then detect signal using chemiluminescence following manufacturer’s instructions. 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions. q g 9. Analyze bands with image analysis software and normalize to loading controls. y g y a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). p b. V5 (tag on exogenous proteins) normalized to Vinculin. p b. V5 (tag on exogenous proteins) normalized to Vinculin. 10. Repeat steps 1–9 independently two additional times. Deliverables: . Data to be collected: . Data to be collected:  Raw counts and infection efficiency (step 1 above).  Full image western blot films of all immunoblots including ladder. (Compare to Figures 2A and 4F.)  Raw data of band analysis and normalized bands for each sample. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Confirmatory analysis plan . Statistical Analysis of the Replication Data: y  One-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MAP3K8  One-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MAP3K8 treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244 with the fol l l d h f  One-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MAP3K8 treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244 with the fo lowing planned comparisons using the Bonferroni correction: & PLX4720 treatment compared to PLX4720 + CI-1040. & PLX4720 treatment compared to PLX4720 + AZD6244.  Two-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MEK1 or DD  Two-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MEK1 or MEK1DD treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244 y p / p g MEK1DD treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244. . Meta-analysis of original and replication attempt effect sizes: y g p p  The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 21 of 32 Registered report Biochemistry Known differences from the original study g y The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the fol- lowing composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and 1% NP-40. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design. Provisions for quality control The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. Infection efficiency will be deter- mined for each replicate. The expression of the kinase of interest will be assessed using antibodies against the V5 tag as well as MAP3K8 and MEK1. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available. Power Calculations For additional details on power calculations, please see analysis scripts and associated files on the Open Science Framework: https://osf.io/sptzv/ Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report h ( , ) Comparisons are between DMSO and all PLX4720 doses (10, 1, and 0.1 mM) Protocol 1 The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original effect size where possible. To identify a suitable sample size, power calculations were performed using different levels of relative variance. Summary of estimated original data reported in Figure 3E: Summary of estimated original data reported in Figure 3E: Cell line PLX4720 concentration (mM) Normalized pMEK/ERK Normalized pERK/MEK A375 0 (DMSO) 1.000 1.000 10 0.00212 0.0147 1 0.00283 0.0254 0.1 0.0981 0.00283 RPMI-7951 0 (DMSO) 1.000 1.000 10 0.827 1.849 1 0.380 1.460 0.1 0.471 1.022 OUMS-23 0 (DMSO) 1.000 1.000 10 0.0579 0.926 1 0.197 1.042 0.1 1.100 1.068 Sharma et al. eLife 2016;5:e11414. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Protocol 1 DOI: 10.7554/eLife.11414 22 of 32 Biochemistry Antibody Cell line F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance pMEK A375 F(1,8) = 20781 0.99962 50.9670 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 2128.4 0.99626 16.31121 80.0% 12 (4 groups) OUMS-23 F(1,8) = 3001.0 0.99734 19.36831 80.0% 12 (4 groups) pERK A375 F(1,8) = 21841 0.99963 52.2504 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 582.85 0.98646 8.535621 80.0% 12 (4 groups) OUMS-23 F(1,8) = 0.8080 0.09173 0.317801 80.0% 12 (4 groups) 15% variance pMEK A375 F(1,8) = 369.44 0.97880 6.79560 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 37.839 0.82547 2.174821 80.0% 12 (4 groups) OUMS-23 F(1,8) = 53.352 0.86960 2.582441 80.0% 12 (4 groups) pERK A375 F(1,8) = 388.28 0.97981 6.96672 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 10.362 0.56431 1.138081 80.0% 12 (4 groups) OUMS-23 F(1,8) = 0.01436 0.001792 0.042371 80.0% 12 (4 groups) 28% variance pMEK A375 F(1,8) = 106.68 0.92984 3.64050 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 10.859 0.57581 1.165091 80.0% 12 (4 groups) OUMS-23 F(1,8) = 15.312 0.65682 1.383451 80.0% 12 (4 groups) pERK A375 F(1,8) = 111.43 0.93302 3.73217 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 2.9737 0.27099 0.609691 80.0% 12 (4 groups) OUMS-23 F(1,8) = 0.04122 0.000515 0.022701 80.0% 12 (4 groups) 40% variance pMEK A375 F(1,8) = 51.952 0.86656 2.54835 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 5.3211 0.39945 0.815561 80.0% 12 (4 groups) OUMS-23 F(1,8) = 7.5026 0.48396 0.968421 80.0% 12 (4 groups) pERK A375 F(1,8) = 54.602 0.87221 2.61252 99.9% 12 (4 groups) RPMI-7951 F(1,8) = 1.4571 0.15408 0.426781 80.0% 12 (4 groups) OUMS-23 F(1,8) = 0.00202 0.000252 0.015891 80.0% 12 (4 groups) 1 This is the calculated effect size using the originally reported value with the indicated variance. Unlike the power calculations to determine sample size, the aim of these sensitivity calculations are not to detect the original effect Registered report Biochemistry Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 23 of 32 Registered report Registered report Biochemistry size, but to understand what effect size could be detected with 80% power and the indicated total sample size of 12. The detectable effect size is 1.29189. size, but to understand what effect size could be detected with 80% power and the indicated total sample size of 12. The detectable effect size is 1.29189. Protocol 1 In order to produce quantitative replication data, we will run the experiment three times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the num- ber of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Protocol 3 The original data is from a single biological replicate. To identify a suitable sample size, power calcu- lations were performed using different levels of relative variance. Summary of estimated original data reported in Figure 3D: Cell line GI50 (mM) A375 0.2276 RPMI-7951 10.862 OUMS-23 8.731 Test family & 2 tailed t test, difference between two independent means, Fisher’s LSD: alpha error = 0.05 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Group 1 Group 2 Effect size d A priori power Group 1 sample size Group 2 sample size 2% variance A375 RPMI-7951 69.2139 99.9% 2 2 A375 OUMS-23 55.3442 99.9% 2 2 15% variance A375 RPMI-7951 68.3898 99.9% 2 2 A375 OUMS-23 7.37923 93.3% 2 2 28% variance A375 RPMI-7951 4.94385 99.2% 3 3 A375 OUMS-23 3.95316 94.4% 3 3 40% variance A375 RPMI-7951 3.46070 88.0% 3 3 A375 OUMS-23 2.76721 90.0% 4 4 In order to produce quantitative replication data, we will run the experiment three times. Each time we will calculate the GI50 value. We will determine the standard deviation of GI50 values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Test family & 2 tailed t test, difference between two independent means, Fisher’s LSD: alpha error = 0.05 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). In order to produce quantitative replication data, we will run the experiment three times. Each time we will calculate the GI50 value. We will determine the standard deviation of GI50 values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. In order to produce quantitative replication data, we will run the experiment three times. Each time we will calculate the GI50 value. Registered report Protocol 3 We will determine the standard deviation of GI50 values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 24 of 32 Biochemistry Protocol 4 The original data does not indicate the error associated with multiple biological replicates. To iden- tify a suitable sample size, power calculations were performed using different levels of relative variance. Summary of original data reported in Figure 3I: Summary of original data reported in Figure 3I: IC50 values performed with R software, version 3.2.1 (Team, 2015). Summary of original data reported in Figure 3I: We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Protocol 5 The original data is from a single biological replicate. To identify a suitable sample size, power calcu- lations were performed using different levels of relative variance. Summary of original data reported in Figure 4E (provided by authors) Cell line Drug(s) Relative viability Technical replicate stdev MEK1 DMSO 100 5.09 PLX4720 (10 mM) 5.78 0.67 PLX4720 (1 mM) 11.32 0.97 PLX4720 (1 mM) + CI-1040 (1 mM) 10.13 0.60 PLX4720 (1 mM) + AZD6244 (1 mM) 10.92 0.91 MEK1DD DMSO 96.96 9.40 PLX4720 (10 mM) 97.56 6.25 PLX4720 (1 mM) 92.10 8.23 PLX4720 (1 mM) + CI-1040 (1 mM) 102.24 8.55 PLX4720 (1 mM) + AZD6244 (1 mM) 42.44 2.74 Continued on next page Registered report Biochemistry Test family & t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha error = 0.025 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). MAP3K8 inhibitor dose Constant (vehicle) Antibody Effect size d A priori power Sample size per group 2% variance 20 mM 0 mM pMEK 88.8889 99.9% 3 pERK 20.4225 99.9% 3 15% variance 20 mM 0 mM pMEK 11.8519 99.9% 3 pERK 2.72300 80.2% 4 28% variance 20 mM 0 mM pMEK 6.34921 95.1% 3 pERK 1.45875 86.6% 8 40% variance 20 mM 0 mM pMEK 4.44444 99.2% 4 pERK 1.02113 81.1% 12 Registered report Biochemistry Test family & t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha error = 0.025 ed report Biochemistry Biochemistry Test family & t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha error = 0.025 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). In order to produce quantitative replication data, we will run the experiment eight times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. Summary of original data reported in Figure 3I: Cell line MAP3K8 inhibitor concentration (mM) Normalized pMEK/ERK Normalized pERK/MEK RPMI-7951 0 (DMSO) 1.00 1.00 1 1.35 1.00 5 1.15 0.92 10 0.70 0.75 20 0.36 0.71 IC50 values performed with R software, version 3.2.1 (Team, 2015). IC50 value of normalized pMEK values IC50 value of normalized pERK values 9.4725 6.4091 Test family & F test, ANOVA: Fixed effects, omnibus, one-way, Bonferroni’s correction: alpha error = 0.025 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). Partial h2 calculated from (Lakens, 2013). Groups Antibody F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance MAP3K8 inhibitor (1, 5, 10, 20 mM) pMEK F(3,8) = 1583.7 0.99832 24.3697 99.9% 8 (4 groups) pERK F(3,8) = 195.33 0.98653 8.55863 99.9% 8 (4 groups) 15% variance MAP3K8 inhibitor (1, 5, 10, 20 mM) pMEK F(3,8) = 28.155 0.91348 3.24931 96.8% 8 (4 groups) pERK F(3,8) = 3.4726 0.56563 1.14114 83.6% 16 (4 groups) 28% variance MAP3K8 inhibitor (1, 5, 10, 20 mM) pMEK F(3,8) = 8.0801 0.75186 1.74070 94.7% 12 (4 groups) pERK F(3,8) = 0.9966 0.27205 0.61133 80.8% 40 (4 groups) 40% variance MAP3K8 inhibitor (1, 5, 10, 20 mM) pMEK F(3,8) = 3.9593 0.59754 1.21849 88.7% 16 (4 groups) pERK F(3,8) = 0.4883 0.15478 0.42793 80.5% 76 (4 groups) Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 25 of 32 Test family & t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha error = 0.025 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). MAP3K8 inhibitor dose Constant (vehicle) Antibody Effect size d A priori power Sample size per group 2% variance 20 mM 0 mM pMEK 88.8889 99.9% 3 pERK 20.4225 99.9% 3 15% variance 20 mM 0 mM pMEK 11.8519 99.9% 3 pERK 2.72300 80.2% 4 28% variance 20 mM 0 mM pMEK 6.34921 95.1% 3 pERK 1.45875 86.6% 8 40% variance 20 mM 0 mM pMEK 4.44444 99.2% 4 pERK 1.02113 81.1% 12 In order to produce quantitative replication data, we will run the experiment eight times. Each time we will quantify band intensity. Summary of original data reported in Figure 3I: We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Protocol 5 The original data is from a single biological replicate. To identify a suitable sample size, power calcu- lations were performed using different levels of relative variance. Summary of original data reported in Figure 4E (provided by authors) Summary of original data reported in Figure 4E (provided by authors) Cell line Drug(s) Relative viability Technical replicate stdev MEK1 DMSO 100 5.09 PLX4720 (10 mM) 5.78 0.67 PLX4720 (1 mM) 11.32 0.97 PLX4720 (1 mM) + CI-1040 (1 mM) 10.13 0.60 PLX4720 (1 mM) + AZD6244 (1 mM) 10.92 0.91 MEK1DD DMSO 96.96 9.40 PLX4720 (10 mM) 97.56 6.25 PLX4720 (1 mM) 92.10 8.23 PLX4720 (1 mM) + CI-1040 (1 mM) 102.24 8.55 PLX4720 (1 mM) + AZD6244 (1 mM) 42.44 2.74 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 26 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Continued Cell line Drug(s) Relative viability Technical replicate stdev MAP3K8 DMSO 100 10.66 PLX4720 (10 mM) 43.44 3.48 PLX4720 (1 mM) 62.81 5.05 PLX4720 (1 mM) + CI-1040 (1 mM) 13.42 2.42 PLX4720 (1 mM) + AZD6244 (1 mM) 12.52 1.65 Registered report Biochemistry Biochemistry Cell line Drug(s) Relative viability Technical replicate stdev MAP3K8 DMSO 100 10.66 PLX4720 (10 mM) 43.44 3.48 PLX4720 (1 mM) 62.81 5.05 PLX4720 (1 mM) + CI-1040 (1 mM) 13.42 2.42 PLX4720 (1 mM) + AZD6244 (1 mM) 12.52 1.65 PLX4720 (1 mM) + AZD6244 (1 mM) 12.52 1.65 Test family & F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MAP3K8 values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(4,25) = 6246.6 0.99900 31.60696 99.9% 10 (5 groups) 15% variance All treatments F(4,25) = 111.04 0.94672 4.21509 99.9% 15 (5 groups) 28% variance All treatments F(4,25) = 31.868 0.83604 2.25807 99.9% 20 (5 groups) 40% variance All treatments F(4,25) = 15.615 0.71416 1.58066 99.9% 25 (5 groups) Test family & 2 tailed t test, difference between two independent means, Bonferroni’s correction: alpha error = 0.0125 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Protocol 5 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(4,50) = 2716.2 0.99542 14.7409 99.9% 20 (10 groups) 15% variance All treatments F(4,50) = 48.287 0.79437 1.96545 99.9% 30 (10 groups) 28% variance All treatments F(4,50) = 13.858 0.52576 1.05292 99.9% 40 (10 groups) 40% variance All treatments F(4,50) = 6.7904 0.35201 0.73704 98.7% 50 (10 groups) In order to produce quantitative replication data, we will run the experiment four times. Each time we will quantify relative viability. We will determine the standard deviation of viability values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if i d t th t th i t h th 80% t d t t th i i l ff t Registered report Biochemistry Test family & F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(4,50) = 2716.2 0.99542 14.7409 99.9% 20 (10 groups) 15% variance All treatments F(4,50) = 48.287 0.79437 1.96545 99.9% 30 (10 groups) 28% variance All treatments F(4,50) = 13.858 0.52576 1.05292 99.9% 40 (10 groups) 40% variance All treatments F(4,50) = 6.7904 0.35201 0.73704 98.7% 50 (10 groups) In order to produce quantitative replication data, we will run the experiment four times. Each time we will quantify relative viability. We will determine the standard deviation of viability values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. Protocol 5 MAP3K8 values Group 1 Group 2 Effect size d A priori power Group 1 sample size Group 2 sample size 2% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 67.45484 99.9% 2 2 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 55.51810 99.9% 2 2 10 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 46.68351 99.9% 2 2 10 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 48.35265 99.9% 2 2 15% variance Continued on next page 27 of 32 Continued Group 1 Group 2 Effect size d A priori power Group 1 sample size Group 2 sample size 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 7.24974 99.4% 3 3 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 7.40241 99.6% 3 3 10 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 6.22447 97.2% 3 3 10 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 6.44702 97.9% 3 3 28% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 3.88379 93.0% 4 4 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 3.96558 94.0% 4 4 10 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 3.33454 82.9% 4 4 10 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 3.45376 85.7% 4 4 40% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 2.71865 82.6% 5 5 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 2.77591 84.3% 5 5 10 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 2.33418 81.5% 6 6 10 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 2.41763 84.5% 6 6 Registered report Biochemistry Biochemistry Biochemistry Continued Group 1 Group 2 Effect size d A priori power Group 1 sample size Group 2 sample size 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 7.24974 99.4% 3 3 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 7.40241 99.6% 3 3 10 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 6.22447 97.2% 3 3 10 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 6.44702 97.9% 3 3 28% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 3.88379 93.0% 4 4 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 3.96558 94.0% 4 4 10 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 3.33454 82.9% 4 4 10 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 3.45376 85.7% 4 4 40% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 2.71865 82.6% 5 5 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 2.77591 84.3% 5 5 10 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 2.33418 81.5% 6 6 10 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 2.41763 84.5% 6 6 Test family & F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Protocol 5 Summary of estimated original data reported in Figure 4F: Cell line Drug(s) Normalized pERK/ERK MEK1 DMSO 1.0000 PLX4720 (1 mM) 0.0799 PLX4720 (1 mM) + CI-1040 (1 mM) 0.0021 PLX4720 (1 mM) + AZD6244 (1 mM) 0.0002 MEK1DD DMSO 1.0000 PLX4720 (1 mM) 1.0203 PLX4720 (1 mM) + CI-1040 (1 mM) 0.3015 PLX4720 (1 mM) + AZD6244 (1 mM) 0.0166 MAP3K8 DMSO 1.0000 PLX4720 (1 mM) 0.7689 PLX4720 (1 mM) + CI-1040 (1 mM) 0.0045 PLX4720 (1 mM) + AZD6244 (1 mM) 0.0063 Test family . F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MAP3K8 values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance Registered report Biochemistry Biochemistry Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Protocol 5 We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Test family & F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(4,50) = 2716.2 0.99542 14.7409 99.9% 20 (10 groups) 15% variance All treatments F(4,50) = 48.287 0.79437 1.96545 99.9% 30 (10 groups) 28% variance All treatments F(4,50) = 13.858 0.52576 1.05292 99.9% 40 (10 groups) 40% variance All treatments F(4,50) = 6.7904 0.35201 0.73704 98.7% 50 (10 groups) MEK1 and MEK1DD values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(4,50) = 2716.2 0.99542 14.7409 99.9% 20 (10 groups) 15% variance All treatments F(4,50) = 48.287 0.79437 1.96545 99.9% 30 (10 groups) 28% variance All treatments F(4,50) = 13.858 0.52576 1.05292 99.9% 40 (10 groups) 40% variance All treatments F(4,50) = 6.7904 0.35201 0.73704 98.7% 50 (10 groups) In order to produce quantitative replication data, we will run the experiment four times. Each time we will quantify relative viability. We will determine the standard deviation of viability values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 28 of 32 Protocol 6 The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original effect size where possible. Protocol 6 We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to sim late the original effect si e We ill se this sim lated effect si e to determine the n mber of Group 1 Group 2 Effect size d A priori power Group 1 sample size Group 2 sample size 2% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 68.8057 99.9% 2 2 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 70.5269 99.9% 2 2 15% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 9.17409 87.8% 2 2 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 9.40358 89.0% 2 2 28% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 4.91469 95.5% 3 3 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 5.03763 96.3% 3 3 40% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 3.44028 93.7% 4 4 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 3.52634 94.7% 4 4 In order to produce quantitative replication data, we will run the experiment three times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect. Protocol 6 The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original effect size where possible. p Summary of estimated original data reported in Figure 4F: Summary of estimated original data reported in Figure 4F: Cell line Drug(s) Normalized pERK/ERK MEK1 DMSO 1.0000 PLX4720 (1 mM) 0.0799 PLX4720 (1 mM) + CI-1040 (1 mM) 0.0021 PLX4720 (1 mM) + AZD6244 (1 mM) 0.0002 MEK1DD DMSO 1.0000 PLX4720 (1 mM) 1.0203 PLX4720 (1 mM) + CI-1040 (1 mM) 0.3015 PLX4720 (1 mM) + AZD6244 (1 mM) 0.0166 MAP3K8 DMSO 1.0000 PLX4720 (1 mM) 0.7689 PLX4720 (1 mM) + CI-1040 (1 mM) 0.0045 PLX4720 (1 mM) + AZD6244 (1 mM) 0.0063 Test family . F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MAP3K8 values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(3,8) = 5024.1 0.99947 43.4257 99.9% 8 (4 groups) 15% variance All treatments F(3,8) = 89.317 0.97101 5.78735 99.9% 8 (4 groups) 28% variance All treatments F(3,8) = 25.633 0.90577 3.10038 99.9% 12 (4 groups) 40% variance All treatments F(3,8) = 12.560 0.82487 2.17026 99.9% 16 (4 groups) Test family . 2 tailed t test, difference between two independent means, Bonferroni’s correction: alpha error = 0.025 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). MAP3K8 values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(3,8) = 5024.1 0.99947 43.4257 99.9% 8 (4 groups) 15% variance All treatments F(3,8) = 89.317 0.97101 5.78735 99.9% 8 (4 groups) 28% variance All treatments F(3,8) = 25.633 0.90577 3.10038 99.9% 12 (4 groups) 40% variance All treatments F(3,8) = 12.560 0.82487 2.17026 99.9% 16 (4 groups) Test family 2 il d diff b i d d B f i’ i l h Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Sharma et al. eLife 2016;5:e11414. Protocol 6 DOI: 10.7554/eLife.11414 29 of 32 Biochemistry Group 1 Group 2 Effect size d A priori power Group 1 sample size Group 2 sample size 2% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 68.8057 99.9% 2 2 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 70.5269 99.9% 2 2 15% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 9.17409 87.8% 2 2 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 9.40358 89.0% 2 2 28% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 4.91469 95.5% 3 3 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 5.03763 96.3% 3 3 40% variance 1 mM PLX4720 1 mM PLX4720 + 1 mM CI1040 3.44028 93.7% 4 4 1 mM PLX4720 1 mM PLX4720 + 1 mM AZD6244 3.52634 94.7% 4 4 Test family & F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values Groups F test statistic Partial h2 Effect size f A priori power Total sample size 2% variance All treatments F(3,16) = 1847.2 0.99712 18.6103 99.9% 16 (8 groups) 15% variance All treatments F(3,16) = 32.840 0.86029 2.48143 99.9% 16 (8 groups) 28% variance All treatments F(3,16) = 9.4247 0.63862 1.32934 99.9% 24 (8 groups) 40% variance All treatments F(3,16) = 4.6181 0.46406 0.93053 99.0% 32 (8 groups) In order to produce quantitative replication data, we will run the experiment three times. Each time we will quantify band intensity. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Acknowledgements g The Reproducibility Project: Cancer Biology core team would like to thank the original authors, in particular Cory Johannessen, for generously sharing critical information as well as reagents to ensure the fidelity and quality of this replication attempt. We thank Courtney Soderberg at the Center for 30 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report Biochemistry Open Science for assistance with statistical analyses. We would also like to thank the following com- panies for generously donating reagents to the Reproducibility Project: Cancer Biology; American Type Culture Collection (ATCC), Applied Biological Materials, BioLegend, Charles River Laborato- ries, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories, LI-COR Biosciences, Mirus Bio, Novus Biologicals, Sigma-Aldrich, and System Biosciences (SBI). Author contributions Author contributions VS, LY, MC, KO, Drafting or revising the article; RP:CB, Conception and design, Drafting or revising the article Reproducibility Project: Cancer Biology Elizabeth Iorns: Science Exchange, Palo Alto, United States; William Gunn: Mendeley, London, United Kingdom; Fraser Tan: Science Exchange, Palo Alto, United States; Joelle Lomax: Science Exchange, Palo Alto, United States; Nicole Perfito: Science Exchange, Palo Alto, United States; Timothy Errington: Center for Open Science, Palo Alto, United States Additional information Group author details References Corcoran RB, Dias-Santagata D, Bergethon K, Iafrate AJ, Settleman J, Engelman JA. 2010. BRAF gene amplification can promote acquired resistance to MEK inhibitors in cancer cells harboring the BRAF V600E mutation. 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https://www.biorxiv.org/content/biorxiv/early/2023/06/24/2023.06.20.545695.full.pdf
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The landscape of microRNA interactions annotation: analysis of three rare disorders as case study
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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint * Presently Astra Zeneca, Data Office, Data Science and AI, UK. Academy House, 136 Hills Road, Cambridge CB2 8PA UK The landscape of microRNA interactions annotation: analysis of three rare disorders as case study Panni Simona 1, Kalpana Panneerselvam,2 Pablo Porras2*, Margaret Duesbury2, Livia Perfetto3, Luana Licata4, Henning Hermjakob2, Sandra Orchard2 Panni Simona 1, Kalpana Panneerselvam,2 Pablo Porras2*, Margaret Duesbury2, Livia Perfetto3, Luana Licata4, Henning Hermjakob2, Sandra Orchard2 1. Università della Calabria, Dipartimento di Biologia, Ecologia e Scienze della Terra, Rende, IT 2. European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL- EBI), Wellcome Genome Campus, Hinxton, Cambridge. CB10 1SD. UK. 3. Department of Biology and Biotechnologies "Charles Darwin", La Sapienza University of Rome, IT 4. Department of Biology University of Rome Tor Vergata, Rome, IT . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Keywords: microRNA, interaction database, rare disease, biocuration, RRI Abstract In recent years, a huge amount of data on ncRNA interactions has been described in scientific papers and databases. Although considerable effort has been made to annotate the available knowledge in public repositories, there are still significant discrepancies in how different resources capture and interpret data on ncRNAs functional and physical associations. In the present paper, we have focused on microRNAs which regulate genes associated with rare diseases, as a case study to investigate data availability. The list of protein-coding genes with a known role in specific rare diseases was retrieved from the Genome England PanelApp, and associated microRNA-mRNA interactions were annotated in the IntAct database, and compared with other datasets. The annotation follows recognised standard criteria approved by the IMEX consortium. RNAcentral identifiers were used for unambiguous, stable identification of ncRNAs. The information about the interaction was enhanced by a detailed description of the cell types and experimental conditions, providing a computer- interpretable summary of the published data, integrated with the huge amount of protein interactions already gathered in the database. Furthermore, for each interaction, the binding sites of the microRNA are precisely mapped on a well-defined mRNA transcript of the target gene. This information is crucial to conceive and design optimal microRNA mimics or inhibitors, to interfere in vivo with a deregulated process. As these approaches become more feasible, high-quality, reliable networks of microRNA interactions are needed to help, for instance, in the selection of the best target to be inhibited and to predict potential secondary off- target effects. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Introduction In recent years, it has become increasingly clear that complex and dynamic interactions between ncRNA molecules or ncRNAs and proteins contribute to virtually any biological process. MicroRNAs are probably the best characterised ncRNAs, as they can be identified with bioinformatics approaches, thanks to the conserved hairpin shape of precursor transcripts, and their possible targets can be predicted [1]. MicroRNAs regulate gene expression by guiding the RNA induced silencing complex (RISC) to those mRNAs which display sequences complementary to the microRNA “seed” i.e. nucleotides 2-7 from the 5’ end [2,3]. Predicted and verified microRNAs from more than 270 species are annotated in miRbase [4], with their precursor and mature sequences and other useful information. The latest release contains 1917 human hairpin precursors and 2654 human mature sequences, of which 26% are high-confidence microRNAs [4]. Several microRNA precursors encode two active microRNAs, named 3p and 5p respectively, which recognise different targets. Identifying the exact function for each miRNA is challenging and time consuming, and only a subset of them have been characterised. As mentioned above, it is possible to predict all possible targets for any microRNA, based on the sequence complementarity, site conservation and other features [1,5]. However, these methods provide a list of hundreds of targets for each miRNAs, many of which are not bona-fide interactors, and need to be experimentally verified [6,7]. To this aim, several techniques have been developed, for low and high throughput interaction detection, which we briefly summarise here. One of the most frequently used assays is the luciferase reporter assay, which is an adaptation of the homonymous test used to identify the regulatory regions on DNA promoters (Figure 1). The method not only permits verification of the interaction, but also the mapping of the precise binding site on the mRNA, through mutagenesis of the predicted sequence [8]. QRT-PCR and western blot allow the quantification of the mRNA and protein levels, respectively, following microRNA over-expression, and can be used to discriminate between mRNA degradation and translation inhibition, which are the two possible results of microRNA interaction (Figure 1). Pull down and immunoprecipitation approaches, commonly used to demonstrate protein binding, have been adapted to demonstrate microRNA interactions. Cross-linking ligation and sequencing of hybrids (CLASH) allows high-throughput identification of RNA-RNA interactions. Introduction In this approach, the RNA is cross-linked to the bait protein, and, after the immunoprecipitation, the ends of co-precipitated, interacting RNAs, are ligated together and sequenced to identify the couples [9]. Other high- In recent years, it has become increasingly clear that complex and dynamic interactions between ncRNA molecules or ncRNAs and proteins contribute to virtually any biological process. MicroRNAs are probably the best characterised ncRNAs, as they can be identified with bioinformatics approaches, thanks to the conserved hairpin shape of precursor transcripts, and their possible targets can be predicted [1]. MicroRNAs regulate gene expression by guiding the RNA induced silencing complex (RISC) to those mRNAs which display sequences complementary to the microRNA “seed” i.e. nucleotides 2-7 from the 5’ end [2,3]. Predicted and verified microRNAs from more than 270 species are annotated in miRbase [4], with their precursor and mature sequences and other useful information. The latest release contains 1917 human hairpin precursors and 2654 human mature sequences, of which 26% are high-confidence microRNAs [4]. Several microRNA precursors encode two active microRNAs, named 3p and 5p respectively, which recognise different targets. Identifying the exact function for each miRNA is challenging and time consuming, and only a subset of them have been characterised. As mentioned above, it is possible to predict all possible targets for any microRNA, based on the sequence complementarity, site conservation and other features [1,5]. However, these methods provide a list of hundreds of targets for each miRNAs, many of which are not bona-fide interactors, and need to be experimentally verified [6,7]. To this aim, several techniques have been developed, for low and high throughput interaction detection, which we briefly summarise here. One of the most frequently used assays is the luciferase reporter assay, which is an adaptation of the homonymous test used to identify the regulatory regions on DNA promoters (Figure 1). The method not only permits verification of the interaction, but also the mapping of the precise binding site on the mRNA, through mutagenesis of the predicted sequence [8]. QRT-PCR and western blot allow the quantification of the mRNA and protein levels, respectively, following microRNA over-expression, and can be used to discriminate between mRNA degradation and translation inhibition, which are the two possible results of microRNA interaction (Figure 1). Pull down and immunoprecipitation approaches, commonly used to demonstrate protein binding, have been adapted to demonstrate microRNA interactions. Introduction Cross-linking ligation and sequencing of hybrids (CLASH) allows high-throughput identification of RNA-RNA interactions. In this approach, the RNA is cross-linked to the bait protein, and, after the immunoprecipitation, the ends of co-precipitated, interacting RNAs, are ligated together and sequenced to identify the couples [9]. Other high- . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint throughput sequencing approaches of immunoprecipitated RNAs, after cross-linking to proteins of the RISC complex (CLIP-seq, PAR-CLIP etc.), provide datasets of potential RNA-RNA interactions [10], largely improving the performance of the binding predictors, although specific couples of miRNA-mRNA should be further verified [11]. While the number of publications describing microRNA interactions is constantly increasing, several databases have begun to collect them, often including high throughput indirect or weak evidence. It is worth noting that miRTarBase [12] and RAID [13,14] allow filtering of collected ncRNAs associations, to reduce false positive rates when selecting “strong evidence” interactions. The inclusion of potentially erroneous targets in network analysis may result in misleading data interpretation, as extensively discussed in Huntley et al., 2018. The UCL Functional Gene Annotation group has focused on the gene ontology annotation of human microRNAs collecting a set of highly reliable microRNA-mRNA pairs [15,16]. g y p [ , ] Since 2002 the HUPO Proteomic Standard Initiative (HUPO-PSI) has provided a standardised annotation system for molecular interactions, and has defined the minimal information requirements and the syntax of terms used to describe an interaction experiment (MIMIx) [17], approved by members of the International Molecular Exchange (IMEx) Consortium [18,19]. Common guidelines help to elude false positives, and increase the level of detail and contextual information captured when describing molecules, such as protein binding [20]. The IntAct database, which is a member of the Consortium, has expanded its activities into the annotation of ncRNA interactions, initially focusing on S. cerevisiae ncRNAs [21], and subsequently on mammalian ncRNAs. Introduction In this paper, we present the collection of microRNA-mRNA interactions annotated in IntAct and we integrate and compare it with other resources. In particular, we have focused on the microRNAs that regulate genes associated with rare diseases, in order to provide information potentially useful for the development of new drugs. A rare disease is a health condition that affects a minority of people compared with other diseases that are prevalent in the population [22]. Although the increased focus on these diseases in the last few years, very little is known on microRNAs involved in rare diseases and their collection may help to gain insights into pathological mechanisms. The interactions can be downloaded from the IntAct database (https://www.ebi.ac.uk/intact/) and also from the RNA central website (https://rnacentral.org/). . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Figure 1. Methods commonly used to detect microRNA-mRNA interactions. A) Luciferase assay comprises of the fusion of the luciferase gene to the 3’ UTR of the microRNA target gene and the subsequent transfection of the construct with or without the microRNA. The approach demonstrates the direct interaction, compared to a mutated copy of the predicted complementary region, which acts as a negative control. Introduction Quantification of the mRNA (qRT-PCR) or protein level (WB) helps to distinguish between the mRNA degradation or the translation inhibition, but are not proof of an interaction when used in isolation. B) CLIP and CLASH. Cross-linking and immunoprecipitation of a RNA binding protein (such as AGO2), followed by RNA sequencing, allows the determination of all the RNAs bound to the protein. These approaches demonstrate direct RNA-RNA binding, if the two interacting RNAs are ligated in a hybrid before sequencing (CLASH, [9]). Data collection and deposition MicroRNA-mRNA interactions were manually annotated in the IntAct database, according to the curation standards established by the IMEx Consortium. We retrieved relevant papers by searching the literature with text-mining seeking for the term “microRNA” (or synonymous) AND a specific gene name in the abstract, and luciferase assay or RIP, or CLASH in the methods. The collected papers were manually filtered to identify those relevant for the curation process. More than 260 papers were selected and annotated. Genes associated with rare diseases were prioritised for annotation. The list of these genes was generated according to green panels from GenomeEngland PanelApp for the following diseases: Aniridia, Anemia Fanconi, Autism, Cakut, Cerebrak Folate Deficiency, Epidermolysis Bullosa, Familial Hirschsprung Disease, Growth Failure in Early Childhood, Early Onset Disease and Mitochondrial Disorders. These last three presented a higher number of genes regulated by microRNAs and were used in the subsequent analysis. The microRNAs are identified with RNAcentral IDs and a short label, which specify the strand of the mature microRNA [23].The database can be queried with Ensembl transcript IDs or gene common names, (then selecting mRNA) or with mRNA common . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint names (mrna_name), as well as with the microRNA name or ID. To retrieve information on mutagenesis analysis, in order to map the binding nucleotides, from the Intact web page, click on the lens in the “Select” column of the displayed interactions, then select “Features” in the following web page. Data are also linked to RNA central [23] in the microRNA entry. Network analysis and databases comparison The molecular interaction network of microRNA-mRNA interactions was downloaded from IntAct using the IntAct app [24]. To build disease- specific networks, the list of Ensembl transcript IDs was used. Networks were built and analysed with Cytoscape [25]. For comparison with other databases, miRTarBase and Raid were chosen to enable the selection of strong evidences of direct binding, comparable to those annotated in IntAct. The data were downloaded, IDs were uniformed, and the tables were uploaded in Cytoscape to build and intersect the networks. Functional enrichment analysis For the “Biological Processes” enrichment analysis of microRNA targets, each transcript ID was converted to the common gene name. The tables were imported in Cytoscape to build the network, and BINGO tool [26] was used to perform the enrichment with 0.003 as significance level for GFEC and EOD related genes, while 0.05 was set for MD because there was no enrichment at lower values. Statistical analysis Correlation analysis: A table was generated containing the number of interacting microRNAs for each gene in each of the three databases. Pearson correlation coefficient was calculated with the ggpairs function in the GGally R package [27,28] Venn Diagram: Intersections among datasets were outlined by ggVennDiagram library [29] in R [28] and Jaccard index was calculated as : J(A, B) = ! ∩ $ ! ∪ $ [30] . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Building microRNA network in IntAct The main molecular function of microRNAs is to down-regulate the target gene by binding to the mRNA through few complementary nucleotides located at the 5’ of the microRNA and commonly (although not always) at the 3’ UTR of the messenger RNA. As discussed in the introduction, several experimental approaches have been developed to investigate microRNA function, but only a subset provides clear evidence of microRNA binding to mRNA, while discrimination between direct binding and causal interaction is crucial for the design of interfering drugs. The partners of the IMEx consortium of interaction databases [18,19] have agreed to consider luciferase assay as a proof of RNA-RNA interaction, when validated with mutagenesis analysis (Figure 1). Papers describing microRNA-mRNA interactions validated with the above-mentioned methods, were selected from the literature and annotated in the IntAct database. As the interaction with mRNA results in the down-regulation of the expression, through the mRNA degradation, or in the inhibition of the translation, the microRNA is featured as “regulator” and a causality statement was used to annotate its effect. Most of the repositories which collect microRNA interactions, identify the targets with the gene name or ID. However, microRNAs bind to messenger RNAs and, in order to map the binding sites on the target, it is necessary to refer to a specific transcript. To this aim, it is worth noting that each gene produces several messengers by alternative splicing of the precursor transcript. The number of the collected transcripts is destined to increase as new sequences will be produced from different cell-types and stages. To avoid ambiguities, we have therefore created one full-detailed entry for each target gene, cross-referred it with its Ensembl transcript ID [31], and, whenever possible, we have mapped all the annotated interactions on this entry. Figure 2 shows this process and its result. For human transcripts, among the possible transcripts listed in the Ensembl database [31] we have selected the reference shown in the GIFT curation tool (https://www.ebi.ac.uk/gifts/). . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . Building microRNA network in IntAct CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Figure 2. MicroRNA binding sites on mRNA_hmga2 entry. A) mRNA_hmga2 entry in IntAct is annotated with the sequence and ID of the main transcript of the gene (identified by GIFT), together with the Ensembl gene ID and a RefSeq. B) Each mutation analysis is mapped on the transcript sequence. C) Hmga2-3’UTR regions identified as necessary for the microRNA binding Figure 2. MicroRNA binding sites on mRNA_hmga2 entry. A) mRNA_hmga2 entry in IntAct is annotated with the sequence and ID of the main transcript of the gene (identified by GIFT), together with the Ensembl gene ID and a RefSeq. B) Each mutation analysis is mapped on the transcript sequence. C) Hmga2-3’UTR regions identified as necessary for the microRNA binding We have mainly annotated human interactions, although mouse entries are also represented. Figure 3A shows a subset of the microRNA-mRNA human interaction network. Similarly to PPI networks, few nodes of both . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint molecular types behave as hubs, presenting a number of interactions above average. The network detail blown up in Fig3B shows two connected hubs: the microRNA hsa-mir-17-5p and the mRNA_cdkn1a. The IntAct database contains thousands of human protein interactions, each with a reliability score, so it is possible to investigate the protein complexes affected by the microRNA regulation and the connections between the protein products of co-regulated genes. Figure 3C shows some high-reliable interactors of hsa-mir-17-5p targets, that may be deregulated by mimics or inhibitors of the microRNA. Figure 3. Human microRNA Network in IntAct. Building microRNA network in IntAct A) Section of the human microRNA Network, showing hubs of both entries type. B) Enlargement of a detail to show two nodes and their interactors. C) PPI network for the genes targeted by hsa-mir-17-5p was downloaded and filtered to a MIscore ⋝0.7 and merged with microRNA-mRNA interactions. Figure 3. Human microRNA Network in IntAct. A) Section of the human microRNA Network, showing hubs of both entries type. B) Enlargement of a detail to show two nodes and their interactors. C) PPI network for the genes targeted by hsa-mir-17-5p was downloaded and filtered to a MIscore ⋝0.7 and merged with microRNA-mRNA interactions. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint MicroRNA regulation of rare diseases associated genes While the amount of microRNA-interaction information is increasing continuously, very little is known about microRNAs involved in rare diseases. Until recently, there has been limited research on the molecular mechanisms that underlie these pathologies, despite their global impact on the population and the undeniable need for optimised treatment. Until recently, there has been limited research on the molecular mechanisms that underlie these pathologies, despite their global impact on the population and the undeniable need for optimised treatment. We collected in IntAct a list of interactions between microRNAs and genes associated with the onset or progression of rare disorders. The list of genes was retrieved from the Genome England PanelApp [32]. Among the genes listed for each disease, few of them have been reported in the literature to bind to microRNAs. We considered several rare diseases (listed in material and methods) and, for the present study, we selected three of them, since they have a considerable number of genes regulated by microRNAs: Growth Failure in Early Childhood (GFEC), Mitochondrial Disorders (MD) and Early Onset Dementia (EOD). Table 1 shows the list of the genes that were found to interact with microRNAs and annotated in IntAct. Growth Failure in Early Childhood is influenced by 48 genes, 17 of which were annotated in IntAct for mRNA- microRNA interactions (Figure 4). Approximately 230 genes are associated to Mitochondrial Disorders, (plus a few mitochondrial tRNA genes we did not consider). We found microRNA interactions suitable to be annotated in IntAct only for 14 of these. Finally, a list of 27 genes are associated with Early Onset Dementia, 13 of which were annotated in IntAct. We collected in IntAct a list of interactions between microRNAs and genes associated with the onset or progression of rare disorders. The list of genes was retrieved from the Genome England PanelApp [32]. Among the genes listed for each disease, few of them have been reported in the literature to bind to microRNAs. We considered several rare diseases (listed in material and methods) and, for the present study, we selected three of them, since they have a considerable number of genes regulated by microRNAs: Growth Failure in Early Childhood (GFEC), Mitochondrial Disorders (MD) and Early Onset Dementia (EOD). Table 1 shows the list of the genes that were found to interact with microRNAs and annotated in IntAct. MicroRNA regulation of rare diseases associated genes Growth Failure in Early Childhood is influenced by 48 genes, 17 of which were annotated in IntAct for mRNA- microRNA interactions (Figure 4). Approximately 230 genes are associated to Mitochondrial Disorders, (plus a few mitochondrial tRNA genes we did not consider). We found microRNA interactions suitable to be annotated in IntAct only for 14 of these. Finally, a list of 27 genes are associated with Early Onset Dementia, 13 of which were annotated in IntAct. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Table 1: genes associated with the rare diseases and regulated by microRNAs. Only interactions confirmed by mutagenesis analysis are considered. Table 1: genes associated with the rare diseases and regulated by microRNAs. Only interactions confirmed by mutagenesis analysis are considered. Early Onset Dementia Growth Failure in Early Childhood Mitochondrial Disorders APP BLM AIFM1 ATXN1 BRCA2 COX10 CSF1R CBL DMPK DNMT1 CDKN1C DNM2 GRN FANCA ISCU HTT FGFR3 MFN2 ITM2B HMGA2 MPC1 MAPT IGF1 NDUFA10 NOTCH3 IGF1R PDHB PRNP IGF2 PDHX PSEN1 KRAS SLC25A1 TARDBP MAP2K1 (MEK1 ) SLC25A12 TBK1 OBSL1 TAZ PIK3R1 UQCC2 PTPN11 SOS1 UBE2T Early Onset Dementia Growth Failure in Early Childhood Mitochondrial Disor We used the BINGO tool to evaluate biological processes statistically overrepresented in the microRNA regulated genes. We found an enrichment of genes involved in positive regulation of cellular processes and cell proliferation, as well as proteins involved in signalling pathways, in genes associated with GFEC and EOD, in relation to non-annotated genes. A small number of genes associated with mitochondrial dysfunctions (MD) are regulated by microRNAs, and the enrichment analysis did not show significant enrichment for any term. MicroRNA regulation of rare diseases associated genes Interestingly, however, there is a strong enrichment of specific metabolic and biosynthetic processes, such as: ATP synthesis coupled electron transport, carboxylic acid metabolic process, generation of precursor metabolites and tRNA metabolic processes, in genes associated with MD and not regulated by microRNAs. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Figure 4. The Interaction Network of the genes associated to Growth Failure in Early Childhood. Interactions within proteins associated to GFEC merged into the microRNA-mRNA network. Proteins regulated by microRNAs are highlighted in orange. The thickness of the edge between proteins is proportional to the MI score (i.e. reliability) of the interaction [37] Figure 4. The Interaction Network of the genes associated to Growth Failure in Early Childhood. Interactions within proteins associated to GFEC merged into the microRNA-mRNA network. Proteins regulated by microRNAs are highlighted in orange. The thickness of the edge between proteins is proportional to the MI score (i.e. reliability) of the interaction [37] We compared our results with two other databases: miRTarBase (https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/in dex.php interactions confirmed by “Luciferase assay”) and RAID (https://www.rna-society.org/raid2/ interactions confirmed by “strong evidence”). The low percentage of microRNA regulated genes was confirmed in the two datasets. Supplementary Table 1 compares genes found in each of the 3 databases to interact with microRNAs. Interestingly there is a very high correlation among the number of interacting microRNAs annotated in the 3 databases for each gene, and frequently those with no interactions in one database are not annotated in the others either (Figure 5A). This suggests that, although the coverage of annotated microRNA interactions may still be low, the networks reflect what is currently known in the literature, with more than half genes of each list not affected by microRNA regulation, many involved in one or few interactions, and a small number targeted by a huge number of . MicroRNA regulation of rare diseases associated genes CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint microRNAs. Despite the high correlation, the three datasets do not contain the same interactions: the Venn diagram shows that only 8% of them (calculated from the union of the three datasets) are present in all databases, while each dataset contains a percentage of 21%, 15% or 13% of interactions not annotated in the others (Figure 5B). This suggests that a combined effort of different resources is necessary to get the full picture and to help advance research in gene regulation (Supplementary Figure 1) . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Figure 5. Comparison of microRNA-mRNA interactions annotated in IntAct, MirTarBase and RAID. MicroRNA regulation of rare diseases associated genes A) Correlation analysis: the number of microRNAs regulating genes associated with the diseases in the 3 datasets was compared (see also Suppl. Table 1). B) Venn Diagram comparing interacting pairs of microRNA-mRNA annotated in the 3 datasets. The Jaccard similarity coefficient (in parenthesis) is calculated as intersection over union and indicates that only 8% of the interactions are annotated in all the datasets, and that each resource contains some exclusive information. Discussion ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint exhaustive information about microRNA binding could accelerate the design of specific mimics or inhibitors, and to predict the effect of the perturbation on the neighbourhood interactors. Since RNA-RNA interactions occur through base pairing, the design and development of interfering molecules is easier than protein inhibitors, as demonstrated by the common use of complementary molecules in scientific papers. exhaustive information about microRNA binding could accelerate the design of specific mimics or inhibitors, and to predict the effect of the perturbation on the neighbourhood interactors. Since RNA-RNA interactions occur through base pairing, the design and development of interfering molecules is easier than protein inhibitors, as demonstrated by the common use of complementary molecules in scientific papers. p y p p We have prioritised the annotation of microRNAs regulating genes involved in rare diseases, hopefully contributing to fill the gap of knowledge in this field. It is worth mentioning that the IntAct database has already collected a dataset of protein-protein interactions directly involved in rare-diseases [35]. Some diseases are directly associated with genetic mutations or variants: the disease is a direct consequence of the mutation, or, more frequently, the mutation increases the probability of the onset or progression. When the mutation directly affects a PPI interaction, the information can be retrieved from the database [36]. Very few papers describe the role of microRNA in rare diseases, and hopefully the list of interactions that affect genes associated to the diseases may help in elucidating their role. Although “rare” the “rare diseases” affect more than 30 million people in Europe, and are considered one of the major public health issues. As the return of investment in research on each individual disease may be limited, no treatment and diagnostic tests have been developed for many of them. The annotation of genes involved in rare diseases has been recognised as a crucial step to expedite the diagnosis and to establish the appropriate treatment [32]. We have expanded this knowledge by collecting existing data on the regulation of genes by microRNAs. The selected interactions are evaluated by IntAct curators and annotated according to approved CV terms. When more evidence supports an edge, a score of reliability is generated which helps the user to interpret the network [37]. Discussion Investigations of gene-disease associations typically focus on protein variants, however, as the relevance of ncRNAs becomes more evident, their involvement in many common disorders is conceivable. For an accurate comprehension of microRNA functions, we need to assemble the entire set of miRNA-mRNA interactions, possibly annotated using standard procedures that are clearly explained to the user. Some resources collect microRNA target interactions using natural language processing to find co-occurrences of terms, followed by manual review. Despite the undeniable relevance and high coverage of these resources, there is no general acceptance of what to consider a bona-fide interactor, versus an indirect regulator. We have filtered the experimental data according to high-quality standards provided by the IMEx consortium, to collect a set of reliable microRNA targets. The IntAct database contains approximately 900 interactions involving more than 200 microRNAs. Currently modest coverage, the data set is expected to increase, as new interactions are continually being added. The target mRNAs were annotated with the Ensembl transcript ID as identifier. In order to expedite the integration with other data identified by gene identifiers, the entries are also linked to the Ensembl gene ID. Referring to a specific transcript sequence allows the user to unequivocally annotate the interacting regions, confirmed by the mutagenesis analysis. Recent advances in RNA molecules stabilisation and delivery methods have renewed interest in RNA-based therapies. Chemical modifications of the nucleotides increase their resistance to nucleases and encapsulation of the microRNA mimics in neutral lipids allows the delivery of the molecules into living cells or organisms [33]. In the last years several microRNA-based therapeutics have been developed, and some have entered phase II or III of clinical trials [34]. The availability of . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. Discussion The collection of “true” interactors will simplify procedures to assess the value and potential unrelated effects of a therapeutic product, and will help the design of competitors. Data availability statement The data underlying this article are available in the IntAct database (https://www.ebi.ac.uk/intact/) Funding This work was supported by European Molecular Biology Laboratory Core Funding, National Human Genome Research Institute (NHGRI), Office of Director (OD/DPCPSI/ODSS), National Institute of Allergy and Infectious Diseases (NIAID), National Institute on Aging (NIA), National Institute of General Medical Sciences (NIGMS), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Eye Institute (NEI), National Cancer Institute (NCI), National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health under Award Number [U24HG007822] (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health), Open Targets; European Union's Horizon 2020 research and innovation program 825575 (EJP RD). Key Points • Rare diseases are considered one of the main public health problems • Little is known about the involvement of microRNAs in rare diseases • We propose a method to identify microRNAs potentially involved in rare diseases • We have collected the interactions in the IntAct database and compared them with other collections . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It The copyright holder for this prepri this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint Supplementary data Legends Supplementary figure 1. Interactions between microRNAs and disease-associated genes. GFEC: Growth Failure in Early Childhood MD: Motochondrial Disorders EOD: Early Onset Dementia. Integration of the interactions from the 3 datasets are shown on the left side of the picture, Intersection on the right. Supplementary Table 1 Number of microRNAs interacting with the listed genes in each of the 3 databases. For Raid database “strong evidence” interactions were considered and for miRTarBase “Luciferase assay”. Data were downloaded in November 2022. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 24, 2023. ; https://doi.org/10.1101/2023.06.20.545695 doi: bioRxiv preprint References 1. Riffo-Campos ÁL, Riquelme I, Brebi-Mieville P. Tools for Sequence-Based miRNA Target Prediction: What to Choose? Int J Mol Sci 2016; 17:1987 2. 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Chronic Kidney Disease and Diabetic Retinopathy in Patients with Type 2 Diabetes
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RESEARCH ARTICLE OPEN ACCESS Citation: Rodríguez-Poncelas A, Mundet-Tudurí X, Miravet-Jiménez S, Casellas A, Barrot-De la Puente JF, Franch-Nadal J, et al. (2016) Chronic Kidney Disease and Diabetic Retinopathy in Patients with Type 2 Diabetes. PLoS ONE 11(2): e0149448. doi:10.1371/journal.pone.0149448 Chronic Kidney Disease and Diabetic Retinopathy in Patients with Type 2 Diabetes Antonio Rodríguez-Poncelas1,2,3, Xavier Mundet-Tudurí3,4*, Sonia Miravet-Jiménez3,5, Aina Casellas2, Joan F. Barrot-De la Puente2,6, Josep Franch-Nadal3,7,8, Gabriel Coll-de Tuero1,2 1 Primary Health Care Center Anglès, Gerència Territorial Girona, Institut Català de la Salut, Girona, Spain, 2 Unitat de Suport a la Recerca Girona, Institut Universitari d’Investigació en Atenció Primària Jordi Gol (IDIAP Jordi Gol), Barcelona, Spain, 3 Unitat de Suport a la Recerca Barcelona Ciutat, Institut Universitari d'Investigació en Atenció Primària Jordi Gol (IDIAP Jordi Gol), Barcelona, Spain, 4 Universitat Autonoma de Barcelona, Bellaterra, Spain, 5 Primary Health Care Center Martorell, Gerència Territorial Metropolitana Sud, Institut Català de la Salut, l’Hospitalet de LLobregat, Barcelona, Spain, 6 Primary Health Care Center Jordi Nadal (Salt), Gerència Territorial Girona, Institut Català de la Salut, Girona, Spain, 7 Primary Health Care Center Raval Sud, Gerència d’Àmbit d’Atenció Primària Barcelona Ciutat, Institut Català de la Salut, Barcelona, Spain, 8 CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain * xavier.mundet@uab.cat Methods This was a population-based, cross-sectional study. A total of 28,344 patients diagnosed with DM2 who had recorded ophthalmologic and renal functional examinations were evalu- ated. Data were obtained from a primary healthcare electronic database of medical records. CKD was defined as an estimated glomerular filtration ratio (eGFR) of <60 ml/min/1.73m2 and/or urine albumin to creatinine ratio (UACR) 30 mg/g. DR was categorized as non- vision threatening diabetic retinopathy and vision threatening diabetic retinopathy. Copyright: © 2016 Rodríguez-Poncelas 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: Due to ethical restrictions, data are available upon request. The data contain identifying human information and are unsuitable for public deposition. Requests may be made to the corresponding author. * xavier.mundet@uab.cat Purpose To explore the relationship between chronic kidney disease (CKD) and diabetic retinopathy (DR) in a representative population of type 2 diabetes mellitus (DM2) patients in Catalonia (Spain). Editor: Florian Kronenberg, Innsbruck Medical University, AUSTRIA Kidney Disease and Diabetic Retinopathy in T2DM Conclusions Competing Interests: The authors have declared that no competing interests exist. These results show that CKD, high UACR and/or low eGFR, appear to be associated with DR in this DM2 population. Introduction Chronic kidney disease (CKD) and diabetes retinopathy (DR) are two major microvascular complications found in long-standing type 1 (DM1) and type 2 diabetic (DM2) patients. In addition to sharing risk factors, such as poor glycaemia control and systolic hypertension, CKD and DR are reflected in the clinical manifestations of similar microvascular lesions in the glomerular and retinal vessels [1–3]. UACR and eGFR are clinically markers for evaluate renal function. Urine albumin to creatinine ratio (UACR) and low estimated glomerular filtration rate (eGFR) are clinical markers of renal function. CKD is often associated with DR in DM1 [4, 5], and in some studies in DM2 [6–8]. However, this relationship has not been properly investi- gated in DM2, and there are few data on which of the two markers of CKD, eGFR or UACR, is more closely related to DR. High UACR is a marker of endothelial dysfunction and may influence the microvasculature of the kidney and retina. However some studies [9–10] found a relationship between higher UACR levels and DR in DM1 patients but not in DM2 patients. Sabanayagam et al. [11] observed that CKD was associated with DR only in the presence of albuminuria, suggesting that CKD is more likely related to diabetes in the presence of albuminuria. Previous studies have shown that UACR not only is an important clinical marker for CKD, but also is closely associated with the progression of DR [12]. Moreover, the relationship between eGFR with DR remains unclear, particularly in DM2. Man et al. [13] observed that lower levels of eGFR were associated with the presence and severity of DR. A recent report showed that both an elevated UACR and decreased eGFR predicted the development of DR in DM2 patients with the former having a greater impact [14]. The aim of this study was to investigate the association between CKD, UACR and/or eGFR, and DR in a representative Catalonian (Spain) DM2 population. Results CKD was associated with a higher rate of DR [OR], 95% confidence interval [CI], 1.5 (1.4– 1.7). When we analyzed the association between different levels of UACR and DR preva- lence observed that DR prevalence rose with the increase of UACR levels, and this associa- tion was significant from UACR values 10 mg/g, and increased considerably with UACR values 300mg/g (Odds ratio [OR], 95% confidence interval [CI], 2.0 (1.6–2.5). This associ- ation was lower in patients with eGFR levels 44 to 30 mL/min/1.73m2 [OR], 95% confidence interval [CI], 1.3 (1.1–1.6). Funding: This study was supported by a grant from the IDIAP J. Gol (SIDIAP-2013 from Fundacio J Gol i Gurina, www.idiapjordigol.com) and the Education and Research in Family Practice Chair (UAB- Novartis). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 10 PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 Assessment of Retinopathy In a large, community-based screening programmer, such as that employed in our study, fun- dus photography is considered to be the preferred option [19]. Photographs are captured from each eye and the severity of DR is categorized according to the international clinical diabetic retinopathy severity scales recommended by the Global Diabetic Retinopathy Project Group [20] as: non-vision threatening DR (non-VTDR) and vision threatening DR (VTDR). The for- mer includes no apparent retinopathy (no DR); mild non-proliferative DR (mild NPDR); and moderate non-proliferative DR (moderate NPDR). The latter is composed of severe non-prolif- erative DR (severe NPDR); proliferative DR (PDR); and diabetic macular edema (DME). In our study, retinal photography was performed by skilled personnel using a non-midriatic cam- era. Subsequently, in the primary health care center, a family physician trained in reading eye fundus photographs registered the result in the patient’s medical records. Each eye was given a DR grade according to the worst result. In the case of patients having more than one fundus photograph during this period, the most recent was employed for analysis. In the study we only included retinal photographs from patients undergoing diabetic retinopathy screening performed at primary health care centers. Measures of Kidney Diabetic Disease Serum creatinine levels and UACR were determined. CKD was defined as UACR >30 mg/g and/or eGFR <60 mL/min/1.73m2. Normoalbuminuria was defined as UACR <30 mg/g; microalbuminuria as UACR 30–299 mg/g; and macroalbuminuria as UACR 300 mg/g. The Chronic Kidney Disease Epidemiol- ogy Collaboration equation was employed to measure eGFR [18]. Clinical variables The following data were obtained from each patient: age, age at diagnosis of diabetes, duration of diabetes, gender, and glycated hemoglobin levels (A1C). Cardiovascular risk factors includ- ing body mass index (BMI), total cholesterol, low-density (LDL) cholesterol, high density lipo- protein (HDL) cholesterol, non-HDL cholesterol, systolic and diastolic blood pressure (SBP and DBP), pulse pressure (PP), and smoking status according to the last condition registered before the index date were collected. Data for clinical variables were gathered from the 15 months prior to the index date with the exception of blood pressure, PP, and BMI, which were obtained from the previous 12 months. Additional data were gathered on medication. The study was approved by the Ethics Committee of the Primary Health Care University Research Institute Jordi Gol (protocol number 13/137). All patient records and information was anonymized and de-identified prior to analysis. Materials and Methods A population-based, cross-sectional study was performed in Catalonia (Spain) with patients aged between 30 and 90 years (at 31st December, 2012), diagnosed with DM2 prior to retinal photography screening, and whose DR category was recorded in their medical records. The most recent retinal photography registered was selected and employed as the index date. The criteria for the diagnosis of type DM2 were those established by the American Diabetes Associ- ation [15] at the time of registration in the electronic database. Data were obtained from the SIDIAP (System for Research and Development in Primary Care) electronic database. The SIDIAP includes data from the primary healthcare electronic medical records (e-CAP) on demographic information, appointment dates with doctors and nurses, clinical diagnoses, clinical variables, prescriptions written, referrals to specialists and hospitals, results from laboratory tests, and medication sold by pharmacies. The quality of the SIDIAP data has been previously documented, and the database has been widely used to study the epidemiology of a number of health outcomes [16, 17]. 2 / 10 Kidney Disease and Diabetic Retinopathy in T2DM Results Prior to retinography 28,344 DM2 patients had their eGFR and UACR values registered. From the total sample, 14.6% had an eGFR <60 mL/min/1.73m2, women had lower rates than men (p<0.001). As can be observed in Table 1, diabetes duration, hypertension, cardiovascular dis- ease (CVD), and treatment with insulin displayed a direct association with decreased eGFR. In contrast, an inverse relationship between eGFR and non-HDL cholesterol, plasma hemo- globin, heart rate, and DBP was reported. With respect to UACR values (Table 2), 16.0% were 30 mg/g with higher levels being found in men (p<0.001). DM2 duration, poor glycemic control (glycated hemoglobin; A1C >7%), SBP, increased heart rate, hypertension, CVD, and treatment with insulin showed a direct relationship with an increase in UACR levels. An inverse relationship, however, was observed between UACR and plasma hemoglobin levels. In Table 3 the characteristics of the patients with respect to the presence or absence of DR can be observed. Duration of diabetes, A1C levels, SBP, heart rate, CVD, and insulin treatment all displayed a direct relationship with DR. An inverse relationship, however, was observed for DR with non-HDL cholesterol, plasma hemoglobin, and DBP. Table 4 shows that CKD was associated with a higher rate of DR (Odds ratio [OR], 1.5; 95% confidence interval [CI], 1.4–1.7). Patients with elevated UACR had higher rates of DR, partic- ularly if UACR values were 300 mg/g (OR, 2.0; 95% CI, 1.6–2.5); this rate was lower in patients with eGFR levels 44 to 30 mL/min/1.73m2 (OR, 1.3; 95% CI, 1.1–1.6). Patients with Table 1. Patients’ characteristics with respect to glomerular filtration rate (eGFR). Statistical analysis A descriptive analysis stratified by renal function (according to eGFR and UACR) and retino- graphy result was performed. The absolute and relative frequencies of the qualitative variables, and their mean and standard deviation (or median and interquartile range according to the characteristics of the variable) were calculated. In order to study whether the clinical variables of interest depended on renal function, Pear- son’s chi square test and analysis of variance (ANOVA) were employed for means and propor- tions, respectively. The hypothesis contrast was bilateral in all cases with a 0.05 level of significance. 3 / 10 PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 Kidney Disease and Diabetic Retinopathy in T2DM In addition, the relationship between renal function values (according to eGFR and UACR) and the presence of retinopathy was assessed by logistic regression models adjusted by age, sex, age at retinography, duration of DM2, systolic blood pressure, A1C 7%, smoking, BMI, and cardiovascular disease. Analyses were performed with Stata/SE version 13 for Windows (Stata Corp., College Sta- tion, Texas, USA). y y A1C, glycated hemoglobin; bpm, beats per minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. aP value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for quantitative ones. bAny cardiovascular disease includes coronary heart disease and/or stroke doi:10.1371/journal.pone.0149448.t001 y y A1C, glycated hemoglobin; bpm, beats per minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. value for comparison of groups by glomerular filtration rate with Pearson s chi square test for qualitative variables y cardiovascular disease includes coronary heart disease and/or stroke g p y g q q q se includes coronary heart disease and/or stroke s by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for quantitative ones y obin; bpm, beats per minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. onary heart disease and/or stroke P value for comparison of groups by glomerular filtration rate with Pearson s chi square test for qualitative variables and t test for quantitative ones. bAny cardiovascular disease includes coronary heart disease and/or stroke y er minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. Results Variable Global eGFR 60 eGFR 59–45 eGFR 44–30 eGFR <30 p-valuea Female, n (%) 12,520 (44.2) 10,363 (42.8) 1,478(51.2) 568 (53.9) 111 (52.6) <0.001 Hypertension, n (%) 22,971 (81.0) 19,001 (78.5) 2,737 (94.8) 1,026 (97.4) 207 (98.1) <0.001 Any cardiovascular diseaseb, n (%) 3,969 (14.0) 3,026 (12.5) 590 (20.4) 286 (27.2) 67 (31.8) <0.001 Coronary Heart disease, n (%) 3,048 (10.8) 2,326 (9.6) 446 (15.5) 221 (21.0) 55 (26.1) <0.001 Stroke, n (%) 1,148 (4.1) 854 (3.5) 186 (6.4) 90 (8.5) 18 (8.5) <0.001 Insulin treatment, n (%) 4,737 (16.7) 3,781 (15,6) 567 (19.6) 302 (28.7) 87 (41.2) <0.001 Age T2DM (years), mean (SD) 58.8(10.5) 57.5(10.2) 65.6 (9.0) 67.2 (9.7) 65.0(10.1) <0.001 Age at retinography (years), mean (SD) 65.7(10.7) 64.3(10.4) 73.7(7.9) 76.1 (7.8) 74.6(8.9) <0.001 Diabetes duration (years), mean (SD) 7.0 (5.2) 6.8 (5.0) 8.2 (5.6) 9.0 (6.0) 9.7(6.1) <0.001 A1C (%), mean (SD) 7.4 (1.4) 7.4(1.4) 7.2 (1.2) 7.3 (1.4) 7.2 (1.3) <0.001 Non-HDL cholesterol (mg/dL), mean (SD) 137.3(35.2) 137.8(35.2) 135.4(34.6) 132.5(35.2) 132.5(38.2) <0.001 Hemoglobin (g/dL), mean (SD) 14.0 (1.5) 14.1 (1.4) 13.3 (1.6) 12.7 (1.6) 12.0 (1.5) <0.001 SBP (mmHg), mean (SD) 134.9(12.6) 134.8(12.5) 136.1(13.1) 135.8(13.5) 133.9(14.0) <0.001 DBP (mmHg), mean (SD) 76.4 (8.3) 76.9 (8.2) 74.2 (8.2) 71.9 (8.3) 70.4 (8.3) <0.001 Heart Rate (bpm), mean (SD) 76.1 (12.1) 76.3 (12.0) 75 (12.5) 74.2 (12.5) 72.5 (12.0) <0.001 aP value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for quantitative ones Patients’ characteristics with respect to glomerular filtration rate (eGFR). PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 4 / 10 y y A1C, glycated hemoglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Threatening Diabetic a P value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for the quantitative ones. b Any cardiovascular disease includes coronary heart disease and/or stroke doi:10.1371/journal.pone.0149448.t003 y ca d o ascu a d sease c udes co o a y ea t d sease a d/o st o e A1C, glycated hemoglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Threatening Diabetic Retinopathy. a P value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for the quantitative ones. b y stolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Threatening Diabetic aP value for comparison of groups by urine albumin to creatinine ratio (ACR) with Pearson’s chi-square test for qualitative variables and t-test for the quantitative ones. Kidney Disease and Diabetic Retinopathy in T2DM Table 2. Patients’ characteristics with respect to urine albumin to creatinine ratio (UACR). Table 2. Patients’ characteristics with respect to urine albumin to creatinine ratio (UACR). Variable Global UACR <30 mg/g UACR = 30–299 mg/g UACR 300 mg/g p-valuea Female, n (%) 12,520 (44.2) 10,886 (45.7) 1,443 (36.4) 191 (33.7) <0.001 Hypertension, n (%) 22,971 (81.0) 18,857 (79.2) 3,573 (90.2) 541 (95.4) <0.001 Any cardiovascular diseaseb, n (%) 3,969 (14.0) 2,996 (12.6) 830 (21.0) 143 (25.2) <0.001 Coronary Heart disease, n (%) 3,048 (10.8) 2,321 (9.7) 616 (15.6) 111 (19.6) <0.001 Stroke, n (%) 1,148 (4.1) 842 (3.5) 268 (6.8) 38 (6.7) <0.001 Insulin treatment, n (%) 4,737 (20.0) 3,512 (17,9) 1,028 (29.0) 197 (39.4) <0.001 Age T2DM (years), mean (SD) 58.8(10.5) 58.6(10.4) 59.8(10.9) 59.1(10.5) <0.001 Age at retinography (years), mean (SD) 65.7(10.7) 65.4(10.6) 67.6(11.0) 67.6(11.0) <0.001 Diabetes duration (years), mean (SD) 7.0 (5.2) 6.8 (5.1) 7.8 (5.4) 8.6 (5.8) <0.001 A1C (%), mean (SD) 7.4 (1.4) 7.3(1.3) 7.7 (1.6) 7.8 (1.7) <0.001 Non-HDL cholesterol (mg/dL), mean (SD) 137.3(35.2) 137.4 (34.7) 136.5(37.6) 136.6(37.3) 0.320 Hemoglobin (g/dL), mean (SD) 14.0 (1.5) 14.0 (1.5) 13.8 (1.7) 13.5 (1.9) <0.001 SBP (mmHg), mean (SD) 134.9(12.6) 134.2(12.2) 138.3(13.7) 141.8(15.8) <0.001 DBP (mmHg), mean (SD) 76.4 (8.3) 76.4 (8.1) 76.4 (9.0) 76.7 (10.0) 0.736 Heart Rate (bpm), mean (SD) 76.1 (12.1) 76.0(12.0) 76.6 (12.8) 77.5 (13.2) 0.002 a aP value for comparison of groups by urine albumin to creatinine ratio (ACR) with Pearson’s chi-square test for qualitative variables and t-test for the quantitative ones UACR values 30 to 299 mg/gr had DR rate slightly higher than those with eGFR values of 30 to 44 mL/min/1.73m2 (OR, 1.5; 95% CI, 1.4–1.7, and OR, 1.3; 95% CI 1.1–1.6, respectively). When we analyzed the association between different levels of UACR and DR prevalence Any cardiovascular disease includes coronary heart disease and/or stroke A1C, glycated hemoglobin; DBP, diastolic blood pressure; SBP, systolic blood pressure. doi:10.1371/journal.pone.0149448.t002 doi:10.1371/journal.pone.0149448.t002 UACR values 30 to 299 mg/gr had DR rate slightly higher than those with eGFR values of 30 to 44 mL/min/1.73m2 (OR, 1.5; 95% CI, 1.4–1.7, and OR, 1.3; 95% CI 1.1–1.6, respectively). a P value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variab b Any cardiovascular disease includes coronary heart disease and/or stroke moglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Thre y y C, glycated hemoglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VT inopathy. PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 q bAny cardiovascular disease includes coronary heart disease and/or stroke When we analyzed the association between different levels of UACR and DR prevalence UACR values 30 to 299 mg/gr had DR rate slightly higher than those with eGFR values of 30 to 44 mL/min/1.73m2 (OR, 1.5; 95% CI, 1.4–1.7, and OR, 1.3; 95% CI 1.1–1.6, respectively). When we analyzed the association between different levels of UACR and DR prevalence Table 3. Patients’ characteristics with respect to diabetic retinopathy. Variable Global Non DR Non-VTDR VTDR p-valuea Female, n (%) 12,520 (44.2) 11,089 (44.3) 1,316 (43.8) 115 (40.2) 0.345 Hypertension, n (%) 22,971 (81.0) 20,141 (80.4) 2,589 (86.1) 241 (84.3) <0.001 Any cardiovascular disease, b n (%) 3,969 (14.0) 3,347 (13.4) 553 (18.4) 69 (24.1) <0.001 Coronary heart disease, n (%) 3,048 (10.8) 2,602 (10.4) 396 (13.2) 50 (17.5) <0.001 Stroke, n (%) 1,148 (4.1) 935 (3.7) 187 (6.2) 26 (9.1) <0.001 Insulin treatment, n (%) 4,737 (20.0) 3,529 (17.1) 1,067 (38.8) 141 (52.8) <0.001 Age DM2 (years), mean (SD) 58.8(10.5) 58.8 (10.4) 58.1 (11.2) 58.4 (11.6) <0.001 Age at retinography (years), mean (SD) 65.7(10.7) 65.6 (10.6) 67.1 (10.8) 67.9 (10.9) <0.001 Diabetes duration (years), mean (SD) 7.0 (5.2) 6.7 (5.0) 9.0 (5.8) 9.5 (5.8) <0.001 A1C (%), mean (SD) 7.4 (1.4) 7.3 (1.3) 7.9 (1.6) 8.2 (1.7) <0.001 Non-HDL cholesterol (mg/dL), mean (SD) 137.3(35.2) 137.8 (35.0) 133.0 (35.5) 133.9 (40.1) <0.001 Hemoglobin (g/dL), mean (SD) 14.0 (1.5) 14.0 (1.5) 13.7 (1.6) 13.7 (1.8) <0.001 SBP (mmHg), mean (SD) 134.9(12.6) 134.6 (12.5) 137.4 (13.6) 138.2 (12.7) <0.001 DBP (mmHg), mean (SD) 76.4 (8.3) 76.5 (8.3) 75.7 (8.5) 75.0 (8.3) <0.001 Heart Rate (bpm), mean (SD) 76.1 (12.1) 76.0 (12.1) 76.9 (12.1) 78.2 (12.3) <0.001 Table 3. Patients’ characteristics with respect to diabetic retinopathy. PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 5 / 10 Kidney Disease and Diabetic Retinopathy in T2DM Table 4. Types of chronic kidney disease and diabetic retinopathy. OR (95% CI) p-value UACR (mg/g)a <30 1.0 30 to 299 1.5 (1.4, 1.7) <0.001 300 2.0 (1.6, 2.5) <0.001 eGFR (mL/min / 1.73m2)a 90 1.0 [60, 89] 1.0 (0.9, 1.1) 0.674 [45, 59] 1.2 (1.0, 1.3) 0.142 [30, 44] 1.3 (1.1, 1.6) 0.009 <30 1.3 (0.9, 1.9) 0.193 aAdjusted by age, sex, age at retinography, duration type 2 diabetes, systolic blood pressure <140 mmHg, A1C  7%, smoking, cardiovascular disease, BMI, UACR and/or eGFR. Model Diabetic Retinopathy (DR): without DR vs. doi:10.1371/journal.pone.0149448.g001 (Fig 1) we observed that DR prevalence rose with the increase of UACR levels, and this associa- tion was significant from UACR values 10 to 29 mg/g, (OR, 1.2; CI, 1.1–1.4), and increased considerably with UACR values 300 mg/g (OR, 2.0; 95% CI, 1.5–2.5). Discussion Our study showed that high UACR level significantly increases the prevalence of diabetic reti- nopathy in type 2 diabetic patients, even after adjustment for variables, and this association was significant from UACR values 10 mg/g. These findings concur with the RIACE study cohort [21]. We observed that individuals with elevated UACR had a higher DR prevalence than those with decreased eGFR. A number of factors could have contributed to the renal and retinal tissue injury observed; including A1C levels, hypertension, dyslipidemia, diabetes dura- tion, age at onset of DM2, smoking, and UACR levels [6, 10, 22, 23]. Patients with CKD are more likely to present with DR VTDR [24]. High UACR doubles the possibility of developing DR with respect to normal UACR levels, a risk that increases considerably with macroalbumi- nuria, even after adjusting for other factors [7, 14, 25]. In addition, an association was observed between low eGFR and greater prevalence of DR. Lu et al. [26] showed that a decrease in eGFR was significantly correlated with DR, after controlling for sex, age, and albuminuria staging. Wu J et al. [27] observed that, after the adjustment of the variables affecting the relationship between eGFR and DR, results of a univariate analysis suggested that eGFR remained signifi- cantly associated with DR. In our study we observed that eGFR levels <45 ml/min/1.73m2 were associated with higher DR prevalence, but we did not find this association with eGFR lev- els 45/ml/min/1.73 m2. This association was not significant with eGFR levels <30 mL/min/ 1.73m2, probably because there were few patients in this group. Penno et al. [28] reported an association between UACR 300 mg/g and DR (OR 2.9; 95% CI, 2.1–4.0) higher than that observed in our work. Such a variation could be due to the differ- ent grouping of DR types. Nevertheless, in both studies elevated UACR had a greater associa- tion with DR than decreased eGFR. CKD in DM2 is more heterogeneous than in type 1 diabetes, retinopathy and albuminuria were both absent in 30% of adults with DM2 and chronic renal insufficiency according to the Third National Health and Nutrition Survey [29]. Patients with diabetes are also susceptible to non diabetic renal disease [30]. A decrease in eGFR with normoalbuminuria in such patients sometimes may be linked to the presence of arteriosclerosis. Recently, Lee et al. [31] showed a direct association between DR and CKD. Kidney Disease and Diabetic Retinopathy in T2DM Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio; VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative DR and moderate non-proliferative DR. Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio; VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative DR and moderate non-proliferative DR. Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio; VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative DR and moderate non-proliferative DR. Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio; VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative DR and moderate non-proliferative DR. doi:10.1371/journal.pone.0149448.g002 any type DR (n = 26,897 patients) DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; OR, odds ratio; UACR, urine albumin to creatinine ratio. doi:10 1371/journal pone 0149448 t004 Table 4. Types of chronic kidney disease and diabetic retinopathy. p-value doi:10.1371/journal.pone.0149448.t004 (Fig 1) we observed that DR prevalence rose with the increase of UACR levels, and this associa- tion was significant from UACR values 10 to 29 mg/g, (OR, 1.2; CI, 1.1–1.4), and increased considerably with UACR values 300 mg/g (OR, 2.0; 95% CI, 1.5–2.5). The prevalence of DR using the KDIGO combination of UACR and eGFR categories is shown in S1 Table. Moreover, Fig 2 shows the association between CKD and DR severity. Patients with eGFR <60 mL/min/1.73m2 and UACR <30 mg/g had an increased non-VTDR rate (OR 1.3; 95% CI 1.1–1.5) and a non-significant increase in VTDR (OR 1.4; 95% CI 1.0– 2.1; p = 0.082). In individuals with UACR 30 mg/g and eGFR 60 mL/min/1.73m2 the rate of VTDR (OR 2.6; 95% CI 1.9–3.5) was higher than non-VTDR (OR 1.6; 95% CI 1.4–1.7). Fig 1. Urine albumin to creatinine ratio and prevalence of diabetic retinopathy. *Adjusted by age, sex, age at retinography, duration type 2 diabetes, systolic blood pressure <140 mmHg, A1C  7%, smoking, cardiovascular disease, BMI, and eGFR. DR, diabetic retinopathy; OR, odds ratio; UACR, urine albumin to creatinine ratio. Fig 1. Urine albumin to creatinine ratio and prevalence of diabetic retinopathy. *Adjusted by age, sex, age at retinography, duration type 2 diabetes, systolic blood pressure <140 mmHg, A1C  7%, smoking, cardiovascular disease, BMI, and eGFR. DR, diabetic retinopathy; OR, odds ratio; UACR, urine Fig 1. Urine albumin to creatinine ratio and prevalence of diabetic retinopathy. *Adjusted by age, sex, age at retinography, duration type 2 diabetes, systolic blood pressure <140 mmHg, A1C  7%, smoking, cardiovascular disease, BMI, and eGFR. DR, diabetic retinopathy; OR, odds ratio; UACR, urine albumin to creatinine ratio. PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 6 / 10 PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 Acknowledgments This study was supported by a grant from the IDIAP J. Gol and the Education and Research in Family Practice Chair (UAB-Novartis). We acknowledge Mònica Gratacòs and Amanda Prowse for providing support in the manuscript preparation and editing. CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM) is an initiative from Instituto de Salud Carlos III. Supporting Information S1 Table. Prevalence of diabetic retinopathy using KDIGO combinations of UACR and eGFR categories. (DOCX) S1 Table. Prevalence of diabetic retinopathy using KDIGO combinations of UACR and eGFR categories. Author Contributions Conceived and designed the experiments: AR-P XM-T GC-T. Performed the experiments: AR- P XM-T SM-J JFB-D JF-N. Analyzed the data: AC. Contributed reagents/materials/analysis tools: AR-P XM-T SM-J JFB-D JF-N. Wrote the paper: AR-P XM-T GC-T. Conceived and designed the experiments: AR-P XM-T GC-T. Performed the experiments: AR- P XM-T SM-J JFB-D JF-N. Analyzed the data: AC. Contributed reagents/materials/analysis tools: AR-P XM-T SM-J JFB-D JF-N. Wrote the paper: AR-P XM-T GC-T. Conclusions CKD appears to be associated with DR in this DM2 population. Elevated UACR had a greater association with DR prevalence than decreased eGFR, but both were associated with DR in DM2 patients. Discussion The presence of CKD identified a group of DM2 patients of being at a greater risk of presenting macro and microvascular complications. Moreover, the presence of CKD and DR was associated with a more rapid reduction in renal function and greater mortality in this group of patients who might benefit from more aggressive treatment. 7 / 10 Kidney Disease and Diabetic Retinopathy in T2DM Our study should be interpreted with consideration of the following limitations. First, this study was a cross-sectional analysis; therefore, the data were recorded from an electronic data- base and diabetic patients with DR-VTDR, who were attended by an ophthalmologist or endo- crinologist, could have been poorly represented. Second, because of the small number of cases in some categories, we could not carry out the final analysis using all KDIGO categories. Despite these limitations, this study used a nationally representative sample of DM2 patients. Moreover, to the best of our knowledge, this is the first large population based study to examine the association between CKD, decreased eGFR and high UACR, and DR among a representa- tive Catalonian, Spain, DM2 population. PLOS ONE | DOI:10.1371/journal.pone.0149448 February 17, 2016 References What is the mechanism of microalbuminuria in diabetes: a role for the glomeru- lar endothelium?. Diabetologia. 2008; 51: 714–25. doi: 10.1007/s00125-008-0961-8 PMID: 18347777 13. 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TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION.
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TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION. Sunil P. Nikhate1, Renuka B. Ikkar2, Narendra D.Yadnik 3, Vijay V. Bhagat4 and Vaidehi V. Raole5. 1 PhD (Scholar) Associate Professor Dept of Kriya Sharir Parul Institute of Ayurved Parul University; Sunil P. Nikhate1, Renuka B. Ikkar2, Narendra D.Yadnik 3, Vijay V. Bhagat4 and Vaidehi V. Raole5. Sunil P. Nikhate1, Renuka B. Ikkar2, Narendra D.Yadnik 3, Vijay V. Bhagat4 and Vaidehi V. Raole5. ( j ) te Professor, Dept of Kriya Sharir, SSAC &H, Panchavati ,Nashik (Maharashtra). j 2. Associate Professor, Dept of Kriya Sharir, SSAC &H, Panchavati ,Nashik (Maharashtra). 3. PhD (Scholar), Assistant Professor, Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University; Limda, Vadodara. (Gujarat) 3. PhD (Scholar), Assistant Professor, Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University; Limda, Vadodara. (Gujarat) j 4. Associate professor, Dept of Samhita & Siddhanta, Parul Institute of Ayurved, Parul University; Limda, Vadodara. (Gujarat). 4. Associate professor, Dept of Samhita & Siddhanta, Parul Institute of Ayurved, Parul University; Limda, Vadodara. (Gujarat). 5. PhD Guide; Professor, PG Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University; Limda, Vadodara. (Gujarat). …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Received: 03 May 2019 Final Accepted: 05 June 2019 Published: July 2019 Key words:- Dhatusara, Types, Twakasara- Characteristics, Importance. Sara is unique concept illuminated in Ayurveda. It is core or excellence part of Dhatu (tissues). Sara Parikshana (Examination) is very important to rule out Bala (strength) of Dhatu. Skin is one of the largest organs of the body. Because of this, caring for skin can directly affect our overall health. Skin acts as a protective shield and is most vulnerable to outside elements. It’s affected by more factors than we may think. So it is necessary to take care of the skin. Dalhanacharya is described Rasasara as a Twakasara because of functions of the Rasa Dhatu is best carried out through Skin. Physical Characteristics of Twakasara is totally concerned with skin on body hair. Physical attractiveness creates a powerful first impression on the mind, so in fact that we may go much beyond looks and simply start generating assumptions about a person's success, status, parenting, and intelligence, even if they prove not to be true. To understand strength or weakness of our look, we need to acknowledge the power of first impressions i.e Twakasarata. It influences the way we see and treat others. TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION. Once an impression is entrenched, it takes a lot to change our attitude about a person. By virtue of Knowledge of skin and Twakasarata we would be able to maintain healthy status of the skin. Copy Right, IJAR, 2019,. All rights reserved. …………………………………………………………………………………………………….... Introduction:- Dosha-Dhatu-Mala is the basic concept of Ayurveda as they are prime constituents of the human body1. They are considered as base of the body like root for the plant. Each and every person is having same elementary entities as mentioned above. Beside that we observed, there are structural and functional variation in components of Sharira. Sara is unique concept illuminated in Ayurveda. It is core or excellence part of Dhatu (tissues). Sara Parikshana (Examination) is very important to rule out Bala (strength) of Dhatu. Skin is one of the largest organs of the body. Because of this, caring for skin can directly affect our overall health. Skin acts as a protective shield and is most vulnerable to outside elements. It’s affected by more factors than we may think. So it is necessary to take care of the skin. Dalhanacharya is described Rasasara as a Twakasara because of functions of the Rasa Dhatu is best carried out through Skin. Physical Characteristics of Twakasara is totally concerned with skin on body hair. Physical attractiveness creates a powerful first impression on the mind, so in fact that we may go much beyond looks and simply start generating assumptions about a person's success, status, parenting, and intelligence, even if they prove not to be true. To understand strength or weakness of our look, we need to acknowledge the power of first impressions i.e Twakasarata. It influences the way we see and treat others. Once an impression is entrenched, it takes a lot to change our attitude about a person. By virtue of Knowledge of skin and Twakasarata we would be able to maintain healthy status of the skin. Copy Right, IJAR, 2019,. All rights reserved. Copy Right, IJAR, 2019,. All rights reserved. Dosha-Dhatu-Mala is the basic concept of Ayurveda as they are prime constituents of the human body1. They are considered as base of the body like root for the plant. Each and every person is having same elementary entities as mentioned above. Beside that we observed, there are structural and functional variation in components of Sharira. 124 Corresponding Author:-Sunil p. Nikhate. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Journal Homepage: -www.journalijar.com Article DOI:10.21474/IJAR01/ 9334 DOI URL: http://dx.doi.org/10.21474/IJAR01/9334 Types: yp Individuals have been classified in various categories, depending upon the predominance of particular Dhatu Sara in the body by virtue of its quantity, quality and functions. Acharya Charak5, Sushruta6 and Vagbhata7 have mentioned eight type of Sara. These are Twak, Rakta, Mansa, Meda, Asthi, Majja, Shukra, and Satvasara. Kashyapa has added another type of Sara i.e. Oja Sara in this way, he mentioned nine types of Sara8. Dhatusara Purusha is classified into three viz. Pravara, Madhyama and Hina Sarata on the basis of Bala (strength)9. Dhatusara Purusha is classified into three viz. Pravara, Madhyama and Hina Sarata on the basis of B TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION. Address:- PhD (Scholar), Associate Professor , Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University; Limda, Vadodara.(Gujarat). . 124 Corresponding Author:-Sunil p. Nikhate. Address:- PhD (Scholar), Associate Professor , Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University; Limda, Vadodara.(Gujarat). . 124 Corresponding Author:-Sunil p. Nikhate. Address:- PhD (Scholar), Associate Professor , Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University; Limda, Vadodara.(Gujarat). 124 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 The state of Dosha Dhatu Mala is dynamic. There equilibrium is health and vitiations lead to development of diseases. Among these entities Dhatu are very important, which provides support to the body. They perform functions of Dharana and Poshana2. It is necessary to know the strength of these important body supporters. In order to evaluate Bala of these Dhatu, Charakacharya mentioned tenfold examination viz; Prakruti, Sara, Samhana, etc3. Out of these, Sara Parikshana is one. By examining Dhatusarata, we can be evaluated Dhatu for its qualitative and functional state. As per Dhatu Poshana, Rasa Dhatu is important as it is foremost one and it gives origin to all Dhatu. In other way we can say it provides nourishment to all remaining Dhatu. Aim & Objectives:- 1. To study Sara & Its Types. 1. To study Sara & Its Types. 2. To study influence of Rasadhatu on Twakasara. 3. To understand characteristics of Twakasara in different samhita. 4. To study importance of Twakasara. SARA Sara is Sthiransha & Bala (strength). In Ayurveda Sara ‘‘word is put for the Vishuddhatara Dhatu. Sara is excellence, essence of part or best part, the supreme quality, superb functional aspect of Dhatu4. Little waning or vitiations of Dhatu is not hampered functions immediately if Dhatu is Uttama Sara. Twaksara Purusha Vivechana No Characteristics Charaka-Samhita Sushruta-Samhita Kashyapa- Samhita 1 Sukha + - - 2 Saubhagya + - - 3 Aishwarya + - - 4 Upbhoga + - + 5 Buddhi + - - 6 Vidya + - - 7 Arogya + - - 8 Praharsha + - - (Body Hair) Alpa + - - Gambhira + - - Mridu + + - Prasanna + + - Other characteristics:- Sr. No Characteristics Charaka-Samhita Sushruta-Samhita Kashyapa- Samhita 1 Sukha + - - 2 Saubhagya + - - 3 Aishwarya + - - 4 Upbhoga + - + 5 Buddhi + - - 6 Vidya + - - 7 Arogya + - - 8 Praharsha + - - Other characteristics:- Twak (Skin) 1. Snigdha: Snigdhata provides moistening and smoothening. Its results into unctuous, oily skin and having soothing look. Snigdha Guna is composed of Apya Mahabhut. It raises Bala (strength) of the body and produces softness in whole body. ( ) 1. Snigdha: Snigdhata provides moistening and smoothening. Its results into unctuous, oily skin and having soothing look. Snigdha Guna is composed of Apya Mahabhut. It raises Bala (strength) of the body and produces softness in whole body. y 2. Slakshana: Mean the skin which possesses the property of smoothness. It has feeling of talcum powder. The smoothness property has power of wound healing (Ropana). p p y p g p 3. Mridu: Mridu mean softness. Person is having a soft skin which gives a feeling like a velvety cloth. This characteristic (Mrudu) is due to predominance Jala & Akash Mahabhuta. 3. Mridu: Mridu mean softness. Person is having a soft skin which gives a feeling like a velvety cloth. This characteristic (Mrudu) is due to predominance Jala & Akash Mahabhuta. 4. Prasanna: Means blooming; Person who has a healthy, energetic, attractive look and creates a feeling of joy to the observer. E.g. good looking male/female models. They are having freshness which observes for long lasting persists for a whole day. 4. Prasanna: Means blooming; Person who has a healthy, energetic, attractive look and creates a feeling of joy to the observer. E.g. good looking male/female models. They are having freshness which observes for long lasting persists for a whole day. p y 5. Sukshma: Person is having thin skin. Since the thickness of skin depends on its site as well as on the amount of subcutaneous fat. Twaksara Purusha Vivechana Twaksara individual are having Sukshma Twak as compare to other. p y 5. Sukshma: Person is having thin skin. Since the thickness of skin depends on its site as well as on the amount of subcutaneous fat. Twaksara individual are having Sukshma Twak as compare to other. 6. Saprabhev Twak: Skin having a natural glow over it. Rasadhatu is Apya, Dravyanusari (fluid in nature). Due to fluidity, it provides nourishment to peripheral tissue and enhances its Prinana Karma. Function of Rasadhatu are physically expressed over the skin that why skin look likes Saprabev without any artificial cosmetic make- up. 7. Twak-Rogarahito: It means free from skin diseases. Rasa Dhatu provides strength, growth and nutrition to the skin (body). Rasa Dhatu Sara person are having Vyadhi Kshamatva (good resistance) against skin disease. By chance disease occurs it may possibly short lasting and easily curable as compare to Hina Rasadhatu sara person. 7. Twak-Rogarahito: It means free from skin diseases. Rasa Dhatu provides strength, growth and nutrition to the skin (body). Rasa Dhatu Sara person are having Vyadhi Kshamatva (good resistance) against skin disease. By chance disease occurs it may possibly short lasting and easily curable as compare to Hina Rasadhatu sara person. p 8. Sadyahchhatpraroh: It means a person having good wound healing power. Whenever Twaksara individual come across the wound or trauma, it‘s duration of healing is earlier as comparison to Rasa- Asara person. It‘s due to Slakshna property15 &Prinana Karma of Rasadhatu. Twaksara Purusha Vivechana ‘Twak-Sara’ means Persons possessing an excellent quality of Rasa Dhatu residing in the skin and Loma i.e. the persons having well-nourished attractive skin and Loma are known as Twaksara Purusha. During the description of Dhatu Sara types, the term ‘Rasa Sara’ is not used in any of the Samhita; it is described as Twak-Sara. Dalhana clarified that in the term ―Twak-Sara’’ the word ‘Twak’ means the Rasa underneath the Twacha10. Probably it is due to the fact that the functions of the Rasa Dhatu i.e. the Prinana11, is best revealed through Twak. ‘Twak-Sara’ means Persons possessing an excellent quality of Rasa Dhatu residing in the skin and Loma i.e. the persons having well-nourished attractive skin and Loma are known as Twaksara Purusha. During the description of Dhatu Sara types, the term ‘Rasa Sara’ is not used in any of the Samhita; it is described as Twak-Sara. Dalhana clarified that in the term ―Twak-Sara’’ the word ‘Twak’ means the Rasa underneath the Twacha10. Probably it is due to the fact that the functions of the Rasa Dhatu i.e. the Prinana11, is best revealed through Twak. The characteristics of Twaksara person are well described in different Ayurveda Samhita-Charaka Samhita, Sushruta Samhita and Kashyapa Samhita. These characteristics can be grouped in to two as- h i l h i i The characteristics of Twaksara person are well described in different Ayurveda Samhita-Charaka Samhita, Sushruta Samhita and Kashyapa Samhita. These characteristics can be grouped in to two as- 1. Physical characteristics y 2. Other characteristics i.e. psycho-somatic characteristics 2. Other characteristics i.e. psycho-somatic characteristics Physical Characteristics:- Body Part Lakshnani Charaka- Samhita12 Sushruta- Samhita13 Kashyapa- Samhita14 Twaka (Skin) Snigdha + - - Slakshana + - - Mridu + + - Prasanna + + + Sukshma + - - Saprabha + - + Twak-Rog-Rahito - - + Sadyah-Kshata-Praroh - - + Loma Sukshma + - - 125 Int. J. Adv. Res. 7(7), 124-129 ISSN: 2320-5407 (Body Hair) Alpa + - - Gambhira + - - Mridu + + - Prasanna + + - Other characteristics:- Sr. Loma (Body Hair) Happiness in Rasa Sara person might be in dietary substances as well as for the substances used for improving the quality of skin. They usually become very happy by taking a delicious food or by getting a good quality of makeup material. As all these activities going to improve Prinana Karma of Twaka. p ya: It means person bless with good luck/ fortune. It also means beauty, charm and grace. g g p 2. Saubhagya: It means person bless with good luck/ fortune. It also means beauty, charm and grace. 3. Aishwarya: It means prosperity. In Twaksara person, Aishwarya flourishes due to his/her attractiveness and good looking (skin and hairs). 3. Aishwarya: It means prosperity. In Twaksara person, Aishwarya flourishes due to his/her attractiveness and good looking (skin and hairs). g g ( ) 4. Upbhoga: It means enjoyment of available stuffs. Twak Sara person enjoys the pleasure of different delicious diet and different cosmetics. 5. Buddhi: It means intelligence (power of understanding). It is a measure of decision making power of the person during tough situations. they can easily take good decision within short period of time. Rasasara person endowed with intelligence and can be assessed with intelligence quotient.(I.Q.test) 5. Buddhi: It means intelligence (power of understanding). It is a measure of decision making power of the person during tough situations. they can easily take good decision within short period of time. Rasasara person endowed with intelligence and can be assessed with intelligence quotient.(I.Q.test) 6. Viddya: It means knowledge of the text. Rasasara person may have knowledge of the different fields like sports, economics, politics etc. 6. Viddya: It means knowledge of the text. Rasasara person may have knowledge of the different fields like sports, economics, politics etc. 7. Arogya: It means health. Twaksara persons are having a good quality, quantity, and functions of Rasa Dhatu as well as Twak. So, they are more resistance against the diseases shared by these two. They generally do not suffer with skin disease. They usually not affected by Rasapradoshaj Vyadhi i.e. Pandu, Hridroga etc .If disease occurs, it will improve faster and cure easily. A Twaksara person can easily tolerate the condition of Rasakshaya or Vriddhi. In such individual the symptoms of the same will be delayed. They can tolerate louder sound; they will be not exhausted easily and can tolerate the thirst and fast easily. Loma (Body Hair) Loma (Body Hair) 1. Sukshma: It means very fine hairs. It is silky in appearance. 2. Alpa: It means such a person will have numerous hair over the body. Since the number of hairs depends on age, sex, and the anatomical site. 2. Alpa: It means such a person will have numerous hair over the body. Since the number of hairs depends on age, sex, and the anatomical site. 3. Gambheer: It means deeply rooted hairs. Rasadhatu nourishes the skin and its appendages (hairs and hair follicles). Twaksara person do not have history of Lomashatana (hair fall) as compare to Hina Rasa sara person. 4. Mridu: Rasasara person possesses Mridu Loma i.e. soft and silky appearance of hair that gives velvet like feelings. 3. Gambheer: It means deeply rooted hairs. Rasadhatu nourishes the skin and its appendages (hairs and hair follicles). Twaksara person do not have history of Lomashatana (hair fall) as compare to Hina Rasa sara person. 3. Gambheer: It means deeply rooted hairs. Rasadhatu nourishes the skin and its appendages (hairs and hair follicles). Twaksara person do not have history of Lomashatana (hair fall) as compare to Hina Rasa sara person. 4. Mridu: Rasasara person possesses Mridu Loma i.e. soft and silky appearance of hair that gives velvet like f li 4. Mridu: Rasasara person possesses Mridu Loma i.e. soft and silky appearance of hair that give feelings. g 5. Prasanna: Person is having lustrous hairs, lucidity in hair & might be dandruff free hairs. 126 Int. J. Adv. Res. 7(7), 124-129 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 Other Characteristics:- Other Characteristics: Only Acharya Charaka has mentioned about other characteristics including physio-psychological characteristics. Ot e C a acte st cs: Only Acharya Charaka has mentioned about other characteristics including physio-psychological characteristics. 1. Sukha: It means person feels happiness after doing some act or work. Happiness in Rasa Sara person might be in dietary substances as well as for the substances used for improving the quality of skin. They usually become very happy by taking a delicious food or by getting a good quality of makeup material. As all these activities going to improve Prinana Karma of Twaka. 1. Sukha: It means person feels happiness after doing some act or work. Importance of Twakasara Parikshana:- Sara examination is important in various aspects. Some are elaborated here Sara examination is important in various aspects. Some are elaborated here 1. For evaluation of Bala- We cannot conclude Rasa Sara person just looking externally. For both aims of Ayurved science, namely, safeguarding of health and if diseased, to cure the disease and come back to homeostatic condition, one needs to know his Bala. Sara examination gives us idea about types of Bala whether it is Pravara, Madhayama or Hina. 1. For evaluation of Bala- We cannot conclude Rasa Sara person just looking externally. For both aims of Ayurved science, namely, safeguarding of health and if diseased, to cure the disease and come back to homeostatic condition, one needs to know his Bala. Sara examination gives us idea about types of Bala whether it is Pravara, Madhayama or Hina. 2. In Chikitsa- Knowledge of Sarata is helpful to decide severity of the disease, prognosis of the disease, pattern of treatment and the drug dose in disease. e.g. Twaka Hina Dhatusara individuals are more susceptible to Rasadhatu Kshaya, & Twaka Vikara. Rasayana therapy can be adapted to improve Dhatusara. In Ayurveda, Rasayana therapy is mentioned to enhance quality of Dhatu. Rasa Dhatu is foremost Dhatu and responsible for Poshana and Prinana (nutrition) of body. 3. In Vyadhikshamatva- Sara is three dimensional Concept of the Dhatu i.e. it put forward qualitative, quantitative as well as functional status of the Dhatu. Twakasara individuals are more resist for any abnormal Vridhi (increase), Kshaya (decrease) or Rasadhatu-Pradushtas Vikara (vitiation of that Dhatu). Person with Uttam Rasadhatusarata can easily tolerate the symptoms of vitiated Dhatu and also easily cure from the same. Rasa Dhatu Sara persons can easily tolerate frequent fasting (Upavasa) without symptoms of Rasadhatukshaya. 4. For better progeny-In gestational periods, Sarata of progeny is influenced by many factors. So that, parent should be examined for their Dhatu Sarata to issue better progeny. After examination we can advise treatment in Hina dhatu sarata for its enhancement. In this way Sara examination is also useful in prevention of hereditary disorders. 5. In occupation- Sara examination is also essential for selection of particular occupation. Each occupation must need well qualified persons with physical and psychological health. e.g. Loma (Body Hair) Hence from the above discussion it is clear that a Twaksara person is comparatively immune for Rasa Kshaya, Rasa Vriddhi, or Rasa Pradoshaj Vyadhi as well as for skin disease. j y 8. Praharsha: It means excessive joyful condition of mind. It is due to Prinana karma of Rasa. It nourishes not only body but also mind of an individual. As Prinana Karma of Rasa Dhatu is expressed in the form of freshness and happiness, an individual looks fresh and happy. 8. Praharsha: It means excessive joyful condition of mind. It is due to Prinana karma of Rasa. It nourishes not only body but also mind of an individual. As Prinana Karma of Rasa Dhatu is expressed in the form of freshness and happiness, an individual looks fresh and happy. Importance of Twakasara Parikshana:- Rasa and Rakta Sara individuals are having beautiful look, and intolerance for heat and physical strain so that, they are not able to hard work & will be best for modeling and so on. In day to day life it is observed that, whenever a person got a job according their physical and psychological capability, He / She do better in their field. By considering all this fact, we understand how much Sara examination is essential for selection of particular occupation. 127 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 Conclusion:- 1. Sara is excellence, superb functional aspect of Dhatu. 1. Sara is excellence, superb functional aspect of Dhatu. 2. Acharya Charak, Sushruta and Vagbhata have mentioned eight type of Sara. These are Twak, Rakta, Mansa, Meda, Asthi, Majja, Shukra, and Satvasara. Acharya Kashyapa has added another type of Sara i.e. Oja Sara. 8 2. Acharya Charak, Sushruta and Vagbhata have mentioned eight type of Sara. These are Twak, Rakta, Mansa, Meda, Asthi, Majja, Shukra, and Satvasara. Acharya Kashyapa has added another type of Sara i.e. Oja Sara. 8 3. In Twakaaara’’ the word ‘Twak’ means the Rasa underneath the Twacha8. Probably it is due to the fact that the functions of the Rasa Dhatu i.e. the Prinana8, is best revealed through Twak. 3. In Twakaaara’’ the word ‘Twak’ means the Rasa underneath the Twacha8. Probably it is due to the fact that the functions of the Rasa Dhatu i.e. the Prinana8, is best revealed through Twak. 4. Examination of Twakasara is mainly advantageous for evaluation of Bala of Rasadhatu and also valuable for planning of Chikitsa, to know Vyadhikshamatva, for getting better progeny and for selection of occupation. 4. Examination of Twakasara is mainly advantageous for evaluation of Bala of Rasadhatu and also valuable for planning of Chikitsa, to know Vyadhikshamatva, for getting better progeny and for selection of occupation. References/ Bibliography:- 1. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and ‘Nyayacandrika’ Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Sutrasthana Chapter15; Verse no. 3; page no.67, published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. 1. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and ‘Nyayacandrika’ Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Sutrasthana Chapter15; Verse no. 3; page no.67, published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. 2. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and Nyayacandrika Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Chapter15; Verse no. 12; page no.62, published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. 2. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and Nyayacandrika Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Chapter15; Verse no. 12; page no.62, published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. p y p 3. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No-94. Page no.276, Published by Chaukhamba Surbharati Prakashan Varanasi. Reprint 2011. 3. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No-94. Page no.276, Published by Chaukhamba Surbharati Prakashan Varanasi. Reprint 2011. 4. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 102 , page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint 2011. 4. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 102 , page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint 2011. y p 5. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. 5. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. 6. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit SansthanaVaranasi, reprint2009. 6. Discussion:- A healthy skin reflects the healthy physiological state of the body which in turn depends on excellence state of Rasa Dhatu16. The main function of Rasa Dhatu is Prinana and responsible for the healthy skin and its appendage. It further helps to maintain nutritional state of the body. Any disturbance in Rasdhatua Formation & its Samhana (metabolism) leads to abnormal changes over Twak. Hence Rasasara and Twaksara are used as synonyms of each other. Twakarasarata is result of superb function of Rasa Dhatu and it reflects in the form of its Characteristics. Strength of Rasa Dhatu is assessed by critical examination of characteristic features mentioned in Twakasara. In the ancient text the characteristics are described in very concise manner. Each and every word has many meaning. Here characteristics are described depending upon reference to context. 12. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. , p 14. Kashyapa Samhita or Vriddhajivakatantra, Preached by Maharshi Marich Kashyapa; Summarized by Acahraya Vriddhajivaka; redacted by vatsya with English commentatory ; edited by Prof. Premvati Tiwari, Sutrasthana chapter 28 verse 37 page no.86; Chaukhamba Vivshvabharati Varanasi reprint 2013 13. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit SansthanaVaranasi, reprint2009. p p g p 15. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar; Sutrasthana chapter 1, verse no.18, page no.12 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 16. Nikhate Sunil P, Sant Sampada S; The comparative study of skin sebum content in Twaksara-asarta; Ayurlog: National Journal of Research in Ayurved Science-2015; 3(3): 67-72. References/ Bibliography:- Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit SansthanaVaranasi, reprint2009. p 7. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar Sharirasthana chapter 3, verse no.117, page no.407 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. p 7. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar Sharirasthana chapter 3, verse no.117, page no.407 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 8. Kashyapa Samhita or Vriddhajivakatantra, Preached by Maharshi Marich Kashyapa; Summarized by Acahraya Vriddhajivaka; redacted by vatsya with English commentatory ; edited by Prof. Premvati Tiwari, Sutrasthana chapter 28 verse 36 page no.86; Chaukhamba Vivshvabharati Varanasi reprint 2013 p p g p 9. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 111-113. , page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint 2011. 10. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit SansthanaVaranasi, reprint 2009. 11. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar; Sutrasthana chapter 11, verse no.4, page no.183 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 128 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 12. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. y p 13. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit SansthanaVaranasi, reprint2009. 13. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit SansthanaVaranasi, reprint2009. , p 14. Kashyapa Samhita or Vriddhajivakatantra, Preached by Maharshi Marich Kashyapa; Summarized by Acahraya Vriddhajivaka; redacted by vatsya with English commentatory ; edited by Prof. References/ Bibliography:- Premvati Tiwari, Sutrasthana chapter 28 verse 37 page no.86; Chaukhamba Vivshvabharati Varanasi reprint 2013 p p g p 15. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar; Sutrasthana chapter 1, verse no.18, page no.12 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. p p g p 15. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar; Sutrasthana chapter 1, verse no.18, page no.12 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 16. Nikhate Sunil P, Sant Sampada S; The comparative study of skin sebum content in Twaksara-asarta; Ayurlog: National Journal of Research in Ayurved Science-2015; 3(3): 67-72. 16. Nikhate Sunil P, Sant Sampada S; The comparative study of skin sebum content in Twaksara-asarta; Ayurlog: National Journal of Research in Ayurved Science-2015; 3(3): 67-72. 129
https://openalex.org/W4375948054
https://www.researchsquare.com/article/rs-2864864/latest.pdf
English
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Insurance approval rates for collagenase clostridium histolyticum prior to discontinuation: a Canada-wide analysis
Research Square (Research Square)
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Introduction Peyronie’s disease (PD) is a condition consisting of an abnormal wound healing response of the tunica albuginea that results in excess scar formation [1]. The resulting signs and symptoms include penile pain, shortening, curvature, narrowing, hinge deformity, palpable plaques, erectile dysfunction, and negative effects on a patient’s emotional state [2, 3]. Though initially thought to be a rare condition, it is now estimated to affect 9% [1, 4] of the male population. Unfortunately, although many patients ultimately experience stabilization of their disease, spontaneous resolution of the condition is rare [1, 2]. While surgical management of PD is associated with high success rates, effective non-surgical management strategies remain limited. Non-surgical treatment options include penile traction, oral medications, and intralesional therapy [4, 5]. However, there are no oral pharmacologic options currently recommended by Canadian Urologic Association (CUA) or American Urologic Association (AUA) guidelines due to a lack of evidence regarding their efficacy [5, 6]. Intralesional therapies such as verapamil are potential options for patients, however the use of these agents remains off-label [5]. Surgical correction remains a viable option for many PD patients, though is associated with its own host of potential complications. In 2013, collagenase clostridium histolyticum (CCh) became the first pharmacologic treatment for PD to be formally approved by the Food and Drug Administration following publication of the landmark IMPRESS I and II trials, which were able to demonstrate the utility of CCh as a safe and efficacious, non-surgical treatment for Peyronie’s Disease [7, 8]. Subsequently, CCh or Xiaflex® (Endo Pharmaceuticals) was included as a treatment option in the most recent CUA and AUA guidelines for the management of PD. Despite the findings from the IMPRESS trials and further studies confirming the efficacy of alternative treatment protocols to reduce the cost of therapy, CCh was discontinued in Australia and Asia in 2019, followed by Europe and Canada in 2020. The primary reason for discontinuation cited by the producer, Endo Pharmaceuticals, was that “The product did not receive government reimbursement approval and demand for the product has remained unsustainably low.” [9] This discontinuation represents a loss to the PD patient, for whom a safe, effective, and relatively non-invasive treatment option is no longer available. A study by Mann et al. 2021 investigating Canadian provider perspectives on CCh found that most providers were proponents of CCh, with the majority of their patients being satisfied with their outcomes [9]. Introduction Moreover, 96% of respondents in this survey study felt that the discontinuation of CCh represented a loss for Canadian patients [10]. Thus, despite its demonstrated efficacy and both patient and provider satisfaction, the United States is now the only market in which intralesional CCh remains available. Given the impact that its discontinuation has left on multiple countries including Canada, we sought to assess insurance approval rates and ultimately usage of CCh across Canada to better understand the factors that led to its withdrawal. Article Version of Record: A version of this preprint was published at International Journal of Impotence Research on August 25th, 2023. See the published version at https://doi.org/10.1038/s41443-023-00749-7. Page 1/13 Abstract Collagenase Clostridium histolyticum (CCh), the first approved non-surgical treatment for Peyronie’s disease (PD), was withdrawn from the European, Canadian, and Asian markets due to poor demand and lack of government reimbursement options. We sought to assess insurance approval rates and usage of CCh across Canada to understand the factors that led to its withdrawal. Data on patients prescribed CCh for PD or Dupuytren's contracture was obtained through collaboration with BioScript Solutions to assess the association of variables with insurance approval and prescription filling. We identified 3297 insurance coverage applications for Xiaflex from April 2018 to June 2020. Of all applications for PD, 92.9% applications were approved while 7.1% were rejected. Despite the withdrawal of CCh from Canadian markets, coverage application approval rates for 2018, 2019, and 2020 were 86.5%, 90.1%, and 89.1%, respectively. Of 2921 approved applications, 88.8% prescriptions were filled. For the 376 rejected applications, 66.4% of prescriptions were filled. Overall, 90% of the cost of Xiaflex was covered in Canada among those with extended health benefits, with an out-of-pocket expense of $210.4. Insurance coverage requests for Xiaflex were approved at a high rate in Canada with approved patients being very likely to proceed with therapy, despite interprovincial variation. Materials and Methods Data regarding all patients who had been prescribed CCh for either PD or Dupuytren’s contracture (DC) were obtained from BioScript Solutions, which served the overwhelming majority of patients (> 95%) being considered for CCh treatment in Canada via the Xiaflex Access Program. BioScript Solutions is a specialty healthcare company that supports a range of complex drug therapies and provides a variety of services including wholesale pharmaceutical distribution as well as clinics and pharmacies. Patients who were deemed potential candidates for CCh by their physicians were forwarded to the Xiaflex Access Program, which would then assist with obtaining Page 2/13 Page 2/13 insurance coverage, navigating reimbursement, dispensing the product itself, and overall patient support. In this role, Bioscript Solutions would obtain information regarding insurance coverage, insurance provider, insurance approval or rejection, coverage amount, and more. For the purposes of this study, Bioscript Solutions was able to provide this data for all patients enrolled in the Xiaflex Access Program from April 2018 to June 2020. Research ethics board approval as per TCPS2 was not required in this study as all data from BioScript Solutions is previously anonymized and no confidential patient information was accessible. insurance coverage, navigating reimbursement, dispensing the product itself, and overall patient support. In this role, Bioscript Solutions would obtain information regarding insurance coverage, insurance provider, insurance approval or rejection, coverage amount, and more. For the purposes of this study, Bioscript Solutions was able to provide this data for all patients enrolled in the Xiaflex Access Program from April 2018 to June 2020. Research ethics board approval as per TCPS2 was not required in this study as all data from BioScript Solutions is previously anonymized and no confidential patient information was accessible. Statistical analysis was performed with SPSS 24 software (Armonk, NY: IBM Corp.). Continuous variables were presented as medians and interquartile ranges [25th − 75th ] in accordance with data distribution on the normality test, and a comparison of continuous variables was performed using the Kruskal-Wallis test. Categorical variables were presented as absolute variables and frequencies and analyzed with Chi-square test. Then, a univariable and multivariable-adjusted logistic regression analysis was performed to determine the association of variables with insurance approval and prescription filling. A p-value < 0.05 was considered statistically significant. Results From April 2018 to June 2020, 3297 insurance coverage applications for Xiaflex were identified. This included prescriptions for both PD and DC. Of these, 2535 (76.9%) of applications were for DC, 748 (22.7%) for PD, and 14 (0.04%) for both conditions. Of all 2921 approved applications, 2594 (88.8%) prescriptions were filled. However, for the 376 rejected applications, 250 (66.4%) of prescriptions were filled. Overall, 90% (80–100) of the cost of Xiaflex was covered in Canada, with an out-of-pocket expense of $210.4 (0- 283.3). Table 1 demonstrates baseline characteristics stratified by applications per Canada Province. Significant differences were seen with respect to application approvals, applications submitted in each calendar year (2018–2020), insurance company and coverage, prescriber volume, out of pocket expense, and whether the prescription was filled. Page 3/13 Table 1 Overall characteristics of the patients that underwent insurance application for xiaflex in accordance with the different provinces in Canada. Results Overall n =  3297 (100%) AB n = 338 (10.3%) BC n = 102 (3.1%) MB n = 14 (0.4%) NB n = 114 (3.5%) NFLD n = 187 (5.7%) NS n = 88 (2.7%) ON n =  1728 (52.4%) PQ n = 715 (21.7%) SK n = 11 (0.3%) p- value Condition                       Dupuytren's contracture 2535 (76.9%) 253 (74.9%) 84 (82.4%) 14 (100%) 114 (100%) 174 (93%) 88 (100%) 1246 (72.1%) 551 (77.1%) 11 (100%)   Peyronie's disease 748 (22.7%) 85 (25.1%) 18 (17.6%) 0 0 13 (7%) 0 477 (27.6%) 155 (21.7%) 0   Both 14 (0.4%) 0 0 0 0 0 0 5 (0.3%) 9 (1.3%) 0 <  0.001 Initial application approval                       Rejected 383 (11.6%) 68 (20.1%) 3 (2.9%) 5 (35.7%) 7 (6.1%) 25 (13.4%) 1 (1.1%) 38 (2.2%) 233 (32.6%) 3 (27.3%)   Approved 2914 (88.4%) 270 (79.9%) 99 (97.1%) 9 (64.3%) 107 (93.9%) 162 (86.6%) 87 (98.9%) 1690 (97.8%) 482 (67.4%) 8 (72.7%) <  0.001 Subsequent application aproval (n =  383)                       Rejected 376 (98.2%) 68 (100%) 3 (100%) 4 (80%) 7 (100%) 25 (100%) 1 (100%) 36 (94.7%) 229 (98.3%) 3 (100%)   Approved 7 (1.8%) 0 0 1 (20%) 0 0 0 2 (5.3%) 4 (1.7%) 0 0.090 Overall approval                       Rejected 376 (11.4%) 68 (20.1%) 3 (2.9%) 4 (28.6%) 7 (6.1%) 25 (13.4%) 1 (1.1%) 36 (2.1%) 229 (32%) 3 (27.3%)   Approved 2921 (88.6%) 270 (79.9%) 99 (97.1%) 10 (71.4%) 107 (93.9%) 162 (86.6%) 87 (98.9%) 1692 (97.9%) 486 (68%) 8 (72.7%) <  0.001 Year                       2018 1246 (37.8%) 100 (29.6%) 25 (24.5%) 9 (64.3%) 40 (35.1%) 102 (54.5%) 39 (44.3%) 680 (39.4%) 251 (35.1%) 0   2019 1564 (47.4%) 164 (48.5%) 57 (55.9%) 5 (35.7%) 56 (49.1%) 78 (41.7%) 38 (43.2%) 789 (45.7%) 366 (51.2%) 11 (100%)   2020 487 (14.8%) 74 (21.9%) 20 (19.6%) 0 18 (15.8%) 7 (3.7%) 11 (12.5%) 259 (15%) 98 (13.7%) 0 <  0.001 Insurance                       Blue Cross 294 (8.9%) 99 (29.3%) 28 (27.5%) 6 (42.9%) 44 (38.6%) 35 (18.7%) 21 (23.9%) 23 (1.3%) 34 (4.8%) 4 (36.4%)   Canada Life 407 (12.3%) 44 (13%) 9 (8.8%) 4 (28.6%) 7 (6.1%) 35 (18.7%) 7 (8%) 270 (15.6%) 29 (4.1%) 2 (18.2%)   Median [Interquartile range 25th – 75th ]. * 2921 patients with insurance approval including patients approved after subsequent applications. Results AB (Alberta) BC (British Columbia) MB (Manitoba) NB (New Brunswick) NFLD (Newfoundland) NS (Nova Scotia) ON (Ontario) PQ Page 4/13 Page 4/13 Overall n =  3297 (100%) AB n = 338 (10.3%) BC n = 102 (3.1%) MB n = 14 (0.4%) NB n = 114 (3.5%) NFLD n = 187 (5.7%) NS n = 88 (2.7%) ON n =  1728 (52.4%) PQ n = 715 (21.7%) SK n = 11 (0.3%) p- value Condition                       ClaimSecure 62 (1.9%) 7 (2.1%) 7 (6.9%) 1 (7.1%) 3 (2.6%) 3 (1.6%) 3 (3.4%) 32 (1.9%) 6 (0.8%) 0   Combination 159 (4.8%) 12 (3.6%) 5 (4.9%) 0 4 (3.5%) 7 (3.7%) 1 (1.1%) 115 (6.7%) 14 (2%) 1 (9.1%)   Desjardins 87 (2.6%) 2 (0.6%) 1 (1%) 0 0 3 (1.6%) 2 (2.3%) 24 (1.4%) 55 (7.7%) 0   Express Scripts 67 (2%) 1 (0.3%) 4 (3.9%) 0 4 (3.5%) 2 (1.1%) 0 56 (3.2%) 0 0   Green Shield 273 (8.3%) 15 (4.4%) 13 (12.7%) 1 (7.1%) 0 5 (2.7%) 3 (3.4%) 223 (12.9%) 12 (1.7%) 1 (9.1%)   Johnson Insurance 76 (2.3%) 0 2 (2%) 0 3 (2.6%) 22 (11.8%) 1 (1.1%) 47 (2.7%) 1 (0.1%) 0   La Capital 59 (1.8%) 0 0 0 0 0 0 0 59 (8.3%) 0   Manulife 434 (13.2%) 28 (8.3%) 14 (13.7%) 0 8 (7%) 16 (8.6%) 7 (8%) 286 (16.6%) 73 (10.2%) 2 (18.2%)   PSHCP 264 (8%) 6 (1.8%) 1 (1%) 1 (7.1%) 12 (10.5%) 30 (16%) 20 (22.7%) 167 (9.7%) 27 (3.8%) 0   RAMQ (Government Plan) 185 (5.6%) 1 (0.3%) 0 0 0 0 0 0 184 (25.7%) 0   SSQ 74 (2.2%) 1 (0.3%) 0 0 0 0 0 8 (0.5%) 65 (9.1%) 0   Sun Life 485 (14.7%) 75 (22.2%) 7 (6.9%) 0 13 (11.4%) 12 (6.4%) 11 (12.5%) 287 (16.6%) 80 (11.2%) 0   Telus 58 (1.8%) 1 (0.3%) 4 (3.9%) 0 4 (3.5%) 0 0 47 (2.7%) 2 (0.3%) 0   Other 313 (9.5%) 46 (13.6%) 7 (6.9%) 1 (7.1%) 12 (10.5%) 17 (9.1%) 12 (13.6%) 143 (8.3%) 74 (10.3%) 1 (9.1%) <  0.001 Prescriptions by provider                       1–20 prescriptions 1345 (40.8%) 152 (45%) 102 (100%) 14 (100%) 30 (26.3%) 47 (25.1%) 63 (71.6%) 637 (36.9%) 289 (40.4%) 11 (100%)   21–40 prescriptions 706 (21.4%) 140 (41.4%) 0 0 21 (18.4%) 29 (15.5%) 25 (28.4%) 423 (24.5%) 68 (9.5%) 0   > 40 prescriptions 1246 (37.8%) 46 (13.6%) 0 0 63 (55.3%) 111 (59.4%) 0 668 (38.7%) 358 (50.1%) 0 <  0.001 Out of pocket expense* 210.4 [0 - 283.3] 144.7 [0– 286] 261.7 [0– 300.3] 270.7 [16.5– 564.9] 50 [15– 267.3] 140.6 [9.2– 275.1] 269 [10– 283.5] 139.1 [0– 278.2] 283.3 [244.9– 494.4] 140 [0– 263.1] <  0.001 Median [Interquartile range 25th – 75th ] * 2921 patients with insurance approval including patients approved after subsequent P 5 13 Median [Interquartile range 25th – 75th ]. Results * 2921 patients with insurance approval including patients approved after subsequent applications. AB (Alberta), BC (British Columbia), MB (Manitoba), NB (New Brunswick), NFLD (Newfoundland), NS (Nova Scotia), ON (Ontario), PQ (Quebec), SK (Saskatchewan) Page 5/13 Page 5/13 Overall n =  3297 (100%) AB n = 338 (10.3%) BC n = 102 (3.1%) MB n = 14 (0.4%) NB n = 114 (3.5%) NFLD n = 187 (5.7%) NS n = 88 (2.7%) ON n =  1728 (52.4%) PQ n = 715 (21.7%) SK n = 11 (0.3%) p- value Condition                       Coverage* 90 [80– 100] 90 [80– 100] 80 [80– 100] 80 [57.8– 85] 95 [80– 100] 80 [80– 99] 80 [80– 100] 90 [80– 100] 80 [80– 90] 80 [52.5– 80] <  0.001 Prescription filled                       No 453 (13.7%) 71 (21%) 5 (4.9%) 0 20 (17.5%) 44 (23.5%) 8 (9.1%) 189 (10.9%) 114 (15.9%) 2 (18.2%)   Yes 2844 (86.3%) 267 (79%) 97 (95.1%) 14 (100%) 94 (82.5%) 143 (76.5%) 80 (90.9%) 1539 (89.1%) 601 (84.1%) 9 (81.8%) <  0.001 Median [Interquartile range 25th – 75th ]. * 2921 patients with insurance approval including patients approved after subsequent applications. AB (Alberta), BC (British Columbia), MB (Manitoba), NB (New Brunswick), NFLD (Newfoundland), NS (Nova Scotia), ON (Ontario), PQ (Quebec), SK (Saskatchewan) AB (Alberta), BC (British Columbia), MB (Manitoba), NB (New Brunswick), NFLD (Newfoundland), NS (Nova Scotia), ON (Ontario), PQ (Quebec), SK (Saskatchewan) Table 2 compares approved and rejected applications. Of all insurance coverage applications for DC, 2214 (87.3%) applications were approved, while 321 (12.7%) were rejected. Of all applications for PD, 695 (92.9%) applications were approved while 53 (7.1%) were rejected. Despite the withdrawal of CCh from Canadian markets in 2021, coverage application approval rates for 2018, 2019, and 2020 were 86.5%, 90.1%, and 89.1% respectively. There was a statistically significant difference in the likelihood of approval by condition (DP vs. PD) and which insurance company was utilized, ultimately affecting whether the prescription was filled. No difference was noted by each subsequent year (2018–2020) or provider volume. Table 2 compares approved and rejected applications. Of all insurance coverage applications for DC, 2214 (87.3%) applications were approved, while 321 (12.7%) were rejected. Of all applications for PD, 695 (92.9%) applications were approved while 53 (7.1%) were rejected. Despite the withdrawal of CCh from Canadian markets in 2021, coverage application approval rates for 2018, 2019, and 2020 were 86.5%, 90.1%, and 89.1% respectively. Results Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Higher volume prescribers were more likely to ascertain insurance approval for their patients and patients were more likely to fill the prescription if insurance approval was accepted. In Table 4, multivariate logistic regression analysis was performed to test for associations between variables and whether the prescription was filled. This demonstrated that patients with PD were more likely to fill their prescriptions for CCh than patients with DC (OR 0.57, p < 0.001). Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Patients were more likely to fill their prescription with insurance approval (OR 4.31, p < 0.001). Rejected n = 376 Approved n = 2921 p-value Condition       Yes 250 (8.8%) 2594 (91.2%) < 0.001 Median [Interquartile range 25th – 75th ]. In Table 3, multivariate logistic regression analysis was performed to test for associations between variables and insurance approval. This demonstrated that patients with PD were more likely to receive insurance coverage for CCh than patients with DC (OR 2.44, p <  0.001). Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Higher volume prescribers were more likely to ascertain insurance approval for their patients and patients were more likely to fill the prescription if insurance approval was accepted. In Table 4, multivariate logistic regression analysis was performed to test for associations between variables and whether the prescription was filled. This demonstrated that patients with PD were more likely to fill their prescriptions for CCh than patients with DC (OR 0.57, p < 0.001). Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Patients were more likely to fill their prescription with insurance approval (OR 4.31, p < 0.001). In Table 3, multivariate logistic regression analysis was performed to test for associations between variables and insurance approval. This demonstrated that patients with PD were more likely to receive insurance coverage for CCh than patients with DC (OR 2.44, p <  0.001). Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Higher volume prescribers were more likely to ascertain insurance approval for their patients and patients were more likely to fill the prescription if insurance approval was accepted. Results There was a statistically significant difference in the likelihood of approval by condition (DP vs. PD) and which insurance company was utilized, ultimately affecting whether the prescription was filled. No difference was noted by each subsequent year (2018–2020) or provider volume. Table 2 compares approved and rejected applications. Of all insurance coverage applications for DC, 2214 (87.3%) applications were approved, while 321 (12.7%) were rejected. Of all applications for PD, 695 (92.9%) applications were approved while 53 (7.1%) were rejected. Despite the withdrawal of CCh from Canadian markets in 2021, coverage application approval rates for 2018, 2019, and 2020 were 86.5%, 90.1%, and 89.1% respectively. There was a statistically significant difference in the likelihood of approval by condition (DP vs. PD) and which insurance company was utilized, ultimately affecting whether the prescription was filled. No difference was noted by each subsequent year (2018–2020) or provider volume. Page 6/13 Page 6/13 Table 2 approval and rejection. Rejected n = 376 Approved n = 2921 p-value Condition       Dupuytren's contracture 321 (12.7%) 2214 (87.3%)   Peyronie's disease 53 (7.1%) 695 (92.9%)   Both 2 (14.3%) 12 (85.7%) < 0.001 Year       2018 168 (13.5%) 1078 (86.5%)   2019 155 (9.9%) 1409 (90.1%)   2020 53 (10.9%) 434 (89.1%) 0.012 Insurance       Blue Cross 65 (22.1%) 229 (77.9%)   Canada Life 35 (8.6%) 372 (91.4%)   ClaimSecure 0 62 (100%)   Combination 6 (3.8%) 153 (96.2%)   Desjardins 0 87 (100%)   Express Scripts 0 67 (100%)   Green Shield 0 273 (100%)   Johnson Insurance 0 76 (100%)   La Capital 0 59 (100%)   Manulife 11 (2.5%) 423 (97.5%)   PSHCP 0 264 (100%)   RAMQ (Government Plan) 182 (98.4%) 3 (1.6%)   SSQ 14 (18.9%) 60 (81.1%)   Sun Life 0 485 (100%)   Telus 0 58 (100%)   Other 63 (20.1%) 250 (88.6%) < 0.001 Prescriptions by provider       1–20 prescriptions 160 (11.9%) 1185 (88.1%)   21–40 prescriptions 65 (9.2%) 641 (90.8%)   > 40 prescriptions 151 (12.1%) 1095 (87.9%) 0.115 Out of pocket expense NA 210.4 [0–283.3] --- Coverage NA 90 [80–100] --- Prescription filled       No 126 (27.8%) 327 (72.2%)   Median [Interquartile range 25th – 75th ]. Rejected n = 376 Approved n = 2921 p-value Condition       Yes 250 (8.8%) 2594 (91.2%) < 0.001 Median [Interquartile range 25th – 75th ]. In Table 3, multivariate logistic regression analysis was performed to test for associations between variables and insurance approval. This demonstrated that patients with PD were more likely to receive insurance coverage for CCh than patients with DC (OR 2.44, p <  0.001). Results In Table 4, multivariate logistic regression analysis was performed to test for associations between variables and whether the prescription was filled. This demonstrated that patients with PD were more likely to fill their prescriptions for CCh than patients with DC (OR 0.57, p < 0.001). Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Patients were more likely to fill their prescription with insurance approval (OR 4.31, p < 0.001). Page 8/13 Page 8/13 Table 3 Univariable and multivariable adjusted logistic regression analysis to insurance approval and analyzed variables. Univariable Multivariable   OR 95% CI p-value OR 95% CI p-value   Condition               Dupuytren's contracture 1     1       Peyronie's disease 1.90 1.40–2.57 < 0.001 2.44 1.69–3.35 < 0.001   Both 0.87 0.19–3.91 0.856 2.73 0.52–14.34 0.236   Year               2018 1     1       2019 1.42 1.12–1.79 0.003 1.70 1.30–2.23 < 0.001   2020 1.28 0.92–1.77 0.145 1.33 0.91–1.95 0.145   Provinces in Canada               Alberta 0.61 0.37–1.01 0.054 0.44 0.26–0.77 0.003   British Columbia 5.09 1.50–17.31 0.009 2.71 0.77 - 9.56 0.121   Manitoba 0.39 0.11–1.33 0.130 0.23 0.07–0.83 0.025   New Brunswick 2.36 0.99–5.65 0.054 2.18 0.89–5.37 0.088   Newfoundland and Labrador 1     1       Nova Scotia 13.43 1.79–100.77 0.012 10.63 1.39–81.36 0.023   Ontario 7.25 4.25–12.39 < 0.001 5.03 2.87–8.82 < 0.001   Quebec 0.33 0.21–0.51 < 0.001 0.20 0.12–0.33 < 0.001   Saskatchewan 0.41 0.10–1.66 0.211 0.25 0.06–1.08 0.064   Prescription by provider               1–20 prescriptions 1     1       21–40 prescriptions 1.33 0.98–1.80 0.065 0.85 0.59–1.21 0.363   > 40 prescriptions 0.98 0.77–1.24 0.862 0.93 0.70–1.25 < 0.001   Prescription filled               No 1     1       Yes 4.00 3.14–5.10 < 0.001 4.67 3.50–6.27 < 0.001   CI: Confidence interval; OR: Odds ratio. Page 9/13 Page 9/13 Page 9/13 Table 4 Univariable and multivariable adjusted logistic regression analysis to prescription filled and analyzed variables. Results Univariable Multivariable   OR 95% CI p-value OR 95% CI p-value   Condition               Dupuytren's contracture 1     1       Peyronie's disease 0.73 0.58–0.91 0.005 0.57 0.44–0.73 < 0.001   Both 0.88 0.20–3.96 0.870 0.67 0.14–3.17 0.614   Year               2018 1     1       2019 1.19 0.96–1.48 0.122 1.12 0.89–1.41 0.336   2020 0.76 0.57–1.002 0.052 0.75 0.56–1.02 0.066   Provinces in Canada               Alberta 1.16 0.76–1.77 0.503 1.71 1.07–2.74 0.026   British Columbia 5.97 2.28–15.59 < 0.001 6.51 2.42–17.47 < 0.001   Manitoba* --     ---       New Brunswick 1.45 0.80–2.61 0.220 1.26 0.69–2.31 0.452   Newfoundland and Labrador 1     1       Nova Scotia 3.08 1.38–6.86 0.006 2.75 1.21–6.25 0.016   Ontario 2.51 1.73–3.63 < 0.001 2.51 1.70–3.73 < 0.001   Quebec 1.62 1.10–2.40 0.016 2.70 1.76–4.14 < 0.001   Saskatchewan 1.39 0.29–6.65 0.684 1.88 0.36–9.80 0.453   Prescription by provider               1–20 prescriptions 1     1       21–40 prescriptions 0.94 0.72–1.22 0.629 1.07 0.81–1.42 0.626   > 40 prescriptions 0.99 0.79–1.24 0.934 1.27 0.98–1.64 0.067   Overall approval               Rejected 1     1       Approved 4.00 3.14–5.10 < 0.001 4.31 3.23–5.73 < 0.001   CI: Confidence interval; OR: Odds ratio. * All patients in Manitoba got their prescription filled. Discussion With the publication of the IMPRESS I and II trials demonstrating the efficacy of CCh and subsequent approval of this medication for the treatment of PD by a variety of national health regulatory bodies, CCh became a popular and important non-surgical treatment option for patients diagnosed with PD. Despite CCh being recommended as a first line therapy by the CUA and AUA [5, 6], Xiaflex® has been removed from Australian, Asian, European, and Canadian European markets. Our study aimed to assess insurance approval rates and usage of CCh in Canada as a potential factor for discontinuation of the medication. Page 10/13 Overall, our study demonstrated high insurance approval rates for use of CCh in Peyronie’s disease, with 92.9% of requests being approved in Canada. Applications were more likely to be accepted for PD compared to DP, however prescriptions for PD patients were less likely to be filled. A potential explanation is the out-of-pocket expense associated with Xiaflex may act as a barrier for patients to access this medication, even with insurance approval. Interestingly, higher volume prescribers were more likely to have accepted applications. However, it is possible that this is due to the fact that providers who have received prior acceptances are more likely to submit more applications and know how to appropriately complete the required application. Although significant interprovincial variation was present, overall insurance coverage application approvals were high for the country as a whole. Once approved, patients were likely to proceed with CCh treatment, with 91.2% of all approved prescriptions being filled. less likely to be filled. A potential explanation is the out-of-pocket expense associated with Xiaflex may act as a barrier for patients to access this medication, even with insurance approval. Interestingly, higher volume prescribers were more likely to have accepted applications. However, it is possible that this is due to the fact that providers who have received prior acceptances are more likely to submit more applications and know how to appropriately complete the required application. Although significant interprovincial variation was present, overall insurance coverage application approvals were high for the country as a whole. Once approved, patients were likely to proceed with CCh treatment, with 91.2% of all approved prescriptions being filled. Despite CCh being the only approved medical therapy for PD, it was unfortunately removed from multiple countries including Canada. Discussion With a lack of effective non-surgical options, this will surely lead to increased surgical intervention rates as the authors of this paper have anecdotally noted. Although a cost-effectiveness analysis by Cordon et al. demonstrated that the cost of penile plication was significantly less than that of the CCh protocol utilized in IMPRESS I and II [11], other considerations should be taken into account. The primary outcome assessed in their study was improvement in curvature [11], and CCh still has utility as a primary non-surgical option not only for achieving a functionally straight penis, but also as a tool to help improve patient satisfaction. Further investigation of alternative CCh protocols were able to demonstrate significantly improved cost effectiveness, with cost being comparable to primary surgery [12]. Additionally, limited operating room time in a single-payer system such as Canada is another consideration that was not accounted for in the US and UK [11, 13] cost effectiveness studies. Aside from the additional stress on the already limited resources of a universal healthcare system, there are several patient factors to consider. Not all patients may be ideal candidates for surgery, and regardless of candidacy, there are inherent risks of surgery as well as post-operative complications that must be discussed with the patient. Among surgical candidates, secondary effects of surgery, such as approximately 2cm of penile shortening associated with penile plication. In contrast, a recent study combining CCh with RestoreX traction device demonstrated 1.9cm improvement in length in addition to curvature improvements [14]. This ~ 4cm difference in penile length between correcting a curve with plication versus CCh + RestoreX traction is dramatic for many patients and must be considered when evaluating patient outcomes and cost-effectiveness. For other techniques such as plaque incision and grafting, a 50% [15] risk of worsening erectile dysfunction is also considered as a non-acceptable risk for many men and leads to a lifetime of adjuvant ED therapies, accruing further expenses. Ultimately, although immediate outcomes for surgical repair are good, patients may express concerns in the long term regarding their quality of life such as satisfaction with penile cosmesis, off-target effects of surgery and new onset ED [16, 17]. The potential effects of the discontinuation of CCh in the Canadian healthcare system was addressed in our prior study, which sought to understand the perception of Canadian providers regarding this issue [9]. Discussion A large majority of the survey participants felt that the discontinuation of CCh would be a significant loss for Canadian PD patients. In addition, few Canadian providers would offer alternative intralesional therapies in the future and would be more likely to offer surgical management for PD. The sentiment among Canadian providers to not offer alternative intralesional therapy may result in a large proportion of PD patients who are not suitable for surgical management to remain untreated. Furthermore, many patients who are unwilling to proceed with surgery due to personal preferences will also remain untreated. This has significant implications given the prevalence of PD as well as its negative impact on mental health [18, 19], with approximately one third of men with PD may suffer from clinical depression [19]. Additionally, PD has been associated with significant emotional and psychosocial distress in domains such as quality of life, self-esteem, social isolation, relationship strain, and stigmatization [18]. Given the mental health difficulties faced by many PD patients, the lack of non-surgical options may further dissuade patients from proceeding with therapy for their condition. This will unfortunately lead to further worsening of both the patient’s physical and mental health. Intralesional interferon has also been discontinued for treatment of Peyronie’s disease in Canada, leaving traction therapy and intralesional verapamil as the only recommended non-surgical options. However, intralesional verapamil has heterogeneous results in the literature [20] and lacks high level data as well as a well-established protocol. Mechanical traction devices may be undesirable to many patients as well, as many treatment regimens recommend use for multiple hours daily. The significant limitations on the efficacy of non- surgical treatments are an important consideration when treating a population where depression is a significant issue [18, 19]. In the future, individuals with Peyronie’s disease may be further dissuaded from proceeding with therapy, putting patients at further risk for worsening of their condition and psychosocial status. This study is not without limitations. Our data was limited to what could be provided by the Canadian distributer, Bioscript Solutions. While relatively granular data was obtained regarding insurance application, acceptance, amount of coverage, and more, data regarding certain baseline information was lacking including age, comorbidities, socioeconomic status, and employment status. Most importantly, detailed explanations for insurance coverage rejection were not available. Conclusion The discontinuation of CCh in Australia, Asia, Europe, and most recently Canada, has further limited the non-surgical options for the treatment of PD. Low rates of government reimbursement approvals and inadequate demand for the product was cited as a reason for withdrawal from these markets. Despite the limitations, this study was able to demonstrate that in Canada, insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to proceed with therapy. Going forward, the lack of conservative management for PD will be to the detriment of PD patients and may act as another barrier for individuals to proceed with therapy. Key Messages - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therapy - Discontinuation of CCh has further limited non-surgical options for treatment of PD - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therapy Di ti ti f CCh h f th li it d i l ti f t t t f PD - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therap - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therapy Discontinuation of CCh has further limited non-surgical options for treatment of PD - Discontinuation of CCh has further limited non-surgical options for treatment of PD - Lack of conservative management strategies for PD may act as another barrier for individuals w - Lack of conservative management strategies for PD may act as another barrier for individuals wanting to proceed with therapy l i Funding No financial assistance was received in support of this study. Ethical Approval Research ethics board approval as per TCPS2 was not required in this study as all data from BioScript Solutions is previously anonymized and no confidential patient information was accessible. Conflicts of Interest RF has received speaking honoraria and an education grant from Boston Scientific and speaking honoraria from Paladin Labs. PP is a consultant for Boston Scientific. The remaining authors have no conflicts of interest to declare. Discussion Additionally, this data is likely biased towards Canadian individuals with extended health coverage, and it is possible that a significant number of patients without extended health benefits did Page 11/13 Page 11/13 not attempt applying for Xiaflex approval. Given that 0.5% of men seek assessment and management of their PD with a healthcare professional, and 11–13% of men likely have PD [21], the total number of prescriptions over a ~ 2-year period of 748 suggests that a significant number of Canadian men suffering with PD are not seeking healthcare evaluation or are not choosing CCH as a treatment method. This reinforces the notion that better public awareness for PD is necessary to engage men suffering in silence and reducing barriers to treatment. not attempt applying for Xiaflex approval. Given that 0.5% of men seek assessment and management of their PD with a healthcare professional, and 11–13% of men likely have PD [21], the total number of prescriptions over a ~ 2-year period of 748 suggests that a significant number of Canadian men suffering with PD are not seeking healthcare evaluation or are not choosing CCH as a treatment method. This reinforces the notion that better public awareness for PD is necessary to engage men suffering in silence and reducing barriers to treatment. Author Contribution Statement DC, BS, DSB, TS, RBB, MG, RF, PP each contributed to the study design, manuscript drafting, approval of the final version, and agree to be accountable for the accuracy of the work. Data Availability Statement The data that support the findings of this study are available from BioScript Solutions but restrictions apply to the availability of this data, which was used under license for the current study, and not publicly available. Data are however available from the authors upon reasonable request and with permission of BioScript Solutions. Declarations Data Availability Statement References Page 12/13 Page 12/13 1. Mulhall JP, Creech SD, Boorjian SA, et al. Subjective and objective analysis of the prevalence of Peyronie’s disease in a population of men presenting for prostate cancer screening. J Urol. 2004;171(6 I):2350-2353. doi:10.1097/01.ju.0000127744.18878.f1 2. Gholami SS, Gonzalez-Cadavid NF, Lin CS, Rajfer J, Lue TF. Peyronie’s disease: A review. J Urol. 2003;169(4):1234-1241. doi:10.1097/01.ju.0000053800.62741.fe 1. Mulhall JP, Creech SD, Boorjian SA, et al. Subjective and objective analysis of the prevalence of Peyronie’s disease in a population of men presenting for prostate cancer screening. J Urol. 2004;171(6 I):2350-2353. doi:10.1097/01.ju.0000127744.18878.f1 men presenting for prostate cancer screening. J Urol. 2004;171(6 I):2350-2353. doi:10.1097/01.ju.0000127744.18878.f1 2. Gholami SS, Gonzalez-Cadavid NF, Lin CS, Rajfer J, Lue TF. Peyronie’s disease: A review. J Urol. 2003;169(4):1234-1241. doi:10.1097/01.ju.0000053800.62741.fe 2. Gholami SS, Gonzalez-Cadavid NF, Lin CS, Rajfer J, Lue TF. Peyronie’s disease: A review. J Urol. 2003;169(4):1234-1241. doi:10.1097/01.ju.0000053800.62741.fe 3. Kadioglu A, Tefekli A, Erol B, Oktar T, Tunc M, Tellaloglu S. A retrospective review of 307 men with peyronie’s disease. J Urol. 2002;168(3):1075-1079. doi:10.1016/S0022-5347(05)64578-8 4. Mulhall JP, Schiff J, Guhring P. An Analysis of the Natural History of Peyronie’s Disease. J Urol. 2006;175(6). doi:10.1016/S0022- 5347(06)00270-9Bella AJ, Lee JC, Grober ED, Carrier S, Benard F, Brock GB. 2018 canadian urological association guideline for peyronie’s disease and congenital penile curvature. Can Urol Assoc J. 2018;12(5):E197-E209. doi:10.5489/cuaj.5255 5. Nehra A, Alterowitz R, Culkin DJ, et al. Peyronie’s disease: AUA guideline. J Urol. 2015;194(3):745-753. doi:10.1016/j.juro.2015.05.098 5. Nehra A, Alterowitz R, Culkin DJ, et al. Peyronie’s disease: AUA guideline. J Urol. 2015;194(3):74 doi:10.1016/j.juro.2015.05.098 6. Gelbard M, Goldstein I, Hellstrom WJG, et al. Clinical efficacy, safety and tolerability of collagenase clostridium histolyticum for the treatment of peyronie disease in 2 large double-blind, randomized, placebo controlled phase 3 studies. J Urol. 2013;190(1):199-207. doi:10.1016/j.juro.2013.01.087 6. Gelbard M, Goldstein I, Hellstrom WJG, et al. Clinical efficacy, safety and tolerability of collagenase clostridium histolyticum for the treatment of peyronie disease in 2 large double-blind, randomized, placebo controlled phase 3 studies. J Urol. 2013;190(1):199-207. doi:10.1016/j.juro.2013.01.087 7. Hellstrom WJG, Tue Nguyen HM, Alzweri L, et al. Intralesional Collagenase Clostridium histolyticum Causes Meaningful Improvement in Men with Peyronie’s Disease: Results of a Multi-Institutional Analysis. J Urol. 2019;201(4):777-782. doi:10.1097/JU.0000000000000032 7. Hellstrom WJG, Tue Nguyen HM, Alzweri L, et al. Intralesional Collagenase Clostridium histolyticum Causes Meaningful Improvement in Men with Peyronie’s Disease: Results of a Multi-Institutional Analysis. J Urol. 2019;201(4):777-782. doi:10.1097/JU.0000000000000032 8. Paladin Labs Inc. Healthcare Professional Information regarding discontinuation of XIAFLEX®. References Published April 30, 2020. https://dupuytrencanada.ca/wp-content/uploads/2020/05/60841-002-Xiaflex_Discontinuation_Notice_04302020.pdf 8. Paladin Labs Inc. Healthcare Professional Information regarding discontinuation of XIAFLEX®. Published April 30, 2020. https://dupuytrencanada.ca/wp-content/uploads/2020/05/60841-002-Xiaflex_Discontinuation_Notice_04302020.pdf 9. Mann U, Shiff B, Jain K, Flannigan R, Elterman D, Patel P. Canadian provider perspectives on Collagenase Clostridium histolyticum for the treatment of Peyronie’s disease and the impact of its discontinuation. Int J Impot Res. 2021. doi:10.1038/s41443-021-00458- z 9. Mann U, Shiff B, Jain K, Flannigan R, Elterman D, Patel P. Canadian provider perspectives on Collagenase Clostridium histolyticum for the treatment of Peyronie’s disease and the impact of its discontinuation. Int J Impot Res. 2021. doi:10.1038/s41443-021-00458- z 10. Cordon BH, Hofer MD, Hutchinson RC, Broderick GA, Lotan Y, Morey AF. Superior Cost Effectiveness of Penile Plication vs Intralesional Collagenase Injection for Treatment of Peyronie’s Disease Deformities. Urol Pract. 2017;4(2):118-125. doi:10.1016/j.urpr.2016.04.005 10. Cordon BH, Hofer MD, Hutchinson RC, Broderick GA, Lotan Y, Morey AF. Superior Cost Effectiveness of Penile Plication vs Intralesional Collagenase Injection for Treatment of Peyronie’s Disease Deformities. Urol Pract. 2017;4(2):118-125. doi:10.1016/j.urpr.2016.04.005 11. Harvey N, Pearce I. At what cost is collagenase clostridium histolyticum viable for treating Peyronie’s disease in a public healthcare system? Andrology. 2020;8(5):1304-1311. doi:10.1111/andr.12830 11. Harvey N, Pearce I. At what cost is collagenase clostridium histolyticum viable for treating Peyronie’s disease in a public healthcare system? Andrology. 2020;8(5):1304-1311. doi:10.1111/andr.12830 12. Wymer K, Hebert K, Kohler T, Trost L. MP65-05 COMPARATIVE COST-EFFECTIVENESS OF SURGERY, COLLAGENASE CLOSTRIDIUM HISTOLYTICUM, AND PENILE TRACTION THERAPY IN THE TREATMENT OF PEYRONIES DISEASE. J Urol. 2019;201(Supplement 4). doi:10.1097/01.ju.0000556917.78697.44 12. Wymer K, Hebert K, Kohler T, Trost L. MP65-05 COMPARATIVE COST-EFFECTIVENESS OF SURGERY, COLLAGENASE CLOSTRIDIUM HISTOLYTICUM, AND PENILE TRACTION THERAPY IN THE TREATMENT OF PEYRONIES DISEASE. J Urol. 2019;201(Supplement 4). doi:10.1097/01.ju.0000556917.78697.44 13. Gamidov S, Shatylko T, Gasanov N, Scherbakov D, Li K, Sukhikh G. Long-term outcomes of surgery for Peyronie’s disease: focus on patient satisfaction. Int J Impot Res. 2021;33(3):332-338. doi:10.1038/s41443-020-0297-6 13. Gamidov S, Shatylko T, Gasanov N, Scherbakov D, Li K, Sukhikh G. Long-term outcomes of surgery for Peyronie’s disease: focus on patient satisfaction. Int J Impot Res. 2021;33(3):332-338. doi:10.1038/s41443-020-0297-6 14. Alom M, Sharma KL, Toussi A, Kohler T, Trost L. Efficacy of Combined Collagenase Clostridium histolyticum and RestoreX Penile Traction Therapy in Men with Peyronie’s Disease. J Sex Med. 2019;16(6):891-900. doi:10.1016/j.jsxm.2019.03.007 14. Alom M, Sharma KL, Toussi A, Kohler T, Trost L. Efficacy of Combined Collagenase Clostridium histolyticum and RestoreX Penile Traction Therapy in Men with Peyronie’s Disease. References J Sex Med. 2019;16(6):891-900. doi:10.1016/j.jsxm.2019.03.007 15. Flores S, Choi J, Alex B, Mulhall JP. Erectile dysfunction after plaque incision and grafting: Short-term assessment of incidence and predictors. J Sex Med. 2011;8(7):2031-2037. doi:10.1111/j.1743-6109.2011.02299.x 15. Flores S, Choi J, Alex B, Mulhall JP. Erectile dysfunction after plaque incision and grafting: Short-term assessment of incidence and predictors. J Sex Med. 2011;8(7):2031-2037. doi:10.1111/j.1743-6109.2011.02299.x 16. Chung E, Clendinning E, Lessard L, Brock G. Five-Year Follow-Up of Peyronie’s Graft Surgery: Outcomes and Patient Satisfaction. J Sex Med. 2011;8(2):594-600. doi:10.1111/j.1743-6109.2010.02102.x 17. Punjani N, Nascimento B, Salter C, et al. Predictors of Depression in Men With Peyronie’s Disease Seeking Evaluation. J Sex Med. 2021;18(4):783-788. doi:10.1016/j.jsxm.2021.02.002 18. Nelson CJ, Mulhall JP. Psychological Impact of Peyronie’s Disease: A Review. J Sex Med. 2013;10(3):653-660. doi:10.1111/j.1743- 6109.2012.02999.x 19. Shirazi M, Haghpanah AR, Badiee M, Afrasiabi MA, Haghpanah S. Effect of intralesional verapamil for treatment of Peyronie’s disease: A randomized single-blind, placebo-controlled study. Int Urol Nephrol. 2009;41(3):467-471. doi:10.1007/s11255-009-9522-4 20. Russo GI, Milenkovic U, Hellstrom W, Levine LA, Ralph D, Albersen M. Clinical Efficacy of Injection and Mechanical Therapy for Peyronie’s Disease: A Systematic Review of the Literature[Figure presented]. Eur Urol. 2018;74(6):767-781. doi:10.1016/j.eururo.2018.07.005 Page 13/13 21. Stuntz M, Perlaky A, Des Vignes F, Kyriakides T, Glass D. The prevalence of Peyronie’s disease in the United States: A population- based study. PLoS One. 2016;11(2). doi:10.1371/journal.pone.0150157 Page 13/13
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Detection of attractors of large Boolean networks via exhaustive enumeration of appropriate subspaces of the state space
BMC bioinformatics
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10,098
* Correspondence: nikolaos.berntenis@roche.com Non-Clinical-Safety, F. Hoffmann – La Roche AG, Grenzacherstrasse 124, 4070, Basel, Switzerland SOFTWARE Open Access Open Access © 2013 Berntenis and Ebeling; 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. Detection of attractors of large Boolean networks via exhaustive enumeration of appropriate subspaces of the state space Nikolaos Berntenis* and Martin Ebeling Nikolaos Berntenis* and Martin Ebeling Abstract It is further argued that these detectable cycles are also the biologically more important ones. Furthermore, lnet also provides standard Boolean analysis features such as node loop detection. inherent in cycle detection is a classification scheme based on the number of non-frozen nodes of the cycle member states, with cycles characterized by fewer non-frozen nodes being easier to detect. It is further argued that these detectable cycles are also the biologically more important ones. Furthermore, lnet also provides standard Boolean analysis features such as node loop detection. Conclusions: lnet is a software package that facilitates the analysis of large Boolean networks. Its intuitive approach helps to better understand the network in question. Keywords: Boolean network, Attractor, Fixed state, Cycle, Regulatory network, State space Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Abstract Background: Boolean models are increasingly used to study biological signaling networks. In a Boolean network, nodes represent biological entities such as genes, proteins or protein complexes, and edges indicate activating or inhibiting influences of one node towards another. Depending on the input of activators or inhibitors, Boolean networks categorize nodes as either active or inactive. The formalism is appealing because for many biological relationships, we lack quantitative information about binding constants or kinetic parameters and can only rely on a qualitative description of the type “A activates (or inhibits) B”. A central aim of Boolean network analysis is the determination of attractors (steady states and/or cycles). This problem is known to be computationally complex, its most important parameter being the number of network nodes. Various algorithms tackle it with considerable success. In this paper we present an algorithm, which extends the size of analyzable networks thanks to simple and intuitive arguments. Results: We present lnet, a software package which, in fully asynchronous updating mode and without any network reduction, detects the fixed states of Boolean networks with up to 150 nodes and a good part of any present cycles for networks with up to half the above number of nodes. The algorithm goes through a complete enumeration of the states of appropriately selected subspaces of the entire network state space. The size of these relevant subspaces is small compared to the full network state space, allowing the analysis of large networks. The subspaces scanned for the analyses of cycles are larger, reducing the size of accessible networks. Importantly, Results: We present lnet, a software package which, in fully asynchronous updating mode and without any network reduction, detects the fixed states of Boolean networks with up to 150 nodes and a good part of any present cycles for networks with up to half the above number of nodes. The algorithm goes through a complete enumeration of the states of appropriately selected subspaces of the entire network state space. The size of these relevant subspaces is small compared to the full network state space, allowing the analysis of large networks. The subspaces scanned for the analyses of cycles are larger, reducing the size of accessible networks. Importantly, inherent in cycle detection is a classification scheme based on the number of non-frozen nodes of the cycle member states, with cycles characterized by fewer non-frozen nodes being easier to detect. Background the activation state, or value, of any node in a network as a function of its activating and/or inhibiting inputs. In general, for a target node with k different input nodes, each of which can again be either “active” or “inactive”, the logical functions assign the resulting values of the target node for each of the 2k possible input patterns. The use of Boolean models I n the study of biological networks was proposed and worked out already in the 1970s [1-5]. A Boolean network model is characterized by the topology of a biological interaction network and a set of qualitative parameters termed “logical functions” by Thomas and D’Ari [6]. Logical functions determine Boolean networks are best suited to analyze and de- scribe steady states of systems (which are independent of kinetic parameters). As demonstrated by Thomas, non-trivial steady states are determined by the presence of negative or positive feedback loops, with the former Page 2 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 characterizing homeostatic or oscillatory processes, and the latter leading to switch-like or differentiation behaviour. the number of activation patterns grows even faster. We refer to this number as the “state complexity” of a network. For every node in a network, the logical parameters determine how it reacts to the input it receives from other nodes, more specifically, to which value (0 or 1) a node will tend under any given input pattern, also re- ferred to as the “image” of the node for this input pat- tern. In general, for a node with k inputs (activating or inhibiting), there are 2k possible input patterns and cor- responding logical parameters. We refer to this as “par- ameter complexity” of a network. For the choice of logical parameters, there are some obvious constraints, e.g., adding an activating input to an already active node should not lead to its inactivation. For simplicity rea- sons, Boolean network analyses often adopt the conven- tion that a node under the influence of at least one inhibitor always tends towards being inactive irrespective of the presence of any activators; in the absence of any inhibiting influences, a single activator will be sufficient to activate it. This effectively eliminates the parameter complexity of the problem. Background In the case of multi-valued Boolean networks, where some of the nodes can have more than 2 values, TF Rep CA1 CA2 T1 Ex1 T2 Ex2 0 0 0 0 K R,CA2 0 KR 0 0 0 0 1 K R 0 KR,CA2 0 0 0 1 0 K R,CA1,CA2 0 KR,CA1 0 0 0 1 1 K R,CA1 0 KR,CA1,CA2 0 0 1 0 0 KCA2 0 K = 0 0 0 1 0 1 K = 0 0 KCA2 0 0 1 1 0 KCA1,CA2 0 KCA1 0 0 1 1 1 KCA1 0 KCA1,CA2 0 1 0 0 0 K TF,R,CA2 0 KTF,R 0 1 0 0 1 K TF,R 0 KTF,R,CA2 1 1 0 1 0 K TF,R,CA1,CA2 = 1 1 KTF,R,CA1 0 1 0 1 1 K TF,R,CA1 0 KTF,R,CA1,CA2 = 1 1 1 1 0 0 K TF,CA2 0 KTF 0 1 1 0 1 K TF 0 KTF,CA2 0 1 1 1 0 K TF,CA1,CA2 0 KTF,CA1 0 1 1 1 1 K TF,CA1 0 KTF,CA1,CA2 0 A C B Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. B: In the absence of detailed knowledge of the underlying interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for the system as depicted in A. Background Here, we deviate from this convention, motivated by biological examples where, for example, both a transcription factor and a co-activator are required for a certain function, or where co- activators and co-repressors compete for a target tran- scription factor (Figure 1). The only assumption we The analysis of Boolean networks comprised of more than a few nodes is feasible thanks to tools developed by various groups. Garg et al. [7] introduced the concept of binary decision diagrams and developed SQUAD [8]. GINsim, a tool implementing Thomas’ program was pro- vided by Gonzalez et al. [9]. Himkelman et al. [10] used algebraic methods to develop ADAM, Helikar et al. [11] developed the simulation platform ChemicalChains and Müssel et al. developed BoolNet [12]. Algorithms were also developed that simplify network architectures without affecting the steady state proper- ties. They eliminate iteratively single nodes that: do not regulate their own function [13,14] or: have one incom- ing and one outgoing edge (simple mediator nodes) or have the same value in all attractors [15,16]. Both ap- proaches preserve the fixed point structure of the net- work. The latter preserves also the cycle attractors, while the former may, in certain cases, introduce spuri- ous ones. For a binary Boolean network with n nodes, there are 2n possible activation patterns that form the state space: every state is represented by an n-dimensional binary vector. Background TF Rep CA1 CA2 T1 Ex1 T2 Ex2 0 0 0 0 K R,CA2 0 KR 0 0 0 0 1 K R 0 KR,CA2 0 0 0 1 0 K R,CA1,CA2 0 KR,CA1 0 0 0 1 1 K R,CA1 0 KR,CA1,CA2 0 0 1 0 0 KCA2 0 K = 0 0 0 1 0 1 K = 0 0 KCA2 0 0 1 1 0 KCA1,CA2 0 KCA1 0 0 1 1 1 KCA1 0 KCA1,CA2 0 1 0 0 0 K TF,R,CA2 0 KTF,R 0 1 0 0 1 K TF,R 0 KTF,R,CA2 1 1 0 1 0 K TF,R,CA1,CA2 = 1 1 KTF,R,CA1 0 1 0 1 1 K TF,R,CA1 0 KTF,R,CA1,CA2 = 1 1 1 1 0 0 K TF,CA2 0 KTF 0 1 1 0 1 K TF 0 KTF,CA2 0 1 1 1 0 K TF,CA1,CA2 0 KTF,CA1 0 1 1 1 1 K TF,CA1 0 KTF,CA1,CA2 0 A C B Fi 1 S l i d l i A S h i i f h h i l l bi l i l k A A B TF Rep CA1 CA2 T1 Ex1 T2 Ex2 0 0 0 0 K R,CA2 0 KR 0 0 0 0 1 K R 0 KR,CA2 0 0 0 1 0 K R,CA1,CA2 0 KR,CA1 0 0 0 1 1 K R,CA1 0 KR,CA1,CA2 0 0 1 0 0 KCA2 0 K = 0 0 0 1 0 1 K = 0 0 KCA2 0 0 1 1 0 KCA1,CA2 0 KCA1 0 0 1 1 1 KCA1 0 KCA1,CA2 0 1 0 0 0 K TF,R,CA2 0 KTF,R 0 1 0 0 1 K TF,R 0 KTF,R,CA2 1 1 0 1 0 K TF,R,CA1,CA2 = 1 1 KTF,R,CA1 0 1 0 1 1 K TF,R,CA1 0 KTF,R,CA1,CA2 = 1 1 1 1 0 0 K TF,CA2 0 KTF 0 1 1 0 1 K TF 0 KTF,CA2 0 1 1 1 0 K TF,CA1,CA2 0 KTF,CA1 0 1 1 1 1 K TF,CA1 0 KTF,CA1,CA2 0 C A C B Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. Background TF Rep CA1 CA2 T1 Ex1 T2 Ex2 0 0 0 0 K R,CA2 0 KR 0 0 0 0 1 K R 0 KR,CA2 0 0 0 1 0 K R,CA1,CA2 0 KR,CA1 0 0 0 1 1 K R,CA1 0 KR,CA1,CA2 0 0 1 0 0 KCA2 0 K = 0 0 0 1 0 1 K = 0 0 KCA2 0 0 1 1 0 KCA1,CA2 0 KCA1 0 0 1 1 1 KCA1 0 KCA1,CA2 0 1 0 0 0 K TF,R,CA2 0 KTF,R 0 1 0 0 1 K TF,R 0 KTF,R,CA2 1 1 0 1 0 K TF,R,CA1,CA2 = 1 1 KTF,R,CA1 0 1 0 1 1 K TF,R,CA1 0 KTF,R,CA1,CA2 = 1 1 1 1 0 0 K TF,CA2 0 KTF 0 1 1 0 1 K TF 0 KTF,CA2 0 1 1 1 0 K TF,CA1,CA2 0 KTF,CA1 0 1 1 1 1 K TF,CA1 0 KTF,CA1,CA2 0 A C B Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. B: In the absence of detailed knowledge of the underlying interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for the system as depicted in A. Fixed states detection Crucial for the development of the algorithm is the realization that the discrete nature of Boolean networks allows us to restrict the search to selected subspaces of the state space in which the fixed states (if any exist) res- ide. These subspaces turn out to be orders of magnitude smaller than the actual state space rendering possible an exact enumeration. This is achieved via a two-step process (Figure 2). For any given network and network state, it is not ob- vious if all nodes satisfy the logical functions, i.e., have values in agreement with their inputs; in fact, it is not even obvious if such a state exists. Node values can be updated by switching them in agreement with the input pattern. Whenever stated that in a given state a node satisfies the equations, it is meant that the node will re- tain its value should we choose to update it (i.e., the value is identical to the image for the current input pat- tern). For simplicity, we refer to such a node as a “con- tent” node. A fixed state of the network is, by definition, a state in which all the node equations are satisfied, i.e. all the nodes are content. In contrast, a node whose current value is in disagreement with its current input, and which will be modified when chosen for an update, is called a “discontent” node. Assume a Boolean network of n nodes. For k of them (typically, k around 10), generate the 2k × k matrix representing all their 2k possible states. Then for each one of the remaining n-k nodes go through the following iterative process: Extend the matrix by first duplicating it and then add- ing to it an extra column with the values of an appropri- ately chosen (k + 1)st node (0 and 1 for each pair of identical rows). The outcome is a 2k+1 × (k + 1) matrix. The choice of the new node is crucial: All its incoming nodes must be present in the initial k-member group (see remark below). Then, for each row, we check whether the value of the added node satisfies its node equation. If it violates it then we remove the row, for it cannot lead to a fixed state. By doing so, all its 2n-(k+1) downstream successors are effectively removed once and for all. Background B: In the absence of detailed knowledge of the underlying interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for the system as depicted in A. Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. B: In the absence of detailed knowledge of the underlying interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for the system as depicted in A. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Page 3 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 format also used in [7]. Background In the input text file each line corresponds to either an activation A →B or to an in- hibition C −| D. make here is that under “optimal” conditions, i.e., pres- ence of all activators and absence of all inhibitors, a node must be activated, and likewise, in the presence of all inhibitors and absence of any activators, it must be inactivated. These minimum assumptions ensure that every node must in principle be able to switch between its two values. We note that state and parameter com- plexity are related and it is often possible to reduce par- ameter complexity by adding symbolic nodes to the network that represent complexes or intermediate steps along a process; however, this comes at the expense of higher state complexity. The source code is freely available from the authors. Fixed states detection The same procedure is followed for any of the k initially selected nodes, if all its incoming nodes are in the matrix. Boolean networks have often been studied using “syn- chronous” update strategies, where the values of all dis- content nodes are switched simultaneously. (Note that this switching can lead to the generation of new discon- tent nodes.) While this approach is technically conveni- ent, it does not properly reflect the characteristics of biological networks, and it may even introduce artifacts in their behavior. “Asynchronous” updates proceed with one node at a time, often selecting them in random order. This method, referred to as “general asynchron- ous” in [15], is the one we choose. Then a (k + 2)nd node is added, further extending the matrix, its equation is tested against all rows, and the process is repeated until all the nodes have been taken into account. At each iteration step, if no remaining node is found having all its incoming nodes already present in the matrix, the one with the maximal out- degree is chosen. The algorithm is simply trying to in- crease the probability that at each step at least one node having all its incoming nodes already present will be available. The choice of the first k nodes is based on this reasoning too: the ones with the highest out-degree values are selected. In the following, we are referring to random asyn- chronous updating and to binary Boolean networks (although the algorithm is also valid for multiple discrete- valued ones). Furthermore, all results were generated from testing on networks having: (i) values of the ratio of edges to nodes between 2 to 3 and (ii) all their nodes with non-zero in- and out-degrees and with at least one of them larger than one (i.e., without simple mediator nodes). The successive removal of equation-violating states (i.e., states containing at least one discontent node) leads to a dramatic decrease of the number of states exam- ined. It can simplify the problem by many orders of magnitude, depending on network size and complexity. In a typical network the number of examined states reaches its maximum value at about or just after the time half of the nodes have been included in the matrix. Implementation We start with the full listing of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix. For this, we also specify the images 1′, 2′, 3′ to which the three nodes tend for any input combination, using logical parameters. In this simple example, only node 3 receives multiple inputs and thus has non-trivial logical functions. Removing the states which cannot match their image state for any valid choice of the logical functions, we obtain two candidates for fixed states. Comparing the states to their image states, we realize that state (1, 1, 0) is a fixed state if 31 equals 0 (i.e., node 3 is not activated by the presence of its activator 1 alone), whereas state (0, 0, 1) is fixed if 32 equals 1 (i.e., node 3 is already activated by the absence of its inhibitor 2 alone). Setting both 31 = 0 and 32 = 1 renders both states fixed. Experimental data in agreement with state (1, 1, 0) but not with (0, 0, 1) thus help in estimating the logical functions. cycles, i.e., sets of states out of which the system cannot escape once it reaches them. Once a system enters a basin of attraction then the equations drive it deeper into it, gradually increasing the number of content nodes. However, for any basin of attraction, it is not triv- ial to find out if it can be reached from any given current state. Then it decreases rapidly as many of the node equations are taken into account during the evaluation. When the full set of nodes has been processed, only the fixed states remain in the final matrix. We note that the approach described here simultan- eously copes with both state and parameter complexity. When testing if the actual value of a node is compatible with the node equations, we accept any states for which this condition holds for at least one choice of the logical parameters. Thus, the algorithm does not only yield fixed states for a concrete combination of logical param- eters, but for all possible ones. The conditions on the lo- gical parameters needed to realize a fixed point can then be read from the final matrix (Figure 2B). This aspect is of key importance for reverse-engineering problems. Implementation We consider a state to be the more unstable/disordered the higher the number of its discontent nodes, and we refer to the state as being 1-, 2- up to n-discontent. Intuitively, the system equations tend to drive the sys- tem towards the more stable/ordered regions of the state space. We note that most of the interesting stable cycles reside in sufficiently ordered space state regions, where a good part of the node values of their members satisfy the equations. Stable cycles including states with a large number of discontent nodes are less relevant which is apparent from the following consi- derations (compare Figure 3). The generalization to multiple-valued case is straight- forward: At each iterative step, instead of just duplicat- ing the rows, the algorithm adds m-1 replicates of the previous matrix when adding a node with m levels, with the corresponding level (from 1 to m-1) appended. Then it proceeds as previously described, namely by removing all equation-violating states. If a state of a system with n nodes is k-discontent, it has k successor states, because each one of the k discon- tent nodes could be updated, and the equations do not change the values of content nodes. A cycle is stable when all its member states flow within the cycle. A single cycle member having a succes- sor that is not part of the cycle renders the cycle meta- stable (Figure 3A). Implementation The software application has been written in ANSI C and it is single-threaded. The only hardware require- ments concern available random access memory. The network topology can be loaded using the straightforward Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 Page 4 of 10 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Page 4 of 10 A B Figure 2 For this simplified account of the algorithm described in the text, we analyze a 3-node network (A). We start with the full listing of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix (B). We start with the full listing of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix. For this, we also specify the images 1′, 2′, 3′ to which the three nodes tend for any input combination, using logical parameters. In this simple example, only node 3 receives multiple inputs and thus has non-trivial logical functions. Removing the states which cannot match their image state for any valid choice of the logical functions, we obtain two candidates for fixed states. Comparing the states to their image states, we realize that state (1, 1, 0) is a fixed state if 31 equals 0 (i.e., node 3 is not activated by the presence of its activator 1 alone), whereas state (0, 0, 1) is fixed if 32 equals 1 (i.e., node 3 is already activated by the absence of its inhibitor 2 alone). Setting both 31 = 0 and 32 = 1 renders both states fixed. Experimental data in agreement with state (1, 1, 0) but not with (0, 0, 1) thus help in estimating the logical functions. A B A B A A B Figure 2 For this simplified account of the algorithm described in the text, we analyze a 3-node network (A). We start with the full listing of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix (B). Cycle detection Preliminaries The system equations direct each state to flow to neigh- boring states and (in asynchronous mode) neighboring states differ by the value of at most a single node. The changing node is set to a value that satisfies the system equations, which increases the number of content nodes by one; however, this change will typically have effects on other nodes, and other discontent nodes can result. If the state space contains basins of attraction then the sys- tem will eventually flow towards them. Basins of attrac- tion signal the existence of fixed states or of stable A stable cycle containing a state with even a moderate number of discontent nodes is necessarily a long cycle (state “c” in Figure 3B is 3-discontent); this state has multiple successors and all of them must be members of the cycle as well as all their successors, etc. On the other hand, cycles whose members have very few discontent nodes are often short. We refer to a k-cycle to indicate that its most discontent member state(s) is (are) Page 5 of 10 Page 5 of 10 Page 5 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 a b c d e g f a b c e f h g d A B Figure 3 Analysis of simple and complex cycles. Filled circles represent states of the system, not to individual nodes. In asynchronous update mode, two neighboring states differ by the value of exactly one node. Thus, the shortest possible cycle (0, 0) → (0, 1) →(1, 1) →(1, 0) →(0, 0) →…, composed of two changing nodes, has length 4 in state space. A: The cycle “abcd” is fixed only in the absence of the dashed transition a →g. In the presence of this transition, the cycle leaks into state g, and the system has only a single fixed point, g. B: In the cycle “abcd”, here state c is 3-discontent and, hence, can transition to three different successor states d, e, and f. For the cycle to be fixed, as shown here, all the successor states of d, e, and f have to return to the cycle. As a consequence, complex fixed cycles are expected to be rare in biologically motivated networks. line with the “standard” graph-theoretical approaches, first finds all parent–child state relationships and then searches for stable cycles. Cycle detection Preliminaries A 1-cycle, in which each state mem- ber has a single successor, is also referred to as a simple cycle (Figure 3A, in the absence of the a →g transition). Otherwise it is a mixed cycle and in such cycles “branch- ing” is present (Figure 3B). Simple cycles are necessarily stable. A typical example is: a 1-cycle that consists of 4 states distinguishable by the 4 combinations (0–0, 0–1, 1–0, and 1–1) of the values of two nodes. The remaining k-2 nodes in the network are all content and shared by all the 4 cycle states. k-discontent states. A 1-cycle, in which each state mem- ber has a single successor, is also referred to as a simple cycle (Figure 3A, in the absence of the a →g transition). Otherwise it is a mixed cycle and in such cycles “branch- ing” is present (Figure 3B). Simple cycles are necessarily stable. A typical example is: a 1-cycle that consists of 4 states distinguishable by the 4 combinations (0–0, 0–1, 1–0, and 1–1) of the values of two nodes. The remaining k-2 nodes in the network are all content and shared by all the 4 cycle states. (ii) Search for cycles within the selected set of states: a. First, identify the basin of attraction of each fixed state. This is done by “walking uphill” the collected information on successor/predecessor relationships, labeling and counting along the way all states belonging to the current basin. Any remaining, non-labeled states belong exclusively to basins of cyclic attractors. In subsequent steps, the search is restricted to these states (apart from the basin size calculations). Absence of non- labeled states clearly implies the absence of cycles in the selected state set; the search is effectively terminated at this point. We note that, in principle, there can be cycles that are long despite the fact that all their states have few discon- tent nodes. b. Starting from state(s) with the lowest k-value, look for directed cycles using again the successor/ predecessor data structures, by creating all di- rected walks in the graph of selected states. If any such cycle is found then it is checked whether any of its member states belong to any of the cur- rently detected basins. If this is the case then the cycle is discarded as unstable. Cycle detection Preliminaries Otherwise, check whether the cycle is “closed”, i.e., whether the children of all its members are also members of the cycle. If this is not the case, the cycle either has members (or successors) belonging to a basin that was not yet found or it is part of a larger mixed cycle. At the current step, such a cycle is simply registered; it will be dealt with at a later stage. If the cycle is found to be closed, then it is stable and constitutes an attractor. All its basin b. Starting from state(s) with the lowest k-value, look for directed cycles using again the successor/ predecessor data structures, by creating all di- rected walks in the graph of selected states. If any such cycle is found then it is checked whether any of its member states belong to any of the cur- rently detected basins. If this is the case then the cycle is discarded as unstable. Otherwise, check whether the cycle is “closed”, i.e., whether the children of all its members are also members of the cycle. If this is not the case, the cycle either has members (or successors) belonging to a basin that was not yet found or it is part of a larger mixed cycle. At the current step, such a cycle is simply registered; it will be dealt with at a later stage. If the cycle is found to be closed, then it is stable and constitutes an attractor. All its basin Cycle detection Preliminaries If and when one is found, it “creates” its basin of attraction via a bottom-up approach. a b c d e g f a b c e f h g d A B Figure 3 Analysis of simple and complex cycles Filled circles a b c d e g f A A B The algorithm goes through two main stages: (i) Determine the appropriate subset of the state space. (i) Determine the appropriate subset of the state space. a. As in the fixed state case, go through iterations adding a node at a time and keeping at each step all states up to and including k-discontent states. Each state is labeled by its number of discontent nodes (in {0, 1, …, k}). The end result is the set of all these states. The current implementation can readily handle sets with up to tens of millions states. Note that all network fixed states are in- cluded and labeled by zero. Figure 3 Analysis of simple and complex cycles. Filled circles Figure 3 Analysis of simple and complex cycles. Filled circles represent states of the system, not to individual nodes. In asynchronous update mode, two neighboring states differ by the value of exactly one node. Thus, the shortest possible cycle (0, 0) → (0, 1) →(1, 1) →(1, 0) →(0, 0) →…, composed of two changing nodes, has length 4 in state space. A: The cycle “abcd” is fixed only in the absence of the dashed transition a →g. In the presence of this transition, the cycle leaks into state g, and the system has only a single fixed point, g. B: In the cycle “abcd”, here state c is 3-discontent and, hence, can transition to three different successor states d, e, and f. For the cycle to be fixed, as shown here, all the successor states of d, e, and f have to return to the cycle. As a consequence, complex fixed cycles are expected to be rare in biologically motivated networks. b. The predecessors and successors of each selected state are recorded in appropriate data structures. All states that do not have any of their successors in the selected set of states (i.e., they flow “outside”) cannot be assessed and are from now on excluded. k-discontent states. Benchmarking F b h For benchmarking purposes we generated programmat- ically a number of sufficiently complex Boolean net- works that have features similar to known, biologically relevant examples. As mentioned above, nodes with a single incoming and a single outgoing edge were ex- cluded. The ratio of edges to nodes was around 2.5 for most of the networks (when not then it is stated so) and no more than 6 incoming edges per node were allowed. We chose to compare lnet to GenYsis and GINsim be- cause these two packages support both true asynchron- ous updating and guarantee detection of all cycles (for The tests were performed on a Linux computer with a Xeon 2.4 GHz processor and 96 GB of physical memory. The GINsim version was 2.4 and it ran on Java 1.5. 40 50 60 70 80 90 100 110 0 5 10 15 20 25 Network nodes (Log2( all states )) Log2( Enumerated states ) Figure 6 Enumerated states versus the total number of states. This log2-log2 plot clearly demonstrates the significant reduction in the number of analyzed states computed to all possible network states. 40 50 60 70 80 90 100 110 0 5 10 15 20 25 Network nodes (Log2( all states )) Log2( Enumerated states ) ous updating and guarantee detection of all cycles (for Discontent nodes 0 1 8 120 80 30 0 Network nodes Grey: lnet + GINsim + GenYsis Green: lnet+ GINsim Burgundy: lnet + GenYsis Blue: lnet Red: GenYsis Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any of the three tools GenYsis, GINsim and lnet. For the region in blue, attractors having at least one discontent node and more than 30 nodes overall, only lnet are applicable. 40 50 60 70 80 90 100 110 0 5 10 15 20 25 Network nodes (Log2( all states )) Log2( Enumerated states ) Figure 6 Enumerated states versus the total number of states. This log2-log2 plot clearly demonstrates the significant reduction in the number of analyzed states computed to all possible network states. Discontent nodes 0 1 8 120 80 30 0 Network nodes Grey: lnet + GINsim + GenYsis Green: lnet+ GINsim Burgundy: lnet + GenYsis Blue: lnet Red: GenYsis Figure 4 Schematic comparison of the three algorithms. Stable cycle finding An iteration process is called which, starting from an appropriately chosen “seed” cycle, keeps enlarging it by adding any new states from cycles with states flowing into it. The iterations continue until this enlarged cycle becomes “closed”. As previously, the corresponding basin is determined and the process continues for any remaining non- labeled states. The time needed for the exact determination of all successor/predecessor relationships and for the enumer- ation of the members of a basin grows linearly with the number of selected states. This fact is instrumental in rendering the algorithm fast. Another crucial element is that the size of the search space is reduced each time an attractor basin is determined. Figure 5 Dependence of the duration of fixed state searches on the number of network nodes. Explanation in the main text. networks up to certain size). lnet and GenYsis share a common input format; for GINsim a compatible version was generated for all relevant runs. Only the lnet test runs, which involved hundreds of networks, were automated The GenYsis runs, as seen below, were limited to smaller-size networks while the GINsim ones were difficult to automate. Therefore, we ran just a few of them that still generate sufficiently rep- resentative results. Stable cycle finding The fixed point search described above is performed in states that satisfy all the system equations. It cannot be used to detect a cycle since each member state of a cycle has to be at least 1-discontent. It follows naturally that relaxing the above restriction can be used for cycle find- ing. Namely, a search in the enlarged subspace that in- cludes all the states up to and including k-discontent states will result in the detection of all j-cycles, where j ≤k). Unavoidably, the enumeration of more states re- sults in a reduction of the size of resolvable networks. The value of k can vary from k = 1 (1-cycle search) to n (search for all cycles). The number of selected states grows very fast as k increases. Cycle finding is feasible only when the number of selected states is manageable because of memory restrictions. In addition, execution running times increase fast because the algorithm, in Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Page 6 of 10 30 40 50 60 70 80 90 100 110 120 −1 0 1 2 3 Network nodes log10( Time in seconds ) Figure 5 Dependence of the duration of fixed state searches on the number of network nodes. Explanation in the main text. states are counted and any non-previously labeled ones are now labeled. (Note that a state can be- long to multiple attractors.) This step is repeated until all registered states have been considered. c. Detected cycles, whose members do not flow towards identified basins, can still remain. Each one of them may be part of a larger mixed cycle. An iteration process is called which, starting from an appropriately chosen “seed” cycle, keeps enlarging it by adding any new states from cycles with states flowing into it. The iterations continue until this enlarged cycle becomes “closed”. As previously, the corresponding basin is determined and the process continues for any remaining non- labeled states. states are counted and any non-previously labeled ones are now labeled. (Note that a state can be- long to multiple attractors.) This step is repeated until all registered states have been considered. until all registered states have been considered. c. Detected cycles, whose members do not flow towards identified basins, can still remain. Each one of them may be part of a larger mixed cycle. Benchmarking F b h Colored regions indicate attractors that can be analyzed using any of the three tools GenYsis, GINsim and lnet. For the region in blue, attractors having at least one discontent node and more than 30 nodes overall, only lnet are applicable. Discontent nodes 0 1 8 120 80 30 0 Network nodes Grey: lnet + GINsim + GenYsis Green: lnet+ GINsim Burgundy: lnet + GenYsis Blue: lnet Red: GenYsis Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any of the three tools GenYsis, GINsim and lnet. For the region in blue, attractors having at least one discontent node and more than 30 nodes overall, only lnet are applicable. Discontent nodes 0 1 8 120 80 30 0 Network nodes Grey: lnet + GINsim + GenYsis Green: lnet+ GINsim Burgundy: lnet + GenYsis Blue: lnet Red: GenYsis Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any of the three tools GenYsis, GINsim and lnet. For the region in blue, attractors having at least one discontent node and more than 30 nodes overall, only lnet are applicable. Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any of the three tools GenYsis, GINsim and lnet. For the region in blue, attractors having at least one discontent node and more than 30 nodes overall, only lnet are applicable. Network nodes (Log2( all states )) Figure 6 Enumerated states versus the total number of states. Figure 6 Enumerated states versus the total number of states. This log2-log2 plot clearly demonstrates the significant reduction in the number of analyzed states computed to all possible network states. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Page 7 of 10 Page 7 of 10 0 1 2 3 4 5 6 7 8 −1 0 1 2 3 4 30−node net log10( Time in seconds) 0 1 2 3 4 −1 0 1 2 3 4 40−node net Discontent nodes 0 1 2 3 −1 0 1 2 50−node net Figure 7 The number of accessible discontent nodes decreases as the number of nodes grows. Computation time for analyses of networks of networks of different size, depending on the maximum allowed number of discontent nodes. Benchmarking F b h For larger networks, there is a gradual reduction of the maximum number of discontent nodes the software can handle. This is also mirrored in an increased use of memory (not shown). For example, for 40-node networks cycles with up to 4 discontent nodes were accessible. Such cycles contain tens or hundreds of states. Discontent nodes Figure 7 The number of accessible discontent nodes decreases as the number of nodes grows. Computation time for analyses of networks of networks of different size, depending on the maximum allowed number of discontent nodes. For larger networks, there is a gradual reduction of the maximum number of discontent nodes the software can handle. This is also mirrored in an increased use of memory (not shown). For example, for 40-node networks cycles with up to 4 discontent nodes were accessible. Such cycles contain tens or hundreds of states. GenYsis was found to be superior in detecting all fixed points and all cycles for networks up to approximately 30 nodes. The execution time for the larger ones can reach 1 hour and the memory requirements are minimal. For even larger networks the execution times were higher than our testing time (1 hour) and memory utilization was becom- ing important. These results are in overall agreement with those of Ferhat et al. [17]. It should be emphasized that GenYsis by design detects all existing cycles, also covering cycles with thousands of member states. lnet detects fixed states for networks up to 150 nodes, detects all cycles for networks up to 20 or so nodes and detects a subset of the cycles for networks with up to 70 nodes. The limiting factor is available computer memory because the size of the space state regions enumerated by lnet grows, inevitably, too large. For cycle detection, up to 20-node networks, lnet’s performance is equivalent to that of GenYsis. From 20 to 30 nodes it is still the same provided, however, that the lnet search is restricted to k-discontent states with low k (the actual value of k depends on the network size). Otherwise, it can be significantly slower, for similar rea- sons as GINsim. The slowdown can be important in the absence of attractors with very low k (k = 0 or 1) because these are always detected first and have their basin states immediately removed from any downstream consider- ation. Benchmarking F b h Elucidating further the different performances of the two algorithms, a 30-node network analysis may take several hours in GenYsis irrespective of the numbers of fixed states or cycles (no matter how large). On the other hand, while lnet may fail to detect a very large cycle, it will find fixed states and small cycles in sub- second time. GINsim detects fixed states for networks up to ap- proximately 120 nodes fast and in a memory-efficient manner as previously reported by Hinkelman [18]. Being multithreaded, GINsim took also advantage of our multi- core testing environment. The execution time for larger networks was, however, higher than our testing time and sometimes seemed not to terminate at all. Its perform- ance when searching for stable cycles was rather poor. It needs already few minutes to analyze 20-node networks and fails for even moderately larger networks. The rea- son is that it attempts exhaustive enumeration of all net- work states. Here it should be noted that GINsim can handle multiple-valued networks too. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Page 8 of 10 2 4 6 8 10 2 3 4 5 6 7 8 Discontent nodes (30−node network) log10(States) Figure 8 Even for cycle searches the numbers of enumerated states remain low. For a 30-node network with approximately 109.03 states, the number of states with no more than 10 discontent nodes still remains a small fraction of the state space. 2 4 6 8 10 2 3 4 5 6 7 8 Di d (30 d k) log10(States) A schematic representation of the comparison of the three algorithms is shown in Figure 4. The remaining figures focus exclusively on lnet results. The first series of tests targeted the fixed states of net- works having nodes ranging from 30 up to 150. For each one node number analyzed, a set of 100 networks was generated, subject to the rules mentioned earlier. Then each one of these networks was analyzed by lnet either searching exclusively for fixed states or for stable cycles as well. The computer execution times were recorded and the results were used to generate the plots shown in Figures 5, 6, 7, 8 9 and 10. Benchmarking F b h In Figure 5 a semi-log plot of the number of nodes versus the computer execution time in seconds is shown. Data are not shown for the 130, 140 and 150 cases be- cause insufficient memory caused termination for 20%, 68% and 68% of them respectively. (For those networks that were successfully analyzed the time duration trends were in agreement with the trend shown in the plot.) Discontent nodes (30−node network) Figure 8 Even for cycle searches the numbers of enumerated states remain low. For a 30-node network with approximately 109.03 states, the number of states with no more than 10 discontent nodes still remains a small fraction of the state space. Next, as has been already explained, the lnet algorithm relies on a drastic reduction of the number of enume- rated states. This becomes evident in Figure 6, where the enumerated states are plotted versus the total number of system space states (for fixed state searches only). Above 30 or so nodes lnet is alone in detecting cycles. For increasing numbers of nodes, we have to decrease the allowed number of discontent nodes for any enu- merated state because of limited computer memory. This results in fewer detectable cycles. Recall, however, that k-cycles with low k values are likely to be the most relevant ones in Boolean networks representing bio- logical systems. In Figure 7, the focus is on cycle searches. The 3 plots show computation times for cycle searches in networks of 30, 40 and 50 nodes, respectively. Data from higher number of nodes (up to 70) are not shown but follow similar trends. 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 Discontent nodes log10(millisecs) Network nodes Figure 9 Overall lnet performance. lnet execution time dependence on network size and number of discontent nodes. Figure 9 Overall lnet performance. lnet execution time dependence on network size and number of discontent nodes. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Page 9 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 100 120 140 160 180 200 220 240 260 280 300 320 −1.5 −1.0 −0.5 0.0 0.5 Network edges (50−node networks) log10( Time in seconds ) Figure 10 Effect of the number of edges on fixed state searches in 50-node networks. Availability and requirements Figure 8, also concerning cycle searches, shows counts of system states having equal or less numbers of discon- tent nodes, for increasing numbers of discontent nodes. For a single 30-node network, states with up to 10 dis- content nodes still cover less than 6% of the entire state space. Enumerating this fraction is sufficient to detect existing cycles with up to a few thousand member states. The lnet executable, a file with instructions on how to use it and examples of input files are provided in Additional file 1. Operating system(s): The source code can be com- piled on Windows, Linux and Apple computers. Programming language: ANSI C Figure 9 summarizes all the lnet results concerning both fixed states and cycles. Restrictions of use by non-academic users: None Finally, Figure 10 shows the effect of increasing the number of edges for networks with a fixed number of nodes (50 nodes were chosen). Benchmarking F b h As the number of edges increases the median lnet execution time increases only slightly but more network instances take substantially longer to analyze (outlying points). available memory. With growing network size, even fo- cusing on small sub-spaces of the entire network state space becomes prohibitively complex at some point. Programming-inspired optimizations will only lead to marginal improvements because the state space of a Bool- ean network doubles in size each time a single node is added. A promising alternative is to apply first one of the existing network reduction methodologies and then sub- mit the resulting network to lnet. Such a scenario should be able to handle networks having (prior to reduction) up to few hundred nodes. During the preparation of the manuscript we became aware of a publication by SQ Zhang et al. [19], where a fixed state detection algorithm, similar to the one pre- sented here, is described. The authors also provide a conceptual outline of a cycle-finding version of their algo- rithm based on a simple periodicity check which, however, could detect reliably only simple cycles. We did not test this algorithm because no sufficient information is avail- able. Finally, there is no discussion of the role of the num- ber of discontent nodes in cycle detection. Network edges (50−node networks) Figure 10 Effect of the number of edges on fixed state searches in 50-node networks. As the number of edges increases the median lnet execution time increases only slightly but more network instances take substantially longer to analyze (outlying points). Additional file Additional file 1: Includes the lnet executable (LINUX), a help file (text format) with detailed instructions on how to run lnet and 4 input network files that are used to illustrate the software in the help file. Authors’ contributions NB wrote the lnet code and performed the comparison tests. NB and ME conceived the algorithm and wrote the manuscript. Both authors read and approved the final manuscript. Key characteristics of the approach are the exact enu- meration of well-defined regions of the network state spaces and the faithful reproduction of the correspond- ing state transition graphs. The result is the detection of all the attractors residing in these regions. Depending on the overall fraction of the enumerated subspaces, it may be possible to deduce information about the size of each attractor basin. Competing interests k l d Competing interests Nikolaos Berntenis and Martin Ebeling are full-time employees of F. Hoffmann–La Roche Ltd. Both authors declare that they have no competing interests. p g Nikolaos Berntenis and Martin Ebeling are full-time employees of F. Hoffmann–La Roche Ltd. h h d l h h h Acknowledgements The authors would like to thank Abhishek Garg, Julien Dorier and Ioannis Xenarios for valuable discussions and Laurent Essioux for his support. Received: 20 March 2013 Accepted: 6 December 2013 Published: 13 December 2013 Received: 20 March 2013 Accepted: 6 December 2013 Published: 13 December 2013 Conclusion We present an algorithm that, based on a build-up ap- proach, greatly reduces the search space for fixed states and fixed cycles in Boolean networks. It allows for the fast and reliable detection of all fixed states in networks of up to 150 nodes, for an edges-to-nodes ratio of up to 3. Highly ordered cycle attractors, which correspond to the biologically most relevant cycles, are detected for networks of approximately half the number of nodes. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 3. Thomas R: Boolean formalization of genetic control circuits. J Theor Biol 1973, 42:563–585. 4. Thomas R, Thieffry D, Kaufman M: Dynamical behaviour of biological regulatory networks-I. Biological role of feedback loops and practical use of the concept of the loop-characteristic state. Bull Math Biol 1995, 57:247–276. 5. Thieffry D, Thomas R: Dynamical behaviour of biological regulatory networks–II. Immunity control in bacteriophage lambda. Bull Math Biol 1995, 57:277–295. 6. Thomas R, Richard D’A: Biological Feedback. Boca Raton: CRC Press; 1990. 7. Garg A, Xenarios I, Mendoza L, DeMicheli G: Efficient methods for dyna analysis of genetic networks and in silico gene perturbation experiments. Lect Notes Comput Sci 2007, 4453:62–76. 8. Di Cara A, Garg A, De Micheli G, Xenarios I, Mendoza L: Dynamic simulation of regulatory networks using SQUAD. BMC Bioinformatics 2007, 8:462. 8. Di Cara A, Garg A, De Micheli G, Xenarios I, Mendoza L: Dynam 9. Gonzalez AG, Naldi A, Sánchez L, Thieffry D, Chaouiya C: GINsim: a software suite for the qualitative modeling, simulation and analysis of regulatory networks. Biosystems 2006, 84(2):91–100. 10. Hinkelmann F, Brandon M, Guang B, McNeill R, Blekherman G, Veliz-Cuba A, Laubenbacher R: ADAM: Analysis of Discrete Models of Biological Systems Using Computer Algebra. BMC Bioinformatics 2011, 12:295. g g 11. Helikar T, Rogers JA: ChemChains: a platform for simulation and analysis of biochemical networks aimed to laboratory scientists. BMC Syst Biol 2009, 3:58. 12. Müssel C, Hopfensitz M, Kestler HA: BoolNet an R package for generation, reconstruction and analysis of Boolean networks. Bioinformatics 2010, 26 (10):1378–1380. 13. Naldi A, Remy E, Thieffry D, Chaouiya C: Dynamically consistent reduction of logical regulatory graphs. Theor Comput Sci 2011, 412(21):2207–2218. 14. Veliz-Cuba A: Reduction of Boolean network models. J Theor Biol 2011, 289C:167–172. 15. Saadatpour A, Albert I, Albert R: Attractor analysis of asynchronous Boolean models of signal transduction networks. J Theor Biol 2010, 266:641–656. 16. Saadatpour A, Wang RS, Liao A, Liu X, Loughran TP, Albert I, Albert R: Dynamical and structural analysis of a T cell survival network identifies novel candidate therapeutic targets for large granular lymphocyte leukemia. PLoS Comput Biol 2011, 7(11):e1002267. 17. Ay F, Fei X, Kahveci T: Scalable Steady State Analysis of Boolean Biological Regulatory Networks. PLoS ONE 2009, 4(12):e7992. 17. Ay F, Fei X, Kahveci T: Scalable Steady State Analysis of Boolean Biological Regulatory Networks. PLoS ONE 2009, 4(12):e7992. 18. References References 1. Kauffman S: Metabolic stability and epigenesis in randomly constructed genetic nets. J Theor Biol 1969, 22:437–467. 2. Glass L, Kauffman SA: The logical analysis of continuous non-linear biochemical control networks. J Theor Biol 1973, 103:103–129. 1. Kauffman S: Metabolic stability and epigenesis in randomly constructed genetic nets. J Theor Biol 1969, 22:437–467. Execution times of lnet are comparable to or faster than other approaches. Its limitation is clearly the 2. Glass L, Kauffman SA: The logical analysis of continuous non-linear biochemical control networks. J Theor Biol 1973, 103:103–129. Page 10 of 10 Page 10 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361 http://www.biomedcentral.com/1471-2105/14/361 Hinkelmann F: Algebraic theory for discrete models in systems biology, PhD thesis. Virginia Polytechnic Institute, Mathematics Department; 2011. 18. Hinkelmann F: Algebraic theory for discrete models in systems biology, PhD thesis. Virginia Polytechnic Institute, Mathematics Department; 2011. 19. Shu-Qin Z, Morihiro H, Tatsuya A, Wai-Ki C, Michael KN: Algorithms for finding small attractors in Boolean networks. EURASIP Journal on Bioinformatics and Systems Biology 2007, 2007:20180. doi:10.1186/1471-2105-14-361 Cite this article as: Berntenis and Ebeling: Detection of attractors of large Boolean networks via exhaustive enumeration of appropriate subspaces of the state space. BMC Bioinformatics 2013 14:361. 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PENGARUH KEMUDAHAN DAN KUALITAS INFORMASI TERHADAP MINAT MAHASISWA DALAM PENGGUNAAN LAYANAN M-BANKING PADA BANK SYARIAH
Jurnal Al-Fatih Global Mulia
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57 Jurnal Al-Fatih Global Mulia PENGARUH KEMUDAHAN DAN KUALITAS INFORMASI TERHADAP MINAT MAHASISWA DALAM PENGGUNAAN LAYANAN M-BANKING PADA BANK SYARIAH Nurajizah1, Rika Mustika Sari2 Sekolah Tinggi Ekonomi dan Bisnis Islam (STEBI) Global Mulia Cikarang1,2 ajizahstebi@gmail.com1, rikamustika54@gmail.com2 Volume 5 Issue 1 (2023), Pages 57-70 Jurnal Al-Fatih Global Mulia pISSN 2580-8036, eISSN 2746-7058 https://jurnalglobalmulia.or.id/index.php/alfatih Volume 5 Issue 1 (2023), Pages 57-70 Jurnal Al-Fatih Global Mulia pISSN 2580-8036, eISSN 2746-7058 https://jurnalglobalmulia.or.id/index.php/alfatih 1 https://www.bankbsi.co.id/news-update/berita/laba-naik-3318-bsi-siap-berlari-raih-pertumbuhan-berkelanjutan. Diakses tanggal 25 agustus 2022 Abstrak Perkembangan teknologi pada era globalisasi yang berkembang pesat ini dapat menarik minat para nasabah yang digunakan sebagai fasilitas pendukung pada aktivitas seluruh lapisan masyarakat. Salah satu teknologi informasi pada perbankan syariah yaitu layanan m-banking yang merupakan pengembangan dari dua bentuk inovasi bank syariah sebelumnya yaitu sms banking dan internet banking. Apabila minat telah muncul pada diri calon nasabah, maka diharapkan calon nasabah dapat memutuskan untuk menggunakan layanan m-banking. Tujuan dari penelitian ini adalah untuk mengetahui tingkat pengaruh kemudahan dan kualitas informasi terhadap minat dalam penggunaan layanan mobile banking pada bank syariah secara partial maupun simultan. Penelitian ini menggunakan metode kuantitatif, data yang digunakan adalah data primer dan sekunder. Teknik pengumpulan data menggunakan teknik sampling rumus slovin dengan 93 responden. Analisis data menggunakan analisis regresi linier berganda dan menggunakan uji validitas, uji reliabilitas, uji asumsi klasik dan uji hipotesis. Pengolahan data menggunakan program SPSS Versi 25.0 for windows. Hasil penelitian ini dapat disimpulkan bahwa secara partial variabel kemudahan berpengaruh secara signifikan terhadap minat mahasiswa dalam penggunaan layanan m-banking pada bank syariah dengan nilai t hitung 4,058 > 1,986 t tabel (df=90) dan nilai sig 0,000 < 0,05. Dan variabel kualitas informasi berpengaruh secara signifikan terhadap minat mahasiswa dalam penggunaan layanan m-banking pada bank syariah dengan nilai t hitung 4,570 > 1986 dan nilai sig 0,000 < 0,05. Selanjutnya pengaruh secara simultan dengan uji F menunjukan bahwa kemudahan dan kualitas informasi berpengaruh bersama-sama secara signifikan terhadap minat mahasiswa dalam penggunaan layanan m-banking pada bank syariah dengan nilai F hitung sebesar 122,177 > 3,10. Berdasarkan uji koefisien determinasi menunjukan bahwa pengaruh minat terhadap kemudahan dan kualitas informasi sebesar 73,1%, sisanya sebesar 26,9% dijelaskan oleh variabel diluar variabel yang diteliti. Kata Kunci: Kemudahan, Kualitas Informasi, Minat Penggunaan Kata Kunci: Kemudahan, Kualitas Informasi, Minat Penggunaan Abstract Technological developments in this rapidly growing era of globalization can attract the interest of customers who are used as supporting facilities for activities of all levels of society. One of the information technology in Islamic banking is m-banking service which is the development of the two previous forms of innovation in Islamic banking, namely SMS banking and internet banking. If interest has arisen in the prospective customer, it is hoped that the prospective customer can decide to use m-banking services. The purpose of this study was to determine the level of influence of the ease and quality of information on interest in the use of mobile banking services at Islamic banks partially or simultaneously. This study uses quantitative methods, the data used are primary and secondary data. The data collection technique used the Slovin formula sampling technique with 93 respondents. Data analysis used multiple linear regression analysis and used validity test, reliability test, classical assumption test and hypothesis test. Data processing using SPSS Version 25.0 for windows. The results of this study can be concluded that partially the convenience variable has a significant effect on student interest in using m-banking services at Islamic banks with a t-count value of 4.058 > 1.986 t table (df=90) and a sig value of 0.000 <0.05. And the information quality variable has a significant effect on student interest in using m-banking services at Islamic banks with a t- count value of 4.570 > 1986 and a sig value of 0.000 <0.05. Furthermore, the simultaneous effect with the F test shows that the ease and quality of information have a significant effect on student interest in using m-banking services at Islamic banks with a calculated F value of 122.177 > 3.10. Based on the coefficient of determination test, it shows that the influence of interest on the ease and quality of information is 73.1%, the remaining 26.9% is explained by variables outside the variables studied. Keywords: Ease, Information Quality, Interest in Use 57 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... PENDAHULUAN Perkembangan teknologi pada era globalisasi yang berkembang pesat ini dapat digunakan sebagai fasilitas pendukung yang digunakan pada aktifitas seluruh lapisan masyarakat. Saat ini teknologi tidak hanya hadir dalam kehidupan masyarakat, bahkan dapat dikatakan sebagai kebutuhan primer sebagian besar kelas sosial. Kemajuan dan perkembangan teknologi informasi yang pesat telah mempengaruhi industri perbankan, seperti halnya m- banking merupakan salah satu wujud dari perkembangan teknologi tersebut. Alat komunikasi merupakan salah satu contoh perkembangan teknologi yang saat ini digunakan dalam industri perbankan. Kehadiran Bank Syariah Indonesia (BSI) telah diresmikan sejak 01 Februari 2021 lalu. BSI melaporkan bahwa pengguna mobile banking-nya sekarang telah menembus 2,5 juta orang. Kemudian, hingga juni 2021 nilai transaksi kanal digital BSI mencapai Rp. 95,13 triliun, dengan kontribusi terbesar berasal dari layanan BSI Mobile yang naik 83,56% dari tahun ke tahun.1 Berdasarkan data - data yang didapat dari Bank Syariah Indoneisa terkait pengguna m- banking pada tahun 2021 tentunya bank syariah bersaing dalam hal peningkatan penggunaan layanan m-banking, namun pada kenyataan yang terjadi pada mahasiswa program studi Perbankan Syariah STEBI Global Mulia Cikarang hal ini berbanding terbalik. Terbukti dengan tidak banyaknya mahasiswa perbankan syariah STEBI Global Mulia Cikarang yang mempunyai rekening Bank Syariah sehingga jarang di temui mahasiswa perbankan syariah 58 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 yang memanfaatkan layanan m-banking bank syariah. Pertanggungjawaban keilmuan yang sudah sepantasnya dilakukan oleh mahasiswa perbankan syariah yang mempraktekan semua teori yang telah dipelajari salah satunya yaitu mempunyai rekening perbankan syariah. Atas bekal ilmu-ilmu yang di dapat semasa kuliah seperti pengetahuan pada dunia perbankan syariah mengenai terbebasnya dari riba, tentu mahasiswa perbankan syariah mempunyai kesadaran untuk ikut andil pada dunia perbankan syariah. Namun fakta yang terjadi, mahasiswa perbankan syariah telah mempunyai rekening perbankan syariah atas dasar keterpaksaan dari mata kuliah yang menganjurkan untuk membuka rekening di bank syariah. karena dirasa mahasiswa perbankan syariah ini mempunyai banyak pengetahuan dalam dunia perbankan syariah. Dengan banyaknya bekal ilmu serta kesadaran yang dimiliki bahwa menggunakan bank syariah dianggap lebih baik, maka penerapannya salah satunya yaitu sudah seharusnya mahasiswa perbankan syariah mempunyai rekening bank syariah. 2 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi), hal. 111 PENDAHULUAN Dengan mempunyai rekening perbankan syariah, tentu hal ini akan memicu mahasiswa untuk menggunakan layanan m-banking sehingga transaksi yang dilakukan akan lebih efektif dan efisien Sebagai lembaga intermediasi, pihak perbankan memang sudah seharusnya memberikan kualitas layanan yang lebih baik kepada nasabahnya, seperti dalam jasa layanan mobile banking, sehingga perhatian bank tidak hanya terbatas dari pelayanan secara tatap muka pada nasabah, tetapi juga pada aspek pelayanan dari jarak jauh melalui m-banking. Dimana hal ini selain untuk memberikan kemudahan bagi nasabah, juga menghemat waktu bagi mahasiswa yang ingin melakukan pembayaran SPP melalui m-banking. Oleh karena itu jika kedua m- banking tersebut memiliki kualitas layanan yang baik, tentu akan mendorong terciptanya publisitas public kepada calon-calon nasabah potensial, dan pada akhirnya akan menciptakan nasabah-nasabah yang setia bagi kedua bank. Kemudahan didefinisikan sebagai sejauh mana seseorang percaya bahwa menggunakan suatu teknologi akan bebas dari usaha. Yang mana dapat memudahakan suatu pekerjaan dari setiap penggunanya. Dirasa dapat menghemat tenaga, biaya dan waktu atas penggunaan objek tertentu. Meskipun pendapat seseorang berbeda-beda mengenai kemudahan, salah satunya kemudahan dalam penggunaan layanan m-banking, dengan adanya aplikasi ini dapat memudahkan setiap transaski perbankan. Kemudahan penggunaan (easeof use) adalah sebagai tingkatan di mana seseorang percaya bahwa Teknologi Informasi (TI) dapat dengan mudah dipahami. Kualitas informasi adalah fitur perlengkapan untuk interaktivitas nasabah, fitur merupakan kriteria penting yang menarik perhatian nasabah di dalam penyampaian jasa mobile banking. Informasi merupakan hasil pengolahan data yang memberikan arti dan manfaat. Kualitas informasi adalah tingkat dimana informasi memiliki karakteristik isi, bentuk, dan waktu, yang memberikannya nilai bagi para pemakai akhir tertentu. Sehingga dapat dikatakan bahwa baik buruknya suatu informasi dapat dinilai melalui kualitas informasi yang didapat, semakin baik kualitas informasi yang didapat akan memberikan kesan positif bagi para pengguna teknologi informasi Teori TAM (Technology Acceptance Model) Technology Acceptance Model (TAM) merupakan perilaku yang pada umumnya digunakan untuk mejelaskan penerimaan individual terhadap penggunaan sistem teknologi. 4 dimana Teori Technology Acceptance Model (TAM) yang dikemukakan oleh (Davis, 1989)5, TAM berpendapat bahwa suatu penerimaan individual terhadap sistem teknologi informasi telah ditentukan oleh dua konsep yang telah diamati. Dua konsep utama ini adalah kegunaan persepsian (perceived usefulness) dan kemudahan penggunaan persepsian (perceived ease of use). ) Menurut Jogiyanto teori ini memiliki beberapa kelebihan, yaitu:6 g y p y a. TAM merupakan model perilaku (behavior) yang bermanfaat untuk menjawab pertanyaan mengapa banyak sistem teknologi informasi yang gagal diterapkan karena pemakaiannya tidak mempunyai minat (intention) untuk menggunakannya. Tidak banyak model-model penerapan sistem teknologi informasi yang memasukkan faktor psikologis atau perilaku (behavior) di dalam modelnya dan TAM adalah salah satu model untuk mempertimbangkannya. b. TAM dibangun dengan dasar teori yang kuat. c. TAM telah diuji dengan banyak penelitian dan hasilnya sebagian besar mendukung dan menyimpulkan bahwasannya TAM merupakan model penelitian yang baik. c. TAM telah diuji dengan banyak penelitian dan hasilnya sebagian besar mendukung dan menyimpulkan bahwasannya TAM merupakan model penelitian yang baik. y p y p p y g d. TAM merupakan model persimoni (persimonious) yaitu model sederhana tetapi valid. Kualitas Informasi d. TAM merupakan model persimoni (persimonious) yaitu model sederhana tetapi valid. Kualitas Informasi M d l T i D L & M L Model Teori DeLone & McLean Model Teori DeLone & McLean Model DeLone dan McLean digunakan untuk mengukur keberhasilan sistem informasi, model ini disebut Sederhana namun dianggap sangat efektif oleh para peneliti (DeLone.W.H & McLean. E.R., 1992).7 D&M Model membuat teori Penelitian teoritis dan empiris berdasarkan sistem informasi yang dibuat oleh para peneliti di sekitarnya 1970-an dan 1980-an. Bahwa Informasi merupakan hasil pengolahan data yang memberikan arti dan manfaat. Kualitas informasi dalah sejauh mana informasi memiliki isi, bentuk, dan karakteristik temporal yang memberikan nilai kepada pengguna akhir tertentu. Sehingga dapat dikatakan bahwa baik buruknya suatu informasi dapat dinilai melalui kualitas informasi yang didapat, semakin baik kualitas informasi yang didapat akan memberikan kesan positif bagi para pengguna teknologi informasi.8 Menurut D&M Model Kualitas dari suatu informasi mencangkup indikator seb 9 Menurut D&M Model Kualitas dari suatu informasi mencangkup indikator sebagai berikut:9 Menurut D&M Model Kualitas dari suatu informasi mencangkup indikator berikut:9 a. Akurat Akurat artinya informasi harus bebas dari kesalahan dan tidak lazim atau menyesatkan. Akurat juga berarti bahwa informasi harus secara jelas mencerminkan maksud dan tujuan. 3 Sauca Ananda Pranidana, “Analisis Faktor-Faktor yang Mempengaruhi Nasabah Bank BCA untuk Menggunakan Klik-BC” 4 Hamzah, 2009, “Penerimaan Teknologi Pengguna Terhadap Sistem Informasi Dengan Metode Technology Acceptance Model”. Jurnal Pilar Nusa Mandiri Vol. IX No.2.Hal 172 5 Davis, F.D. 1989. Perceived usefulness, perceived ease of use, and user acceptance of information technology. MIS Quarterly. 13(3): 319-339 6 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi), 7 DeLone.W.H & McLean. E.R., 1992. Informatiom System Success: The Quest For The Dependent Variable. Pp.60-95. 8 Azhar Susanto, Sistem Informasi Manajemen:Konsep dan Pengembangannya, (Bandung: Linggar Jaya, 2004), hal. 40. 9 Azhar Susanto, Sistem Informasi Manajemen:Konsep dan Pengembangannya, (Bandung: Linggar Jaya, 2004), hal. 43. Kemudahan Kemudahan diartikan sampai mana seseorang merasa yakin dan percaya jika menggunakan suatu teknologi akan bebas dari usaha.2 Yang mana dapat memudahakan suatu pekerjaan dari setiap penggunanya. Dianggap dapat menghemat tenaga, biaya dan waktu atas penggunaan objek tertentu. Meskipun pendapat seseorang berbeda-beda mengenai kemudahan, salah satunya kemudahan dalam penggunaan layanan mbanking, dengan adanya aplikasi ini dapat memudahkan setiap transaksi perbankan. Pranidana berpendapat bahwa “Kemudahan 59 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... penggunaan (ease of use) adalah sebagai tingkatan dimana seseorang percaya bahwa teknologi informasi (TI) dapat dengan mudah dipahami”.3 penggunaan (ease of use) adalah sebagai tingkatan dimana seseorang percaya bahwa teknologi informasi (TI) dapat dengan mudah dipahami”.3 3 Sauca Ananda Pranidana, “Analisis Faktor-Faktor yang Mempengaruhi Nasabah Bank BCA untuk Menggunakan Klik-BC” 10 Slameto, 2010, “Belajar & Faktor-Faktor yang mempengaruhi”, (Jakarta:PT Rineka Cipta) 11 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi) 12 Undang-Undang Nomor 21 tahun 2008 tentang perbankan syariah ayat 1 Teori TAM (Technology Acceptance Model) Informasi harus akurat karena dapat terjadi banyak gangguan (noise) a. Akurat Akurat artinya informasi harus bebas dari kesalahan dan tidak lazim atau menyesatkan. Akurat juga berarti bahwa informasi harus secara jelas mencerminkan maksud dan tujuan. Informasi harus akurat karena dapat terjadi banyak gangguan (noise) 60 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 yang dapat mengubah atau merusak informasi dari sumber informasi ke penerima informasi. yang dapat mengubah atau merusak informasi dari sumber informasi ke penerima informasi. b. Tepat pada waktunya Tepat waktu artinya informasi yang sampai pada penerima tidak boleh terlambat. Informasi yang terlupakan tidak akan lagi memiliki nilai karena informasi merupakan dasar pengambilan keputusan. Keputusan untuk datang terlambat dapat berakibat fatal bagi organisasi atau asosiasi. Cepatnya teknologi yang harus didapat itu disebabkan tingginya nilai informasi, sehingga dibutuhkan teknologi-teknologi yang modern untuk mendapatkan, mengolah dan mengirimkannya. Sehingga ketepatan waktu pada sistem teknologi informasi sangat diperlukan. c. Relevan Relevan berarti informasi tersebut memiliki manfaat untuk penggunanya. Relevansi informasi untuk tiap-tiap orang dari satu dengan yang lainnya berbeda-beda. Misalnya informasi mengenai sebab kerusakan mesin produksi kepada akuntan perusahaan adalah kurang relevan dan akan lebih relevan apabila ditunjukkan kepada ahli teknik perusahaan. Sehingga informasi tepat sasarannya. d. Lengkap Lengkap artinya informasi yang diberikan harus diterima sepenuhnya oleh penerima. Informasi yang diterima tidak boleh setengahsetengah. sehingga penerima informasi dapat menggunakan informasi tersebut semaksimal mungkin. Informasi yang lengkap akan memberikan dampak positif bagi penggunanya. Ketika informasi dapat memuat semua indikator di atas, maka kualitas informasi dapat dikatakan baik dan layak untuk digunakan. Dalam lingkup ini, kualitas informasi yang dimaksud adalah semua informasi yang dimuat dalam layanan m-banking khususnya pada Bank Syariah. Minat Minat merupakan sutau keinginan perasaan yang dimiliki setiap individu tentu berbeda- beda, hal ini dijadikan pengaruh dalam terbentuknya minat terhadap sesuatu. Hal ini tentu akan menentukan seseorang tersebut aktif tidaknya dalam melakukan suatu pekerjaan dan dalam situasi kondisi apapun. Perasaan senang maupun sedih akan mempengaruhi minat dari individu itu sendiri. Selain itu, minat juga akan mempengaruhi sesuatu yang dilihat atau diamati. Hal ini tentu ada hubungannya dengan keinginan atau kebutuhan dari individu itu sendiri. Ada beberapa pendapat minat menurut ahli a. Minat adalah suatu rasa lebih suka dan rasa karakteristik pada suatu hal atau aktivitas, tanpa ada yang menyuruh. Minat pada dasarnya adalah menerima hubungan antara diri sendiri dengan hal-hal eksternal. Semakin kuat atau dekat hubungannya, semakin besar minatnya.10 y b. Minat perilaku (behavioral intention) merupakan kecenderungan untuk mendorong seseorang malakukan suatu tindakan. 11 b. Minat perilaku (behavioral intention) merupakan kecenderungan untuk mendorong seseorang malakukan suatu tindakan. 11 c. Menurut Salahudin Minat dapat diartikan suatu keadaan dimana apabila seseorang melihat ciri-ciri atau arti sementara situasi yang dihubungkan dengan keinginan atau kebutuhan sendiri. minat sebagai perhatian yang mengandung unsur perasaan. B k S i h c. Menurut Salahudin Minat dapat diartikan suatu keadaan dimana apabila seseorang melihat ciri-ciri atau arti sementara situasi yang dihubungkan dengan keinginan atau kebutuhan sendiri. minat sebagai perhatian yang mengandung unsur perasaan. g y ( ) g p ( gy ) 12 Undang-Undang Nomor 21 tahun 2008 tentang perbankan syariah ayat 1 11 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi) 10 Slameto, 2010, “Belajar & Faktor-Faktor yang mempengaruhi”, (Jakarta:PT Rineka Cipta) Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi) 12 Undang-Undang Nomor 21 tahun 2008 tentang perbankan syariah ayat 1 Bank Syariah Menurut Pasal 1 UU Nomor 21 Tahun 200812, bank adalah badan usaha yang menghimpun dana masyarakat dalam bentuk simpanan dan menyalurkannya kepada masyarakat dalam bentuk kredit atau lainnya untuk menaikkan standar kehidupan masyarakat luas. Bank memiliki dua jenis, yaitu bank konvensional dan bank syariah. Bank Konvensional adalah bank yang melakukan bisnis dengan cara Konvensional. Bank Konvensional meliputi Bank Umum Konvensional (BUK) dan Bank Perkreditan Rakyat (BPR). Bank syariah adalah bank yang menjalankan usaha sesuai dengan prinsip syariah. Bank syariah antara lain Bank 61 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... Umum Syariah (BUS) dan Bank Pembiayaan Rakyat Syariah (BPRS). Prinsip dasar syariah adalah dasar hukum islam. Berlandaskan penetapan fatwa dibidang syariah, lembaga memiliki kewenangan dan kebijakan dalam kegiatan perbankan.13 BUS merupakan bank syariah yang kegiatannya menyediakan jasa pembayaran. BPRS adalah bank syariah yang menjalankan kegiatan usahanya dan tidak memberikan jasa lalu lintas pembayaran. Unit Usaha Syariah (UUS) adalah unit kerja kantor pusat Bank Umum Konvensional, sebagai kantor utama dari kantor atau unit yang melakukan kegiatan usaha sesuai dengan prinsip syariah, atau sebagai unit kerja kantor cabang. Bank yang melakukan kegiatan usaha di luar negeri, biasanya sebagai kantor pusat cabang unit usaha syariah. g y g p g y Di dalam Pasal 2 UU Nomor 21 Tahun 2008, kegiatan usaha pada perbankan syariah diatur berdasarkan prinsip syariah, demokrasi ekonomi, dan prinsip kehati-hatian. Selanjutnya mengenai tujuan Perbankan Syariah, Pasal 3 Undang-Undang No 21 Tahun 2008, menyatakan bahwa Perbankan Syariah bertujuan untuk mendukung terselenggaranya pembangunan nasional dalam rangka meningkatkan keadilan, kebersaman dan pemerataan masyarakat. Sejarah Berdirinya Bank Syariah di Indonesia Lahirnya Bank Syariah pertama di Indonesia yaitu terbentuknya PT Bank Muamalat Indonesia (BMI) yang merupakan hasil kerja tim perbankan MUI yang berdirinya ditandatangani pada tanggal 1 November 1991. Untuk saat ini BMI sudah memiliki beberapa cabang di kota besar seperti Jakarta, Surabaya, Bandung, Makassar, dan beberapa kota-kota besar lainnya.14 Di Indonesia, Bank Syariah yang pertama kali didirikan pada tahun 1992 adalah bank Muamalah Indonesia. Walaupun terbilang lambat dalam perkembangannya tetapi jika dibandingkan dengan negara-negara muslim yang ada di dunia, perbankan syariah di Indonesia akan terus berkembang dan bersaing dalam dunia perbankan negara lainnya. Bank Syariah hanya memiliki satu unit bank pada periode tahun 1992-1998, kemudian pada tahun 2004 telah bertambah menjadi 20 unit bank syariah di Indonesia, ditahun 2005 bertambah 88 unit bank syariah, terakhir menurut data OJK pada tahun 2016 jumlah Bank Syariah di Indonesia mencapai 166 unit. p ( p , ), 14 Heri Sudarsono, Bank dan Lembaga Keuangan Syariah (Yogyakarta: Ekonisia, 2008), h.30 g j g ( ) 16 Ikatan Bankir Indonesia (IBI) dan Lembaga Sertifikasi Profesi Perbankan (LSPP), Mengenal Operasional Perbankan 1, (Jakarta: PT Gramedia Pustaka Utama, 2014), hal. 59. 13 Rizal Yaya, Aji Erlangga Martawireja, Ahim Abdurahim, Akuntansi Perbankan Syariah Teori Dan Praktik Kontemporer (Jakarta: Salemba Empat, 2016), hal.48. g g y gy 15 Otoritas Jasa Keuangan, 2015, Bijak Ber-Electric Banking. (Jakarta:OJK) 14 Heri Sudarsono, Bank dan Lembaga Keuangan Syariah (Yogyakarta: Eko Kelebihan m-banking g 1. Mobile banking menawarkan beberapa jasa-jasa yang cukup menarik untuk diminat 2. Selain bisa menghemat waktu, m-banking juga bisa menghemat biaya. 3. Akses nasabah dengan bank bisa dilakukan dengan jasa-jasanya kapanpun dan dimanapun. 4. Nasabah bisa mengetahui jika telah terjadi penarikan uang bukan dari nasabah itu sendiri dan bisa langsung melakukan pemblokiran ATM. 1. Strategi kompetitif dari pihak bank pada layanan m-banking bisa memberikan nilai tambah kepada nasabahnya. 2. Biaya pengurusan nasabah dapat berkurang. 18 Kekurangan M-banking18 a. Jenis OS handphone Kekurangan M-banking18 a. Jenis OS handphone c. Jenis kartu yang digunakan d. Kecepatan data/jaringan suatu wilayah e. batasan limit transaksi dalam sehari f. Aktifasi setiap bulan g. gangguan pada sistem layanan m-banking Bank Syariah Ini menunjukkan bahwa Bank Indonesia mampu bersaing dengan bank- bank konvensional, dan semakin tahun mengalami kemajuan yang lumayan pesat.15 Tujuan Berdirinya Bank Syariah Setelah mengetahui tentang suatu bank konvensional yang dirasa mengalami kegagalan menjalani suatu peran utamanya yang menghubungkan pemilik modal dengan pihak yang memerlukan dana, maka Bank Syariah dibentuk untuk tujuan sebagai berikut.16 a. Untuk mengarahkan suatu kegiatan ekonomi dalam bermuamalah secara syariat islam, seperti terhindarnya dari praktek riba dalam dunia perbankan. b. Kegiatan Investasi dibuat untuk meratakan pendapatan dan menciptakan suatu kead dibidang ekonomi. b. Kegiatan Investasi dibuat untuk meratakan pendapatan dan menciptakan suatu keadilan dibidang ekonomi. d Untuk peningkatan taraf hidup umat muslim peluang usaha yang besar akan dibuka untuk d. Untuk peningkatan taraf hidup umat muslim, peluang usaha yang besar akan dibuka u mereka kelompok miskin agar diarakahkan ke berwirausaha. e. Untuk mengurangi tingkat kemiskinan, merupakan program utama dari negara-ne muslim yang sedang berkembang. f. Untuk menjaga keadaan yang stabil pada ekonomi/moneter pemerintah. Diharap mampu menghindari inflasi akibat penerapan sistem bunga pada aktivitas bank syari p g p p g p y g. Untuk mengurangi minat umat muslim menjadi nasabah pada Bank Konvensional. 62 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 17 Syamsul Hadi Novi, “Faktor-Faktor Yang Mempengaruhi Layanan Mobile Banking”, Universitas Islam Indonesia, h. 58. M-Banking Mobile banking atau yang sering disebut m-banking ialah suatu layanan perbankan yang dapat diakses melalui handphone GSM (Global for Mobile Communication). m-banking adalah suatu layanan perbankan yang menerapkan suatu teknologi informasi.17 Layanan perbankan ini akan menjadikan peluang bagi bank untuk menawarkan nilai tambah kepada pengguna atau nasabah. M-banking juga memberikan suatu layanan untuk mendukung kelancaran dan kemudahan dalam aktivitas perbankan. Keefektifan dan keefisienan nasabah yang akan melakukan berbagai transasksi di perbankan jika tidak memiliki fasilitas pendukung seperti handphone dan internet akan menjadi kesulitan bagi nasabah. Jika setiap pengguna memiliki handphone dan internet maka mereka bisa bertansaksi dimanapun dan kapanpun. Ada beberapa jenis transaksi Mobile banking, sebagai berikut: 1. Mengetahui informasi saldo, Transfer uang, mutasi rekening, informasi nilai tukar. 2. Pembayaran (kartu kredit, PLN, telepon, handphone,listrik, asuransi) 3. Pembelian (pulsa isi ulang, saham). Kegiatan transaksi mobile banking bisa dilakukan dimana saja dan kapan saja, salah satu keuntungan diperolehnya nasabah selain menghemat waktu, yaitu nasabah juga bisa mengontrol rekening dan melakukan transaksi perbankan lainnya dengan cara menggunakan ponsel. Habsari, wawancara dengan penulis, Bank Syariah Mandiri KC Bandar Jaya, Lampung, 31 Mei 2018. 18 Santi Habsari, wawancara dengan penulis, Bank Syariah Mandiri KC Bandar Jaya, Lampung, 31 Mei 2018. 19 Sarwono Jonathan, Rumus-Rumus Populer dalam SPSS 22 untuk Riset Skripsi, Yogyakarta: C.V Andi Offset, 2015. hal. 101. METODE PENELITIAN Penelitan ini menggunakan metode kuantitatif. Jenis dan sumber data menggunakan data primer dan data sekunder. Populasi yang diambil adalah mahasiswa angkatan 2018-2021 program studi perbankan syariah STEBI Global Mulia Cikarang. Dengan teknik pengambilan sampling menggunakan rumus slovin yang di dapat yaitu 93 responden dari 120 mahasiswa. Teknik pengumpulan data pada penelitian ini menggunakan metode perpustakaan dan kuesioner dengan instrumen pengukuran menggunakan skala likert. 63 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... Peneliti ini menggunakan metode analisis regresi linier berganda dengan menggunakan program komputer yaitu Software Statistical Package For The Social Science (SPSS) versi 25.0. Penerapan metode ini akan menghasilkan tingkat hubungan antara variabel-variabel bebas (independen) terhadap variabel terkaitnya (dependen) serta arah hubungan yang terjadi (hubungan negative atau positive). Yaitu dengan menggunakan Uji Validitas, Uji Reliabilitas Uji Asumsi klasik meliputi uji normalitas, uji multikolinearitas dan uji heteroskedastisitas, lalu juga menggunakan uji hipotesis meliputi uji t (partial), uji F (simultan),uji koefisien determinasi dan menggunakan uji anaisis regresi linier berganda. adapun persamaan regresinya adalah sebagai berikut : Y= a + b₁X₁ + b₂X₂ + e Keterangan : Y = Variabel terikat (minat penggunaan layanan m-banking) a, b₁, b₂ = Bilangan Konstanta X₁ = Kemudahan X₂ = Kualitas Informasi e = Residual Eror dari masing-masing variabel HASIL DAN PEMBAHASAN 20 Paturohman, I. (2019). SPSS 25 Main Subject: Linear Regression Analysis. Bandung. HASIL DAN PEMBAHASAN 1. Hasil Uji Validitas 1. Hasil Uji Validitas Korelasi product moment pearson merupakan pengukuran parametik, akan menghasilkan koefisien yang berfungsi untuk mengukur kekuatan hubungan linier antara dua variabel, jika hubungan dua variabel tidak linier, maka koefisien korelasi pearson tersebut tidak mencerminkan kekuatan hubungan dua variabel yang sedang diteliti, meski dua variabel mempunyai hubungan kuat.19 Tabel 1 Hasil Koefisien Pearson Product Moment No Pertanyaan Pearson Correlation Sig. Keterangan Kemudahan (X₁) 1 0,842 0,000 Valid 2 0,850 0,000 Valid 3 0,841 0,000 Valid 4 0,789 0,000 Valid 5 0,774 0,000 Valid 6 0,762 0,000 Valid 7 0,877 0,000 Valid 8 0,729 0,000 Valid Kualitas Informasi (X₂) 1 0,790 0,000 Valid 2 0,706 0.000 Valid 3 0,714 0,000 Valid 4 0,831 0,000 Valid 5 0,824 0,000 Valid 6 0,859 0,000 Valid 7 0,749 0,000 Valid 8 0,732 0,000 Valid 64 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 Minat Penggunaan (Y) 1 0,811 0,000 Valid 2 0,581 0,000 Valid 3 0,729 0,000 Valid 4 0,858 0,000 Valid 5 0,819 0,000 Valid 6 0,852 0,000 Valid 7 0,865 0,000 Valid 8 0,861 0,000 Valid 9 0,793 0,000 Valid Sumber : Hasil Pengelolahan SPSS 2022 Berdasarkan Tabel 1 menunjukan bahwa seluruh variabel mempunyai 25 item pernyataan yang diberikan kepada 93 responden mempunyai nilai r hitung yang lebih besar dari r tabel yaitu 0.2039 dan nilai signifikansinya lebih kecil dari 0,05, sehingga dapat dikatakan bahwa semua indikator pertanyaan pada variabel penelitian adalah valid. 2. 2. Hasil Uji Reliablitas Hasil Uji Reliablitas Tabel 2 Hasil Uji Reliabilitas Variabel Alpha Cronbanch’s Kesimpulan Kemudahan 0,924 Reliabel Kualitas Infromasi 0,905 Reliabel Minat Penggunaan 0,925 Reliabel Sumber : Hasil Pengelolahan SPSS 2022 Tabel 2 menunjukan bahwa nilai cronbach’s alpha atas variabel kemudahan sebesar 0,924, kualitas informasi sebesar 0,905 dan minat penggunaan sebesar 0,925. Dengan demikian, dapat disimpulkan bahwa pernyataan dalam kuesioner ini reliabel karena mempunyai nilai cronbach’s alpha > 0,60. Hal ini menunjukan bahwa setiap item pernyataan yang digunakan akan mampu memperoleh data yang konsisten berarti bila pernyataan itu diajukan kembali akan diperoleh jawaban yang relatif sama dengan jawaban yang sebelumnya. 65 Jurnal Al-Fatih Global Mulia 3. Uji Asumsi Klasik a. Uji Normalitas Uji normalitas digunakan untuk menguji apakah dalam model regresi, data variabel dependen dan independen yang dipakai terdistribusi normal atau tidak. Sebuah data penelitian yang baik adalah yang datanya berdistribusi normal (Ghozali, 2016). Pada penelitian ini uji normalitas menggunakan metode analisa grafik. Dasar pengambilan keputusan untuk mendeteksi kenormalan adalah jika data menyebar di sekitar garis diagonal dan mengikuti arah diagonal, maka model regresi memenuhi asumsi normalitas dan begitu juga dengan sebaliknya.20 1) Metode P-P Plot of Regression Standardized Residual. Berikut ini output perhit ngan SPSS ersi 25 0 a. Uji Normalitas Uji normalitas digunakan untuk menguji apakah dalam model regresi, data variabel dependen dan independen yang dipakai terdistribusi normal atau tidak. Sebuah data penelitian yang baik adalah yang datanya berdistribusi normal (Ghozali, 2016). Pada penelitian ini uji normalitas menggunakan metode analisa grafik. p j gg g Dasar pengambilan keputusan untuk mendeteksi kenormalan adalah jika data menyebar di sekitar garis diagonal dan mengikuti arah diagonal, maka model regresi memenuhi asumsi normalitas dan begitu juga dengan sebaliknya.20 p j gg g Dasar pengambilan keputusan untuk mendeteksi kenormalan adalah jika data menyebar di sekitar garis diagonal dan mengikuti arah diagonal, maka model regresi memenuhi asumsi normalitas dan begitu juga dengan sebaliknya.20 g j g g y 1) Metode P-P Plot of Regression Standardized Residual. Berikut ini output perhitungan SPSS versi 25.0. 1) Metode P-P Plot of Regression Standardized Residual. Berikut ini output perhitungan SPSS versi 25.0. 65 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... Berdasarkan gambar 4.2 di atas dapat disimpulkan bahwa grafik normal P- Plot terlihat titik-titik menyebar disekitar garis diagonal dan penyebarannya mengikuti arah diagonal, maka grafik menunjukan bahwa model regresi layak dipakai karena memenuhi asumsi normalitas. 2) Metode One Sample Kolmogorov Smirnov. Berikut output perhitungan SPSS Versi 25.0. Berdasarkan gambar 4.2 di atas dapat disimpulkan bahwa grafik normal P- Plot terlihat titik-titik menyebar disekitar garis diagonal dan penyebarannya mengikuti arah diagonal, maka grafik menunjukan bahwa model regresi layak dipakai karena memenuhi asumsi normalitas. p 2) Metode One Sample Kolmogorov Smirnov. Berikut output perhitungan SPSS Versi 25.0. One-Sample Kolmogorov-Smirnov Test Unstandardized Residual N 93 Normal Parametersa,b Mean .0000000 Std. Deviation 2.72222596 Most Extreme Differences Absolute .088 Positive .082 Negative -.088 Test Statistic .088 Asymp. Sig. (2-tailed) .073c a. Test distribution is Normal. b. Calculated from data. c. Lilliefors Significance Correction. 66 Jurnal Al-Fatih Global Mulia b. Uji Multikolinearitas p p j p g 2) Metode Uji Glejser, berikut output hasil SPSS Versi 25.0 hasil uji Heteroskedastisitas metode Glejser, diketahui nilai signifikansi variabel X₁ adalah 0,969, dan nilai signifikansi variabel X₂ adalah 0,730. Nilai signifikansi kedua variabel ini > 0,05, maka disimpulkan bahwa data tidak mengalami Heteroskedastisitas, dengan kata lain tidak terjadi keragaman varian dari nilai residual terhadap semua pengamatan yang ada pada model regresi. 2) Metode Uji Glejser, berikut output hasil SPSS Versi 25.0 hasil uji Heteroskedastisitas metode Glejser, diketahui nilai signifikansi variabel X₁ adalah 0,969, dan nilai signifikansi variabel X₂ adalah 0,730. Nilai signifikansi kedua variabel ini > 0,05, maka disimpulkan bahwa data tidak mengalami Heteroskedastisitas, dengan kata lain tidak terjadi keragaman varian dari nilai residual terhadap semua pengamatan yang ada pada model regresi. b. Uji Multikolinearitas b. Uji Multikolinearitas Multikolinearitas adalah ditemukan adanya korelasi yang sempurna atau mendekati sempurna antar variabel independen dan model regresi (Ghozali I. , 2016). Jika nilai tolerance > 0,05 maka artinya tidak terjadi multikolinearitas. Dan jika nilai VIF < 5,00 maka tidak terjadi multikolinearitas. Hal ini bisa dikatakan dengan model regresi yang baik karena tidak terjadi multikolinearitas. j Multikolinearitas adalah ditemukan adanya korelasi yang sempurna atau mendekati sempurna antar variabel independen dan model regresi (Ghozali I. , 2016). Jika nilai tolerance > 0,05 maka artinya tidak terjadi multikolinearitas. Dan jika nilai VIF < 5,00 maka tidak terjadi multikolinearitas. Hal ini bisa dikatakan dengan model regresi yang baik karena tidak terjadi multikolinearitas. Dari data yang telah di uji, nilai tolerence tiap variabel lebih besar dari 0,05 dan nilai VIF lebih kecil dari nilai 5,00. pada analisi data output uji multikolinearitas bisa disimpulkan : 1) Nilai Tolerence variabel X₁ dan X₂ = 0,282 > 0,05 Artinya : tidak terjadi multikolinearitas pada variabel bebas 2) Nilai VIF Variabel X₁ dan X₂ = 3,552 < 5,00 Artinya : tidak terjadi multikolinearitas pada variabel bebas c. Uji Heteroskedastisitas Uji Heteroskedastisitas adalah keadaan yang mana dalam model regresi terjadi ketidaksamaan variansi dan residual pada satu pengamatan ke pengamatan yang lain. Model regresi yang baik adalah tidak terjadi heteroskedastisitas (Ghozali I. , 2016 edisi 8). c. Uji Heteroskedastisitas Uji Heteroskedastisitas adalah keadaan yang mana dalam model regresi terjadi ketidaksamaan variansi dan residual pada satu pengamatan ke pengamatan yang lain. Model regresi yang baik adalah tidak terjadi heteroskedastisitas (Ghozali I. , 2016 edisi 8). 1) Metode Scatterplot, berikut output hasil SPSS Versi 25.0 Terlihat bahwa distribusi data tidak teratur dan tidak membentuk pola tertentu, serta tersebar diatas maupun dibawah angka nol pada sumbu Y. Hal ni dapat disimpulkan tidak terjadi heteroskedastisitas pada model regresi. 2) Metode Uji Glejser, berikut output hasil SPSS Versi 25.0 hasil uji Heteroskedastisitas metode Glejser, diketahui nilai signifikansi variabel X₁ adalah 0,969, dan nilai signifikansi variabel X₂ adalah 0,730. Nilai signifikansi kedua variabel ini > 0,05, maka disimpulkan bahwa data tidak mengalami Heteroskedastisitas, dengan kata lain tidak terjadi keragaman varian d i il i id l t h d t d d d l i 1) Metode Scatterplot, berikut output hasil SPSS Versi 25.0 Terlihat bahwa distribusi data tidak teratur dan tidak membentuk pola tertentu, serta tersebar diatas maupun dibawah angka nol pada sumbu Y. Hal ni dapat disimpulkan tidak terjadi heteroskedastisitas pada model regresi. 3. Uji Asumsi Klasik Berdasarkan Tabel 4.13 output SPSS di atas, diketahui bahwa nilai signifikasi Asymp Sig sebesar 0,073 > 0,05, maka sesuai dengan dasar pengambilan keputusan dalam uji normalitas one sample kolmogorov smirnov di atas, dapat disimpulkan bahwa data berdistribusi normal. Dengan demikian asumsi atau persyaratan normalitas dalam model regresi sudah terpenuhi. 66 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 b. Uji Multikolinearitas Multikolinearitas adalah ditemukan adanya korelasi yang sempurna atau mendekati sempurna antar variabel independen dan model regresi (Ghozali I. , 2016). Jika nilai tolerance > 0,05 maka artinya tidak terjadi multikolinearitas. Dan jika nilai VIF < 5,00 maka tidak terjadi multikolinearitas. Hal ini bisa dikatakan dengan model regresi yang baik karena tidak terjadi multikolinearitas. Dari data yang telah di uji, nilai tolerence tiap variabel lebih besar dari 0,05 dan nilai VIF lebih kecil dari nilai 5,00. pada analisi data output uji multikolinearitas bisa disimpulkan : 1) Nilai Tolerence variabel X₁ dan X₂ = 0,282 > 0,05 Artinya : tidak terjadi multikolinearitas pada variabel bebas 2) Nilai VIF Variabel X₁ dan X₂ = 3,552 < 5,00 Artinya : tidak terjadi multikolinearitas pada variabel bebas 4. Uji Hipotesis 4. Uji Hipotesis a. Uji Partial (Uji-t) Uji t dilakukan untuk menunjukan seberapa jauh pengaruh setiap variabel bebas Kemudahan (X₁) dan Kualitas Informasi (X₂) terhadap Minat Penggunaan (Y) (Ghozali I. , 2016). Berdasarkan hasil uji partial, diketahui bahwa nilai t hitung variabel kemudahan (X₁) sebesar 4,058 > nilai t tabel sebesar 1,986 (df = n - k) atau df = 93 – 3 = 90). Selain itu, nilai signifikasi t bernilai 0,000 < 0,05, sehingga H0 ditolak dan H₁ diterima. Hal ini menunjukan bahwa variabel kemudahan (X₁) berpengaruh secara signifikan terhadap minat penggunaan (Y). Kemudian bahwa nilai t hitung variabel kualitas informasi (X₂) sebesar 4,570 > nilai t tabel sebesar 1,986 (df=90). Selain itu. Nilai signifikansi t bernilai 0,000 < 0.05, sehingga H0 ditolak dan H₂ diterima. Hal ini menunjukan bahwa variabel Kualitas Informasi (X₂) berpengaruh secara signifikan terhadap minat penggunaan (Y). 4. Uji Hipotesis a. Uji Partial (Uji-t) Uji t dilakukan untuk menunjukan seberapa jauh pengaruh setiap variabel bebas Kemudahan (X₁) dan Kualitas Informasi (X₂) terhadap Minat Penggunaan (Y) (Ghozali I. , 2016). Berdasarkan hasil uji partial, diketahui bahwa nilai t hitung variabel kemudahan (X₁) sebesar 4,058 > nilai t tabel sebesar 1,986 (df = n - k) atau df = 93 – 3 = 90). Selain itu, nilai signifikasi t bernilai 0,000 < 0,05, sehingga H0 ditolak dan H₁ diterima. Hal ini menunjukan bahwa variabel kemudahan (X₁) berpengaruh secara signifikan terhadap minat penggunaan (Y). Kemudian bahwa nilai t hitung variabel kualitas informasi (X₂) sebesar 4,570 > nilai t tabel sebesar 1,986 (df=90). Selain itu. Nilai signifikansi t bernilai 0,000 < 0.05, sehingga H0 ditolak dan H₂ diterima. Hal ini menunjukan bahwa variabel Kualitas Informasi (X₂) berpengaruh secara signifikan terhadap minat penggunaan (Y). p g g p p gg b. Uji Simultan (Uji F) Uji F dilakukan untuk menunjukan apakah variabel bebas Kemudahan (X₁) dan Kualitas Informasi (X₂) yang dimasukan dalam model regresi secara bersama-sama berpengaruh terhadap variabel terikat Minat Penggunaan (Y) (Ghozali I. , 2016) b. Uji Simultan (Uji F) Uji F dilakukan untuk menunjukan apakah variabel bebas Kemudahan (X₁) dan Kualitas Informasi (X₂) yang dimasukan dalam model regresi secara bersama-sama berpengaruh terhadap variabel terikat Minat Penggunaan (Y) (Ghozali I. , 2016) b. Uji Simultan (Uji F) Uji F dilakukan untuk menunjukan apakah variabel bebas Kemudahan (X₁) dan Kualitas Informasi (X₂) yang dimasukan dalam model regresi secara bersama-sama berpengaruh terhadap variabel terikat Minat Penggunaan (Y) (Ghozali I. 4. Uji Hipotesis , 2016) Berdasarkan hasil Uji ANOVA atas Ftest pada tabel didapatkan f hitung sebesar 122,177 dengan tingkat signifikan 0,05, karena F hitung > F tabel (122,177 > 3,10) dan tingkat signifikasi 0,000 < 0,05, maka model regresi dapat digunakan untuk memprediksi minat penggunaan (Y) atau dikatakan bahwa variabel kemudahan (X₁) dan kualitas 67 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... informasi (X₂) secara bersama-sama berpengaruh secara nyata terhadap variabel minat penggunaan (Y). c. Uji Koefisien Determinasi (R Square) Koefisien determinasi menunjukkan sejauh mana tingkat hubungan antara variabel dependen dengan independen atau sebaliknya sejauh mana konstribusi variabel independen mempengaruhi variabel dependen. c. Uji Koefisien Determinasi (R Square) Koefisien determinasi menunjukkan sejauh mana tingkat hubungan antara variabel dependen dengan independen atau sebaliknya sejauh mana konstribusi variabel independen mempengaruhi variabel dependen. p g p Dari hasil uji koefisien determinasi menjelaskan besarnya nilai korelasi/hubungan (R) yaitu sebesar 0,855 (85,5%). Dari output tersebut diperoleh koefisien determinasi (R Square) sebesar 0,731 (73,1%). Angka ini mengandung pengertian bahwa 73,1% Minat Penggunaan (Y) dapat dijelaskan melalui variabel Kemudahan (X₁) dan Kualitas Informasi (X₂). Sisanya sebesar 26,9% dijelaskan oleh variabel lain diluar variabel yang diteliti. d Uji Analisis Regresi Linier Berganda Dari hasil uji koefisien determinasi menjelaskan besarnya nilai korelasi/hubungan (R) yaitu sebesar 0,855 (85,5%). Dari output tersebut diperoleh koefisien determinasi (R Square) sebesar 0,731 (73,1%). Angka ini mengandung pengertian bahwa 73,1% Minat Penggunaan (Y) dapat dijelaskan melalui variabel Kemudahan (X₁) dan Kualitas Informasi (X₂). Sisanya sebesar 26,9% dijelaskan oleh variabel lain diluar variabel yang diteliti. d. Uji Analisis Regresi Linier Berganda Uji regresi linier berganda adalah analisis untuk mengukur besarnya pengaruh antara dua atau lebih variabel independen terhadap variabel dependen. Berikut adalah hasil uji analisis regresi liniar berganda dengan perhitungan SPSS Versi 25.0. Berdasarkan Uji Analisis Regresi Linear Berganda, maka pengaruh variabel kemudahan (X₁) dan variabel Kualitas Informasi (X₂) terhadap minat penggunaan (Y) memiliki persamaan regresi sebagai berikut : p g g Y= 0,533 + 0,503X₁ + 0,585X₂ + e Y= 0,533 + 0,503X₁ + 0,585X₂ + e Penjelasan persamaan regresi di atas adalah sebagai berikut : Penjelasan persamaan regresi di atas adalah sebagai berikut : 1) Nilai konstanta sebesar 0,533 yang berarti nilai minat penggunaan adalah 0,533, saat X₁ dan X₂ adalah nol (0). 4. Uji Hipotesis 2) Nilai koefisien variabel kemudahan mengalami peningkatan satu satuan sedangkan variabel kualitas informasi konstanta atau tidak ada atau sebesar 0,503 maka Y (minat penggunaan) akan mengalami peningkatan sebesar 0,503 dengan asumsi variabel kemudahan (X₁) bernilai tetap. Nilai koefisien variabel kualitas informasi mengalami peningkatan satu satuan sedangkan variabel kemudahan konstan atau tidak ada atau sebesar 0,585 maka Y (minat penggunaan) akan mengalami peningkatan sebesar 0,585 dengan asumsi variabel kualitas informasi (X₂) bernilai tetap. 68 Jurnal Al-Fatih Global Mulia KESIMPULAN Berdasarkan hasil penelitian yang telah dilakukan dan telah melalui tahap pengumpulan data, pengolahan data, analisis data, dan interpretasi hasil analisis mengenai pengaruh kemudahan dan kualitas informasi terhadap minat mahasiswa dalam penggunaan layanan m- banking pada Bank Syariah, dengan menggunakan data yang terdistribusi normal dan tidak terdapat multikolinearitas, serta tidak adanya masalah heteroskedastisitas maka dihasilkan kesimpulan sebagai berikut: 1. Variabel Kemudahan Hasil uji t nilai t hitung variabel kemudahan (X₁) sebesar 4,058 > nilai t tabel sebesar 1,986 (df=90). Selain itu, nilai signifikasi bernilai 0,000 < 0,05. sehingga H0 ditolak dan H₁ diterima. Hal ini menunjukan bahwa kemudahan (X₁) berpengaruh secara signifikan terhadap minat penggunaan (Y). 2. Variabel Kualitas Informasi Hasil uji t nilai t hitung variabel kualitas Informasi (X₂) sebesar 4,570 > nilai t tabel sebesar 1,986 (df=90). Selain itu, nilai signifikasi bernilai 0,000 < 0,05, sehingga H0 ditolak dan H₂ diterima. Hal ini menunjukan bahwa Kualitas Informasi (X₂) berpengaruh secara signifikan terhadap minat penggunaan (Y). 3. Hasil Uji F hitung sebesar 122,177 dengan tingkat signifikan 0,000, karena F hitung > F tabel (122,177 > 3,10) dan tingkat signifikasi 0,000 < 0,05, sehingga H0 ditolak dan H₃ diterima. 68 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 Hal ini menunjukan bahwa variabel kemudahan (X₁) dan kualitas informasi (X₂) secara bersama-sama berpengaruh secara signifikan terhadap variabel minat penggunaan (Y). Berdasarkan uji koefisien determinasi menunjukan kemudahan (X₁) dan kualitas informasi (X₂) secara bersama-sama berpengaruh terhadap minat dalam penggunaan layanan m- banking pada bank syariah (Y) sebesar 73,1%. Hal ini menunjukan bahwa variabel kemudahan (X₁) dan kualitas informasi (X₂) secara bersama-sama berpengaruh secara signifikan terhadap variabel minat penggunaan (Y). Berdasarkan uji koefisien determinasi menunjukan kemudahan (X₁) dan kualitas informasi (X₂) secara bersama-sama berpengaruh terhadap minat dalam penggunaan layanan m- banking pada bank syariah (Y) sebesar 73,1%. 69 Jurnal Al-Fatih Global Mulia 70 Jurnal Al-Fatih Global Mulia DAFTAR PUSTAKA Azhar Susanto, Sistem Informasi Manajemen:Konsep dan Pengembangannya, (Bandung: Linggar Jaya, 2004), hal. 40. gg y Azhari, s. B. (2005). Analisis Statistik dengan MIcrosoft Excel dan SPSS . Yogyakarta: Azhari, s. B. (2005). Analisis Statistik dengan MIcrosoft Excel dan SPSS . Yogyakarta: ANDI Azis, M. (2019). Analisis Faktor-Faktor Yang Mempengaruhi Minat Menabung Di Bank Syariah . jurnal skripsi, 3. Azis, M. (2019). Analisis Faktor-Faktor Yang Mempengaruhi Minat Menabung D Syariah . jurnal skripsi, 3. Davis, F.D. 1989. Perceived usefulness, perceived ease of use, and user acceptance of information technology. MIS Quarterly. 13(3): 319-339 DeLone.W.H & McLean. E.R., 1992. Informatiom System Success: The Quest For The Dependent Variable. Pp.60-95. DeLone.W.H & McLean. E.R., 2003. The Delone And McLean Model Of Information System Success: A Ten-Year Update. Managemen Information, Pp.9-30 Dengan Metode Technology Acceptance Model”. Jurnal Pilar Nusa Mandiri Vol. IX No.2.Hal 172 Heri Sudarsono, Bank dan Lembaga Keuangan Syariah (Yogyakarta: Ekonisia, 2008), h.30 Ikatan Bankir Indonesia (IBI) dan Lembaga Sertifikasi Profesi Perbankan (LSPP), Mengenal Operasional Perbankan 1, (Jakarta: PT Gramedia Pustaka Utama, 2014), hal. 59. Imam Ghozali, Aplikasi Analisis Multivariate dengan Program SPSS (Semarang: Universitas Diponegoro, 2013) Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi), hal. 111 Kasmir, Bank dan Lembaga Keuangan Lainnya, (Jakarta: Rajawali Pers, 2012), h. 166 g y ( ) g p , ( gy ), Kasmir, Bank dan Lembaga Keuangan Lainnya, (Jakarta: Rajawali Pers, 2012), h. 166 Kasmir, Dasar-Dasar Perbankan (Jakarta: PT RajaGrafindo Persada, 2002), h.215 Muhammad, Metodologi Penelitian Ekonomi Islam: Pendekatan Kuantitatif, (Jakarta: Rajawali Press, 2013), hal. 161. Narbuko, C. (2009). Metodologi Penelitian . Jakarta : PT BumiAksara Paturohman, I. (2019). “SPSS 25 Main Subject: Linear Regression Analysis”. Bandung. 69 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... 70 Jurnal Al-Fatih Global Mulia
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Low-head pumped hydro storage: A review of applicable technologies for design, grid integration, control and modelling
Renewable & sustainable energy reviews
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Citation for the original published paper (version of record): Hoffstaedt, J., Truijen, D., Fahlbeck, J. et al (2022). Low-head pumped hydro storage: A review of applicable technologies for design, grid integration, control and modelling. Renewable and Sustainable Energy Reviews, 158. http://dx.doi.org/10.1016/j.rser.2022.112119 N.B. When citing this work, cite the original published paper. research.chalmers.se offers the possibility of retrieving research publications produced at Chalmers University of Technology. It covers all kind of research output: articles, dissertations, conference papers, reports etc. since 2004. research.chalmers.se is administrated and maintained by Chalmers Library research.chalmers.se offers the possibility of retrieving research publications produced at Chalmers University of Technology. It covers all kind of research output: articles, dissertations, conference papers, reports etc. since 2004. research.chalmers.se is administrated and maintained by Chalmers Library A R T I C L E I N F O Keywords: Low-head pumped hydro storage Energy storage Grid stability Renewables integration Energy transition Reversible pump-turbine To counteract a potential reduction in grid stability caused by a rapidly growing share of intermittent renewable energy sources within our electrical grids, large scale deployment of energy storage will become indispensable. Pumped hydro storage is widely regarded as the most cost-effective option for this. However, its application is traditionally limited to certain topographic features. Expanding its operating range to low- head scenarios could unlock the potential of widespread deployment in regions where so far it has not yet been feasible. This review aims at giving a multi-disciplinary insight on technologies that are applicable for low-head (2-30 m) pumped hydro storage, in terms of design, grid integration, control, and modelling. A general overview and the historical development of pumped hydro storage are presented and trends for further innovation and a shift towards application in low-head scenarios are identified. Key drivers for future deployment and the technological and economic challenges to do so are discussed. Based on these challenges, technologies in the field of pumped hydro storage are reviewed and specifically analysed regarding their fitness for low-head application. This is done for pump and turbine design and configuration, electric machines and control, as well as modelling. Further aspects regarding grid integration are discussed. Among conventional machines, it is found that, for high-flow low-head application, axial flow pump-turbines with variable speed drives are the most suitable. Machines such as Archimedes screws, counter-rotating and rotary positive displacement reversible pump-turbines have potential to emerge as innovative solutions. Coupled axial flux permanent magnet synchronous motor-generators are the most promising electric machines. To ensure grid stability, grid-forming control alongside bulk energy storage with capabilities of providing synthetic inertia next to other ancillary services are required. Low-head pumped hydro storage: A review of applicable technologies for design, grid integration, control and modelling Low-head pumped hydro storage: A review of applicable technologies for design, grid integration, control and modelling J.P. Hoffstaedt a,∗, D.P.K. Truijen b, J. Fahlbeck c, L.H.A. Gans d, M. Qudaih e, A.J. Laguna a, J.D.M. De Kooning b, K. Stockman b, H. Nilsson c, P.-T. Storli d, B. Engel e, M. Marence f, J.D. Bricker g,h a Faculty of Mechanical, Maritime and Materials Engineering, Department of Maritime and Transport Technology, Delft University of Technology, Mekelweg 2 2628 CD Delft The Netherlands a Faculty of Mechanical, Maritime and Materials Engineering, Department of Maritime and Transport Technology, Delft University of Technology, Mekelweg 2, 2628 CD Delft, The Netherlands f Electromechanical Systems & Metal Engineering, Ghent University & FlandersMake@UGent - Corelab EEDT-MP, Sint-Martens-Latemlaan rijk, Belgium b Department of Electromechanical Systems & Metal Engineering, Ghent University & FlandersMake@UGent - Corelab EEDT-MP, Sint-Martens-Latemlaan 2B, 8500 Kortrijk, Belgium c Department of Mechanics and Maritime Sciences, Division of Fluid Dynamics, Chalmers University of Technology, 412 96 Gothenburg, Sweden d Department of Energy and Process Engineering, Waterpower Laboratory, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway e Elenia Institute for High Voltage Technology and Power Systems, Technische Universität Braunschweig, Schleinitzstraße 23, 38106 Braunschweig, Germany f IHE Delft Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands g Department of Hydraulic Engineering, Hydraulic Structures and Flood Risk, Delft University of Technology, The Netherlands h Department of Civil and Environmental Engineering, University of Michigan, 2350 Hayward, Ann Arbor, MI 48109-2125, USA Contents lists available at ScienceDirect Contents lists available at ScienceDirect 1364-0321/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). p // g/ /j Received 8 September 2021; Received in revised form 17 December 2021; Accepted 9 January 2022 E-mail address: J.P.Hoffstaedt@tudelft.nl (J.P. Hoffstaedt). (article starts on next page) Renewable and Sustainable Energy Reviews 158 (2022) 112119 Renewable and Sustainable Energy Reviews 158 (2022) 112119 1364-0321/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:/ Received 8 September 2021; Received in revised form 17 December 2021; Accepted 9 January 2022 A R T I C L E I N F O Abbreviations: ADRC, Active Distribution Rejection Control; AF-PMSM, Axial Flux Permanent Magnet Synchronous Machine; ANN, Artificial Neural Network; AS, Ancillary Services; CAES, Compressed Air Energy Storage; CRPT, Counter-Rotating Pump-Turbine; DSO, Distribution System Operator; DSSR, Double-Stator Single-Rotor; DTC, Direct Torque Control; EIA, Energy Information Administration; ESHA, European Small Hydropower Association; ESOEI, Energy Storage On Energy Invested; ESS, Energy Storage System; FOC, Field Oriented Control; IRES, Intermittent Renewable Energy Source; LCOS, Levelised Cost Of Storage; MEPT, Maximum Efficiency Point Tracking; MMD, Modular Machine Drive; MPC, Model Predictive Control; MPPT, Maximum Power Point Tracking; MTPA, Maximum Torque Per Ampere; PAT, Pump As Turbine; PHS, Pumped Hydro Storage; PLL, Phase Locked Loop; PM, Permanent Magnet; PMSM, Permanent Magnet Synchronous Machine; PTO, Power Take-Off; PWM, Pulse-Width Modulation; RPT, Reversible Pump-Turbine; SSDR, Single-Stator Double-Rotor; SSSR, Single-Stator Single-Rotor; SVM, Space Vector Modulation; TSO, Transmission System Operator ∗Corresponding author. Received 8 September 2021; Received in revised form 17 December 2021; Accepted 9 January 2022 1. Introduction and analyse technologies based on their applicability for low-head utilisation. First, an overview of PHS and its historical development is given. Based on this, recent trends leading to further innovation in the field are identified, and the potential and necessity of storage technolo- gies are discussed. Finally, technological and economic challenges are explored, and the key advancements that could contribute to economic and technical viability are isolated. Building on this, in the major tech- nological fields – pump-turbine design and configuration, control and electric machinery, as well as modelling – the most promising technolo- gies are compared and their fitness for low-head application is assessed. Additionally, implications of grid integration are discussed, including further elaboration on the significance of integrating large-scale energy storage, such as low-head PHS into world-wide grids. In a global effort to reduce greenhouse gas emissions, renewables are now the second biggest contributor to the world-wide electricity mix, claiming a total share of 29% in 2020 [1]. Although hydropower takes the largest share within that mix of renewables, solar photo- voltaics and wind generation experience steep average annual growth rates of 36.5% and 23%, respectively, since 1990 [2]. Both of these technologies, however, significantly differ in their generation char- acteristics when compared to traditional thermal power plants. This trend towards an increase in intermittent generation, coupled with a reduction in spinning reserves, could undermine grid stability. To counteract these effects, grid-scale deployment of energy storage is indispensable. There are complementary approaches to balance demand and sup- ply in an electricity grid, such as an increase in flexible generation, demand management, or exporting and importing electricity. Nonethe- less, at certain penetration levels of renewables, to reduce the risk of grid instability, a heterogeneous pool of storage solutions is needed. A wide variety of such storage technologies – including capacitors, flywheels, electro-chemical batteries, compressed air energy storage (CAES), molten-salt or hydrogen storage – is available to balance the grid in the timescale from seconds up to seasonal variations. Crucial factors for large-scale balancing include energy and power capacity as well as fast response times while maintaining high efficiencies. Aside from fulfilling these criteria, the major driver towards commercial deployment is the levelised cost of storage (LCOS); leading in this are pumped hydro storage (PHS) and CAES [3]. 2. Overview and historical development of pumped hydro storage Pumped hydro storage is an amended concept to conventional hydropower as it cannot only extract, but also store energy. This is achieved by converting electrical to potential energy and vice versa in the form of pumping and releasing water between a lower and a higher reservoir. The energy conversion occurs by using pumps and turbines either combined in a reversible (binary set) or separate configuration (ternary and quaternary sets). The power of such a system, as well as the amount of energy that can be extracted or stored, is proportional to the product of head and water flow or volume, respectively. Hence, a higher head results in a reduced flow for a given desired power and smaller reservoirs for a given storage capacity. It does not, therefore just correlate with scaled-down reservoirs but also smaller remaining civil structures and machinery, historically leading to reduced cost and a significant economic advantage of utilising high-head differences [7]. Of today’s bulk energy storage integrated into the world-wide grids, over 90% is comprised of PHS of which the vast majority are high-head applications. According to the International Hydropower Association, in 2019, the global installed capacity reached 158 GW with the biggest contributors being China making up 30.3 GW of the share, Japan 27.6 GW, and the United States 22.9 GW [8]. Fig. 2 shows the distribu- tion of global storage capacity that is operational, under construction, planned, and announced as of 2021. Pumped hydro storage is a mature and well-known technology that has been used since the beginning of the 20th century. In 2020, it contributed with 90.3% of the world’s energy storage capacity [5]. However, while some regions reach the limits of economically viable PHS that can be implemented, others lack entirely the necessary to- pographic features. Traditional PHS relies on high heads to realise the expected power and storage capacity. Most of the plants produce in the order of 1000–1500 MW of power, with round-trip efficiencies which are commonly in the range of 70%–85% [6]. In comparison, the next largest contributors to bulk energy storage are electro-chemical battery storage – rapidly growing with a total capacity of 14.2 GW – and thermal storage with 2.9 GW in 2020 [5]. To explain the historic market dominance of PHS and understand recent trends, several factors have to be taken into account. 2. Overview and historical development of pumped hydro storage Pumped hydro storage utilising reversible pump-turbines has been available as a mature and cost-effective solution for the better part of a century with an estimated energy based capital cost of 5–100 $/kWh [10]. To- day, compressed air energy storage is considered mature and reliable, offering similarly low capital cost between 2–50 $/kWh, and electro- chemical batteries offer high energy density with higher costs, and experience drastic growth while the impact of hydrogen-based storage in the energy transition is largely expected to be substantial [10]. Aside from its use to store energy, hydropower is regarded as the foremost renewable generation method when it comes to flexibility and improving grid stability. Due to the proven advantages of hydro- electric power generation, wide-ranging research efforts have focused on conceptual adaptations and technological advancements utilising low- and ultra-low-head scenarios. Some of these technologies, such as wastewater, run-of-river hydropower, or tidal barrages have seen pro- totyping and commercial deployment. However, theoretical attention and practical implementation towards low-head PHS has been limited. Fig. 1 shows a conceptual drawing of what such a system may consist of when deploying a reversible pump-turbine coupled to a motor- generator that is connected to the grid via an AC-DC-AC converter for variable speed operation. However, PHS‘s dominance is not only due to its historic lead but can also be attributed to its technical, economic, and sustainability advantages. These include high efficiencies, large achievable capacities, and long lifetimes. Compared to rapidly expanding battery storage that can be used wherever it is most needed, one clear advantage is this durability. It is currently assumed that a battery system will last around 15–20 years, but on the other hand, the oldest hydropower plant in Norway has been operating for over 120 years [11,12]. Prolonged lifetimes are one factor improving the sustainability of PHS compared to other storage solutions. Others include maturity, low capital and operating cost, as well as low energy and carbon dioxide density. Based on these and other factors regarding economic, performance, technological, and environmental considerations, Ren et al. ranked PHS as the most sustainable storage technology [13]. The lack of attention on low-head PHS can be partly explained through high levelised cost of storage (LCOS) caused by extensive civil structures, enlarged machinery, lower round-trip efficiencies, and lim- ited flexibility to provide ancillary services (AS). https://doi.org/10.1016/j.rser.2022.112119 J.P. Hoffstaedt et al. J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 1. Introduction An alternative approach is based on the so-called energy stored on energy invested (ESOEI), which gives an estimate of the relation between the stored energy during the lifetime of a system and the energy required to construct the system. Also for this metric, PHS and CAES are, by far, in the lead [4]. 2. Overview and historical development of pumped hydro storage The predicted increase in demand for energy balancing and AS in the upcoming decades will likely justify increased LCOS. Additionally, technological advancements could significantly contribute to a reduction in LCOS. Addressing the technological challenges and overcoming economic barriers of low- head PHS could unlock the potential of integrating large-scale energy storage into the grids of regions where it has not been feasible so far. For the given reasons, research and development towards shifting the operating range of PHS to low heads is scarce. Using a multi- disciplinary approach, the main goal of this research is to review 2 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 2. Global PHS Capacity [9]. ther advantages of PHS include suitability for long term storage become the norm since the middle of the 20th century [6] Experie Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 2. Global PHS Capacity [9]. Fig. 2. Global PHS Capacity [9]. Further advantages of PHS include suitability for long-term storage – since hardly any storage losses occur other than seepage and evap- oration – and quick availability due to short switch-on and switch-off times. With these factors ensuring a significant share within a heteroge- neous pool of storage technologies, one major disadvantage of PHS has historically been its topographic constraints. A switch from river-based to closed-loop off-river systems could overcome some of the constraints and increase the potential for deployment [14,15]. Nonetheless, regions with flat topographies still do not offer viable sites. become the norm since the middle of the 20th century [6]. Experiencing a major boom in Europe, parts of Asia, and North America, a large portion of today‘s installed PHS capacity was constructed in the 1960s, 1970s, and 1980s; in most countries, this occurred alongside rapidly in- creasing nuclear power. The gained flexibility that PHS plants provided allowed them to match a varying demand with the baseload generation of nuclear power plants. 2. Overview and historical development of pumped hydro storage An example where this was particularly rele- vant is Japan due to its lack of interconnections to other countries as well as their strong strategy towards nuclear energy. In the United States, another reason for the growing capacity of PHS during that period was the energy crisis in the 1970s, leading to an increased cost of fossil fuels allowing PHS to grow as a substi- tute for peak balancing [16]. After that period, development slowed down in most regions with the major exception of China. Its rapidly growing economy and correlated energy demand largely satisfied by non-flexible coal plants required major energy storage. 2.3. Potential of deployment and scalability in Europe Resulting from the rapid transition that grids are experiencing worldwide, the need for energy storage is evident. However, there are a variety of factors influencing the actual storage demand and its ex- pected progression during the coming decades. First and foremost, this is the growth in intermittent and converter coupled renewables. While a direct correlation between renewable penetration levels and storage demand can be assumed, mitigating factors such as improved genera- tion forecasting and the continuous development of renewables able to provide AS will allow for later deployment of energy storage. Further factors to consider include the flexibility of remaining generators in the grids and the emerging need for additional operating reserves, improved demand management, as well as further grid expansion and interconnection. The second major trend is expanding the operating range and application. One of the most limiting factors in the potential use of large-scale PHS has been the fact that not many locations could offer economically viable deployment. These were traditionally mountainous regions accessing water with enough space to construct extensive civil structures. There is a large potential in Europe to deploy further mini and small hydropower plants to counteract the effects of higher renew- able penetration levels. However, this does not apply to countries with flat topographies, such as Denmark, the Netherlands, or Belgium [21]. Additionally, to achieve the balancing capabilities of pumped storage systems, larger plants typically provide better economies of scale. Suitable locations for such are rare in Europe and some countries like Japan are considered to have used nearly all available sites [22]. This limited availability of appropriate locations drives the development of new approaches. Examples for a promising change of approach are underwater PHS or gravity energy storage. Bearing these factors in mind, it becomes clear that storage demand will heavily depend on individual grid characteristics and may vary in different regions. Based on Germany as an example, additional short-term storage can be expected at renewable shares between 40% and 60% and long-term storage between 60% and 80%. Above 80% and towards a fully renewable generation, bulk energy storage on all timescales is not only required in order to avoid extensive renewable energy curtailing, ensure grid stability and power quality, but will be a cost-effective solution in the GW range [29]. J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 to low and ultra low-head applications, including the potential use of seawater, while improving its capability to provide AS. If technological advancements allow for economic viability, large-scale low-head PHS could be integrated in regions where PHS so far was not a feasible solution. Up until this point, pump-turbines were coupled to fixed speed motor-generators. The next significant development occurred in the 1990s when variable speed operation was introduced in Japan. The ability to adjust the angular velocity of the runners allowed for higher efficiencies under changing conditions, reduced the switching time between pump and turbine mode, and facilitated higher ramp rates and quicker response times [17]. Later trends for PHS show the usage of ternary units. With ternary units, a separate pump and turbine are connected on a single shaft to an electric machine that can work either as a motor or a generator [26]. This configuration presents a very flexible and fast response range, shows higher efficiencies than reversible machines, and can utilise hydraulic short circuits for optimal power in- or outtake [27,28]. The major drawback with ternary units is that they requires higher investment and maintenance costs compared to a reversible unit [28]. In a low-head scenario, the increase in investment cost would be even greater since a high-power, low-head machine needs to be large in order to handle a high flow rate. Thus, it is suggested as a less attractive alternative for low-head PHS. 2.2. Recent trends After an initial reduction of PHS deployment around the turn of the millennium, the rapidly growing share of intermittent renewable energy sources (IRES) in the last couple of decades sparked new interest in sustainable flexible generation as well as large-scale energy storage solutions. This caused increasing attention towards the rehabilitation of old hydropower plants and an expansion of PHS [18]. While PHS experienced a much longer development process than competitive tech- nologies and could hence be considered mature, two major trends can be identified pushing further innovation in the field. The first one is derived from the change in grid characteristics caused by a reduction in spinning reserves. A growing number of converter coupled renewables raise the necessity for external provision of AS. To provide these using PHS, research efforts focus on devel- oping improved control and machinery but also novel concepts, such as hybrid storage solutions. Examples of such concepts could be the coupling of conventional PHS with flywheels for frequency control or supercapacitors providing virtual inertia [19]. Hybrid storage solutions incorporating PHS, such as hybrid pumped and battery storage, are also particularly suited for off-grid applications [20]. 2.1. Early deployment and progression in the 𝟐𝟎th century Not long after hydropower began to generate electricity, the first small-scale PHS plants were constructed in the mountainous regions of central Europe in the beginning of the 20th century. Initially using separate pumps and turbines, combined reversible pump-turbines have 3 J.P. Hoffstaedt et al. 2.4. Technological and economic challenges Radial- or mixed-flow machines are preferable for pumps with a specific speed of 𝛺s < 2.7, and as the specific speed increases (2.6 < 𝛺s < 11.6), an axial configuration is more suitable [45]. Here, 𝛺is the runner rotational speed in rad/s, 𝑄is the volumetric flow rate in m3/s, and 𝐻is the head in metre. Stepanoff [42] explained in 1948 that a higher specific speed results in a smaller, and thus cheaper, machine. With a higher flow rate, the blade design differs significantly, as illustrated in Fig. 3. On the other hand, machines with low rotational speeds and small shear forces (e.g. Archimedes screw and positive displacement machines) are more fish-friendly [43,44]. Radial- or mixed-flow machines are preferable for pumps with a specific speed of 𝛺s < 2.7, and as the specific speed increases (2.6 < 𝛺s < 11.6), an axial configuration is more suitable [45]. Based on these challenges, three main areas can be identified where significant progress could contribute to making low-head PHS techni- cally and economically competitive. These are pump-turbine design and configuration, grid integration, and electrical machines and control. Research in these fields will be discussed in the chapters following. Additionally, modelling approaches that may aid the development are compared. Carravetta et al. [46] postulate that axial-flow pumps can be used as PAT for heads between 1–5 m and flow rates up to 1000 l/s. They also claim that mixed-flow PATs can be used for heads in the region of 5–15 m and flow rates of 50–150 l/s. Bogenrieder [47] stated that radial pump-turbines are suitable to use for heads that are above 60 me- tre, with a power exceeding 50 MW. Typically, radial- or mixed-flow machines work best for high heads and low flow rates. For exam- ple, regular Francis-like pump-turbines (mixed-flow) are the common choice when it comes to mid- to high-head applications, but the head variations at low-head operation would greatly affect efficiency [48]. Mixed-flow machines can be used as low-head PHS if the flow rate is low. However, according to Eq. (1), this implies that the power will also be low. Multiple machines could be used in parallel to increase the total power. Breeze [48] suggests that a Deriaz, mixed-flow machine can be used for heads between 20–100 m. 2.4. Technological and economic challenges The definition of low-head is not unanimous among different coun- tries and researchers. For example, the U.S. Energy Information Admin- istration (EIA) considers low-head when the head is less than 30 metre and Okot [36] classifies it as when 5 < 𝐻< 15 metre. In this work, the European Small Hydropower Association (ESHA) classification defines low-head, and states that low-head hydropower plants have a head of 2-30 metre [37]. Low-head PHS has not yet seen deployment on a significant scale within our grids. This is largely due to the increased upfront cost required. While highly dependent on the individual project and site, the major contributor to the initial CAPEX of PHS projects in general are civil structures, including the reservoir, penstock, and lining [31]. Due to larger masses of water being stored in the reservoir and flowing through the penstock when it comes to low-head applications, their contribution to the overall economic viability can only be assumed to be significant. All aspects regarding civil components as well as detailed economic analyses of low-head PHS systems are, however, outside of the scope of this review. The overall efficiency of a low-head power plant is more sensitive to head losses than a high-head alternative, and low-head PHS requires that the pipelines are short to be economically feasible [38]. This is because head losses are proportional to the pipeline length and the flow velocity squared, which is a further incentive for not using ternary units in a low-head case since they require more pipelines and would thus decrease the plant‘s overall efficiency. With the higher flow rate of high-power low-head PHS, larger reservoirs are required to store the same amount of energy as a corresponding high-head application [34]. This is because the energy storage capacity is a function of the water mass and head. Apart from that, other conditions such as the type of machine (radial-, mixed-, or axial-flow), operation (variable or fixed speed), and reservoir configuration may apply when choosing the best reversible pump-turbine configuration [39]. Chapallaz et al. [40] stated that, in practice, almost any hydro pump can also be used as a turbine. The reverse is, however, not the case. As an example, impulse turbines (Pelton or Turgo) cannot be used as pumps. Low-head PHS would be most competitive utilising a storage ca- pacity able to provide balancing in the timescale of hours to days. 2.4. Technological and economic challenges This places it in the middle between lithium-ion batteries appropri- ate for shorter and hydrogen storage appropriate for longer periods. The LCOS of new high-head PHS systems ranges from 50 e/MWh to 80 e/MWh [32]. Initially low-head plants may not be able to compete with this. However, changing demand characteristics of the electricity markets, as well as further development and improvement of techno- logical aspects, significantly influence economic viability and thereby the potential of large-scale deployment. The increase in renewables in world-wide grids will lead to rising demands, not just for short- and long-term balancing but also the provision of AS increasing the value of both. Additional revenue from the provision AS could hence increase economic viability. The design and characteristics of any hydro pump and turbine are determined by its conditions of operation. In turbomachinery, the specific speed is one key parameter to select the most appropriate reversible pump-turbine or using a pump as turbine (PAT). In this paper, it is defined in accordance with Dixon and Hall [41] as Eq. (2), and Table 1 shows ranges of specific speeds for various machines. Further drivers making PHS economically more attractive are grow- ing interconnections to other grids opening up additional markets as well as technical advances such as higher efficiencies across a broader operating range [22]. Technological progression can help fa- cilitate these drivers. A reduction of switching times between pump and turbine mode together with higher power ramp rates will allow for enhanced capabilities to provide AS as well as maximise balancing. Aside from regulatory changes, such as carbon taxation, electricity price margins have been identified as one of the major drivers towards PHS utilisation. Improved round-trip efficiencies directly correlate with higher revenues for the operator and therefore result in increased utilisation [33]. 𝛺s = 𝛺𝑄1∕2 (𝑔𝐻)3∕4 (2) 𝛺s = 𝛺𝑄1∕2 (𝑔𝐻)3∕4 (2) Here, 𝛺is the runner rotational speed in rad/s, 𝑄is the volumetric flow rate in m3/s, and 𝐻is the head in metre. Stepanoff [42] explained in 1948 that a higher specific speed results in a smaller, and thus cheaper, machine. With a higher flow rate, the blade design differs significantly, as illustrated in Fig. 3. On the other hand, machines with low rotational speeds and small shear forces (e.g. Archimedes screw and positive displacement machines) are more fish-friendly [43,44]. 2.4. Technological and economic challenges This is because its design is closer to an axial machine compared to a conventional Francis-like pump- turbine and the Deriaz design also presents adjustable blades [48,49]. Breeze further expresses the necessity of variable speed drives to extend the operational region at high efficiency. 3. Pump-turbine design and configuration for low-head pumped hydro storage The choices when selecting the type of reversible pump-turbine (RPT) unit, or evaluating using a pump as turbine (PAT), are governed by a number of factors. The first thing to evaluate is the power of the hydropower plant, which is a function of head and flow rate and the general formula is given by Eq. (1). (1) 𝑃= 𝜌𝑔𝐻𝑄𝜂 (1) 2.3. Potential of deployment and scalability in Europe Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 high volumetric flow rate are also regarded as having great potential to unleash new opportunities for pumped hydro storage [35]. 2.3. Potential of deployment and scalability in Europe In less flexible grids, for example utilising large-scale nuclear power to cover the base load, the need for extensive storage will be reached at much lower renewable levels. The former is a recently developed and tested concept based on submerging a hollow sphere offshore and using the static pressure difference for energy storage. The surrounding sea acts as the upper reservoir and the sphere as the lower which can be filled to generate electricity or emptied to store it. Initial model-scale tests have been successful; it is a freely scalable technology without issues regarding land use and considered cost competitive with PHS and compressed air storage [23]. Using seawater in general for PHS is so far an un- common practice, but has been investigated as a solution for isolated grids [24]. If technical, environmental, and economic challenges are overcome, utilising seawater could be another promising expansion of PHS’s potential deployment. An update to the European Green Deal has raised the ambition to reduce greenhouse gas emission by 55% until 2030 compared to the standard of 1990 paving the way for a carbon neutral energy supply by 2050. Consequently, the share of intermittent renewables will need to increase faster. Recent estimates see a growth towards 70% renewable power generation in 2030 [30]. Depending on the flexibility of this share of renewables in different regions, a rapid increase in demand for storage and the provision of AS can be expected with PHS being a promising candidate to fill the gap. For countries with a flat topography, economically viable low- head PHS could bear a huge potential to cover the growing demand. This is especially relevant if a large coastline is available, opening up the possibility for seawater application. Coastal applications also come with the additional benefit of proximity to large-scale IRES, such as offshore wind farms. The latter similarly decouples the fundamental principle of PHS from its topographic restrictions. Storage is done via gravitational potential energy. However, energy is stored or extracted respectively by moving a piston of large mass up and down using water powered by a pump-turbine for conversion. While still under development, initial economic evaluations show it to have a potentially attractive LCOS compared to other storage technologies [25]. An alternative that is not less promising and will potentially suit both these trends is to extend the operating range of conventional PHS 4 J.P. Hoffstaedt et al. J.P. Table 1 stated that centrifugal machines have preferable efficiency as a function of flow rate; however, axial machines have a flatter efficiency curve as a function of head. This further suggests that an axial machine is preferable in a low-head scenario due to the fact that part-load operations will be less influenced by the large variation in head of a low-head PHS application. Ranges of specific speed for various machines [45]. Lately, axial-flow pump-turbines with two runners, rotating in op- posite direction from one another, have been proposed as an alternative for low-head PHS. They are usually referred to as counter-rotating pump-turbines (CRPT) due to the rotation of the individual runners, as illustrated in Fig. 4. According to Furukawa [55], the advantages of those machines are that they can be of smaller size, have a more stable head-flow rate characteristic curve, and have a wider range of high efficiency with individual speed control of the runners when compared to a single runner axial machine. Several numerical studies predict that a well designed low-head counter-rotating pump-turbine may achieve efficiencies of up to 80%–90% in both pump and turbine mode [56–58]. Fahlbeck et al. [59] showed numerical results for a prototype counter- rotating pump-turbine in pump mode with a peak efficiency of 91%, heads up to 12 metre, flow rates between 60–160 m3/s, and a maximum power of almost 14 MW. bValues should be regarded only as a reference number, since it is not common to indicate a specific speed for these machines. for a machine with high power at low heads. An axial machine also allows for cheaper civil structures. In turbine mode, the runner rotates due to the torque that is generated by the flow-induced runner blade pressure and suction sides. The electric generator extracts power by a balancing counteracting torque at the particular rotational speed. In pump mode, an electric motor adds power to the runner in the form of torque at the particular rotational speed. A flow is developed due to the rotating runner blade pressure and suction sides, causing a balancing counteracting torque. The pressure change is in an axial machine primarily due to the change of relative flow velocity [45]. This is because the tangential velocity of the runner and the cross- sectional area are constant along a streamline in an axial machine. Table 1 In a centrifugal machine, the flow must change direction from axial to radial (pump mode), or radial to axial (turbine mode), as shown in Fig. 3. The principles for the head rise in a centrifugal machine are here described in pump mode for brevity. As the flow goes through the machine, the cross-sectional area and the tangential velocity of the runner increase with the radius through the machine. The absolute flow velocity will decrease as the cross-sectional area increases, due to continuity. According to Bernoulli’s principle, the static pressure will increase with the change of absolute velocity squared [50]. The main cause of the static pressure rise is, however, due to centrifugal effects caused by the increase of the runner’s tangential velocity, and passage diffusion, due to a reduction in the relative flow velocity, through the machine [45]. The result is that the exit blade velocity needs to be small in order to limit the pressure rise in a low-head application. This means that the machine needs to be small and that the entrance-to- exit diameters decrease with the decreasing head [51]. The smaller size further limits the flow rate and thus the power. Additional non-conventional machines have also been studied as low-head PHS. The Archimedes screw, depicted in Fig. 4, is a viable option for heads between 2–10 m, discharge ranges up to 15 m3/s, and power output of up to 355 kW [36,43,60]. The Archimedes screw enables lower installation and maintenance costs compared to other conventional pump-turbines and can reach efficiencies of up to 90% in turbine mode [61,62]. An additional benefit is that the Archimedes screw presents better conditions for fish-friendliness when compared to conventional bladed pump-turbines [43,61,63]. Positive displacement (PD) pumps are usually chosen when the system requires low specific speeds [45]. Some PD pumps can also represent a good alternative when reversible flows must be taken into account, thus resembling a PAT [64]. Positive displacement pumps are self-priming, typically produce low flow rates, can handle big variations in head without significantly changing their efficiency, and are often regarded as a good choice for viscous fluids or fluids with the presence of solids or precipitates that need to be handled [45,50]. Rotary positive displacement machines have already been studied as micro hydro turbines in water supply pipelines with pressures up to 5 bar (hydraulic head equivalent to 51 metre) and presented efficiencies between 60%– 80% [65–68]. 𝑃= 𝜌𝑔𝐻𝑄𝜂 Here, 𝜌is the density of water, 𝑔is the gravity acceleration, 𝐻is the head, 𝑄is the volumetric flow rate, and 𝜂is the overall efficiency of the power plant. The gravity acceleration and water density can be regarded as constant. The equation shows that if the head is low, the flow rate must be large in order to produce high power [34]. With a large flow rate, the diameter of pipelines and the runner need to be large as well to limit the flow velocity, and thus hydraulic losses in the system. High-head conditions are usually preferable to build pump storage hydropower plants. However, low-head solutions with The reason why an axial machine is preferable in a low-head application is that it allows for a higher flow rate, which is necessary 5 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Renewable and Sustainable Energy Reviews 158 (2022) 112119 J.P. Hoffstaedt et al. Fig. 3. Principle view of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles shown in [40–42,46]. Fig. 3. Principle view of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles shown in [40 42 46] Fig. 3. Principle view of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles shown in [40–42,46]. iew of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is bas mp-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles sh Table 1 Ranges of specific speed for various machines [45]. Technology Specific speed 𝛺𝑠 Axiala 2.6–11.6 Mixed 0.6–2.7 Radial 0.1–0.8 Archimedes screwb 0.03–0.39 Positive displacementb 0.01–0.13 aCRPT is classified as an axial machine. bValues should be regarded only as a reference number, since it is not common to indicate a specific speed for these machines. 4. Grid integration of energy storage systems A reliable electrical power grid is a balanced system. As generation and demand fluctuate perpetually, transmission system operators (TSO) and distribution system operators (DSO) have to keep the system balance everywhere in the electrical grid. This balance ensures that the grid operates at its nominal frequency (50 or 60 Hz) and that voltage and power load remain within a certain limit at all times. However, the higher the penetration of intermittent renewable energy sources, the more insecure this balance. Thus, the increasing penetration of IRES is a challenge that TSO and DSO have to handle [71,72]. At the present time, power systems rely on conventional power plants utilising synchronous generators contributing significantly to the stabilisation of the electrical power system, using the rotating masses in their generators (rotors). The synchronous generators keep the frequency steady at its nominal value due to their large flywheel masses and thereby assure system stability. In the case of generation or load fluctuation leading to sudden grid frequency deviations, the rotor’s combined inertia keeps the generators rotating and consequently supports the grid stability [73]. On the contrary, not all IRES have large rotating masses and most are integrated into the grid via converters, subsequently decoupling the rotating masses from the grid frequency. Therefore, they do not have any natural inertia (spinning reserve) and thus operate in an entirely different way than synchronous generators. As of today, the grid-connected converters for IRES follow the grid frequency by using a phase locked loop (PLL). This tracks the grid frequency in order to keep the IRES converters synchronised to the grid. The PLL control concept is known as grid-following control [73,74]. 𝑃m −𝑃e = 𝐽𝜔0 d𝜔 d𝑡 (3) 𝜔= d𝜗 d𝑡 (4) 𝑃m −𝑃e = 𝐽𝜔0 d𝜔 d𝑡 (3) 𝜔= d𝜗 d𝑡 (4) 𝑃m −𝑃e = 𝐽𝜔0 d𝜔 d𝑡 (3) 𝜔= d𝜗 d𝑡 𝜔= d𝜗 d𝑡 (4) Here, 𝑃m is the mechanical power, 𝑃e is the electrical power, 𝜔0 is the nominal angular frequency, 𝜔is the output angular frequency, 𝜗 is the rotation angle, and J is the total moment of inertia of the rotor mass. Fig. 5 shows a simplified block diagram for a synthetic electrical inertia control system. As illustrated, ESS are needed along the grid- forming control to provide the necessary synthetic electrical inertia. Table 1 From all the available PD alternatives, the lobe and gear pump configurations – illustrated in Fig. 4 – are the most suitable options to handle reversible flow. Given the low specific speed, PD pumps could most likely be regarded as a fish-friendly technology [44]. On the other hand, only the lobe design seems to handle fish and solid without extra mitigation measures. A few small-scale projects have tested PD RPTs [64]. However, further investigations and real-scale In general, pump-turbines are worse at part-load conditions com- pared to a pure pump or turbine since the pump-turbine design is often a trade-off to reach acceptable performance at design condi- tions [52,53]. Delgado et al. [54] reported that it is hard to predict part-load performance for PATs, especially in turbine mode since pump manufacturers usually do not supply any data of this. Stepanoff [42] 6 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Renewable and Sustainable Energy Reviews 158 (2022 Hoffstaedt et al. Fig. 4. Principle view of non-conventional pump-turbine configurations. The drawing is based on principles showed in [45,55,56]. tions. The drawing is based on principles showed in [45,55,56]. Fig. 5. Simplified block diagram for synthetic electrical inertia control [80,81]. Here, 𝛥f is the deviation of system frequency, ROCOF is the rate of change of frequency, J is the virtual inertia control constant, 𝛥𝑃imitate is the active power of the converter, and 𝛥𝑃inertia is the emulated virtual inertia power that could be imitated into the system. Fig. 4. Principle view of non-conventional pump-turbine configurations. The drawing is based on principles showed in [45,55,56]. Fig. 5. Simplified block diagram for synthetic electrical inertia control [80,81]. Here, 𝛥f is the deviation of system frequency, ROCOF is the rate of change of frequency, J is the virtual inertia control constant, 𝛥𝑃imitate is the active power of the converter, and 𝛥𝑃inertia is the emulated virtual inertia power that could be imitated into the system. prototypes are still needed to validate the use of PD RPTs in low-head pumped storage application. Pump-turbines in PHS applications can operate at fixed or vari- able rotational speed. Variable speed machines take advantage of a wider operating range at high efficiency and can thus produce power in a wider spectrum [38,69]. Moreover, variable speed units ensure greater penetration and bring more flexibility to PHS operations, es- pecially for smaller machines [70]. Table 1 Despite the technical advantages, this technology is about 30% more expensive than fixed speed units. Thus, the choice between the two speed control options relies on both techno-economic and demand aspects [38,70]. Fig. 5. Simplified block diagram for synthetic electrical inertia control [80,81]. Here, 𝛥f is the deviation of system frequency, ROCOF is the rate of change of frequency, J is the virtual inertia control constant, 𝛥𝑃imitate is the active power of the converter, and 𝛥𝑃inertia is the emulated virtual inertia power that could be imitated into the system. controlled converter acts as an AC voltage source with stated voltage, phase, and frequency. By controlling the voltage magnitude and fre- quency, the converter behaves very similar to a synchronous generator. The fundamental difference between grid-following and grid-forming is the way of synchronisation. By applying the swing Eqs. (3) and (4) [77], the grid-forming control strategy calculates the voltage an- gle and amplitude deviation, using current power transfer. It is thus self-synchronising. Therefore, a converter using grid-forming control coupled with an ESS is currently being discussed as a viable alternative to imitate the synchronous generator‘s behaviour regarding frequency control, especially its ability to provide synthetic electrical inertia [73, 74,78]. In power plants with rotating mass and consequent inertia that are decoupled from the grid frequency, an additional control loop is required that gives a power reference proportional to the derivative of grid frequency. To provide the additional power requested by the synthetic inertia, the plant may still rely on the physical inertia present but due to its decoupled nature depends on said synthetic inertia control. 4. Grid integration of energy storage systems This shows that ESS are an important factor in the energy transi- tion and will play a key role in the future. Energy storage systems will provide inertia for local grid stability as well as other necessary AS, such as steady state voltage control, fast reactive current injec- tions, short-circuit current, black start capability, and island operation capability [79]. To tackle the challenge of increasing IRES and decreasing natural system inertia without affecting the system stability, two approaches are feasible. The first is to maintain a minimum number of rotat- ing machines. Among other purposes, the contribution of short-circuit power and voltage support can provide the necessary inertia to the transmission system in a case of disturbances in the grid [75,76]. The second solution is through IRES itself. This occurs by using the capabilities of the power electronics, or energy storage systems (ESS), to provide and ensure a stable grid frequency without any synchronous rotating machines. For this purpose, a grid-forming control mode is cur- rently being developed and tested in many research projects. Here, the Moreover, ESS will also need to compensate for weather and sea- sonal fluctuations in the power supply from IRES, specially from wind and solar power. For all the previous reasons, ESS are becoming in- creasingly important. New possibilities for medium and long-term ESS with sufficient storage capacity and flexibility, in accordance with 7 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 the respective requirements, are needed to meet the growing demand from IRES. Low-head PHS system is a power generation system and serves at the same time as an ESS. This makes the integration of PHS (low-head or high-head) via grid-forming controlled converter a vitally important milestone of the energy transition in order to provide the necessary storage capacity needed for grid stability and flexibility. The grid integration of low-head PHS via a grid-forming, controlled converter will not only be of great significance for countries with flat topographies such as Denmark, Belgium, and the Netherlands. It will also be essential for countries with a high share of offshore wind energy as these could enable the concept of energy islands. of a stator yoke and the corresponding iron losses can be averted. This can be achieved by using a north–south PM arrangement of the rotors. Then, the flux path is completely axial, obviating the magnetic function of the yoke. 5.1. Electric machines In traditional high-head, high-power PHS, synchronous machines with excitation winding and direct grid connection are used. However, doubly-fed induction machines have been adopted in Europe since 2006 for lower power applications. Doubly-fed induction machines are coupled to a partially rated converter with rotor winding to in- crease the operating range, which increases turbine efficiency at lower speeds [82]. As can be seen from Eq. (2), RPT operation at low head and high power reduces the nominal rotational speed for a fixed specific speed. Therefore, the power take-off (PTO) in low-head PHS needs to be designed to operate at high efficiency for low rotational speeds. Furthermore, variable speed RPTs require a highly efficient PTO over a wide operating range. Doubly-fed induction machines with a gearbox were the classical choice for such low-speed applications. However, with the recent decrease in cost of power electronics, permanent mag- net synchronous machines (PMSM) with a fully rated converter are opted for instead [83–90]. Advantages include a high power density, high efficiency, and controllability over a wide operating range [91, 92]. Furthermore, PMSMs with a large pole number avert the use of reduction gearing, which reduces energy losses and increases reliabil- ity [93,94]. However, the increased cost of permanent magnets (PM) and converter losses limits its application for high-power installations. It can be concluded that the PMSM is currently the most sensible electric machine technology for modern low-head PHS due to its high efficiency and direct-drive capability, although the use of rare earth materials is a drawback. The principles of axial flux design, modularity for fault tolerance and data-driven condition monitoring are likely to play a role in the further improvement of the PMSM. A more recent development in PMSMs is the axial flux PMSM (AF-PMSM), which has a magnetic flux direction parallel to the axis of rotation, in contrast to their radial counterparts. These disc-type machines have a high diameter-to-length ratio, can accommodate high pole numbers, and are suitable for high-torque low-speed applica- tions [95–97]. They have a higher power density and use less core iron, leading to a lower weight [96,98,99], which in turn results in a higher torque-to-weight ratio. The possible topologies are single-stator single-rotor (SSSR), double-stator single-rotor (DSSR), or single-stator double-rotor (SSDR). 5.1. Electric machines Furthermore, different concepts can be differ- entiated on the use of surface mounted or interior PMs, slotted or slotless armature, presence or absence of stator core, concentrated or distributed windings, etc. [100]. 4. Grid integration of energy storage systems The single-stator double-rotor topology has already been adopted in wind and tidal turbine applications [104,105]. An SSDR with coreless stator maximises efficiency, while averting cogging torque and torque ripple [106–108]. Since the flux path is axial, grain-oriented material – which has greater magnetic permeability in one direction – can be used in the stator slots. This results in significantly lower iron losses compared to non-oriented material [109], while reducing PM use compared to coreless alternatives [100]. Especially in high-voltage electric machines, the vast majority of oc- curring faults are stator faults, followed by rotor and bearing faults [110]. Therefore, AF-PMSMs with concentrated windings can offer a significant advantage by adopting a Modular Machine Drive (MMD) design. If a fault arises in one of the stator windings, the MMD can compensate this with the other modules and remain functional albeit the maximum power is reduced [111,112]. This fault-tolerant design improves the reliability of the electric machine, which is a considerable advantage for a low-head PHS system providing grid support. The reliability can be further increased by means of condition monitoring techniques and fault or anomaly detection methods [113, 114]. Thanks to the drastic increase in computational power in the past years (both local and in the cloud), these techniques have become more data-driven, relying on, e.g., machine learning [115,116], including artificial neural networks [117], support vector machines [118], and deep learning [119,120]. The use of digital twins for predictive main- tenance of mechanical components [121] or the full drivetrain [122] shows promising results and offer a perspective for the future of condition monitoring [123]. These techniques can be applied on the electric machine, and in extension on the whole drivetrain. Current, voltage, magnetic flux, speed, temperature, and vibration signals can be captured on the electric machine and serve as inputs for the condition monitoring system. 5.3.1. Maximum power point tracking algorithms Maximum power point tracking (MPPT)-based algorithms are used to find the optimal speed setpoint for a certain power setpoint in tur- bine mode. In a low-head turbine, this power setpoint is the maximum available power, hence the name MPPT. In grid-supporting PHS, this may not be the case, as the power setpoints depend on grid frequency. Therefore, adjustments need to be made to the existing algorithms. The MPPT algorithms can be divided into direct and indirect meth- ods. Direct MPPT algorithms are based on iterative extremum-seeking control algorithms. These algorithms require limited knowledge of the system, but are inherently slow due to their iterative behaviour, making them less suitable for grid-supporting PHS. However, they can still be used in storage systems with lower dynamic requirements because of their simplicity. Indirect methods are based on a model of the system, making them more dynamic but less flexible. Most of the existing MPPT control methods rely on flow rate measurement. However, a flow rate sensor is costly and has a certain error. In low-head RPTs, the accuracy can further decrease due to a non-uniform flow, a short intake, and high turbulence [133]. To evade these drawbacks, Borkowski and Dariusz [90] presented a flow rate estimator. The estimator is based on an artificial neural network (ANN), which is trained by experimental data. Active distribution rejection control (ADRC) is used in torque and speed control to account for known and unknown electrical, hydrauli- cal, or mechanical disturbances in the system, increasing performance and robustness. Guo et al. [84] applies a first order ADRC for a PMSM in a hydropower application, where the known disturbances are mechanical friction and hydraulic torque. A second order state observer is used to estimate the rotational speed and hydraulic torque. ADCR is especially useful in low-head high-power PHS, since any change in the system tubes has a significant influence on the head losses, because of the high flow rate at low head. Direct torque control (DTC) is an alternative control method to FOC. In DTC, the electromagnetic torque and stator flux are controlled by switching between a discrete number of stator voltage vectors, which in turn form the stator flux vector interacting with the rotor flux. Based on the torque and flux linkage reference and the current flux vector position, a lookup table is consulted to select the optimal voltage vector. If e.g. 5.2. Torque and speed control 7. Control schematic of DTC. Fig. 7. Control schematic of DTC. Fig. 6. Decoupled field oriented control of a PMSM with estimated back-EMF feedforward. estimator based on phase voltages and currents is used (bottom). These estimated values are compared to torque and flux setpoints. Hysteresis controllers then determine the proper voltage vector from a lookup table, resulting in the switching signals. Direct torque control has a slightly better torque response compared to FOC and does not require a position sensor [128,129]. However, DTC relies on an accurate estima- tor. Especially at low speeds, an estimator based on phase voltages and currents cannot accurately estimate the stator flux [130] which makes it less suitable for low-head PHS. Although some improved estimator algorithms have been studied [130], this drawback is best averted by using a position sensor in the estimator. Disadvantages of DTC include variable switching frequency, high harmonic current distortion, and torque ripple [128,129,131]. To achieve a smoother dynamic response and thus less torque and flux ripple, space vector modulation (SVM) is used instead of the lookup table [128,129,132]. Here, 𝑝is the pole pair number and 𝛹PM is the constant flux of the permanent magnets. Fig. 6 shows the control scheme of a field oriented controlled PMSM. On the bottom right, the stator currents are measured and transformed to the rotating 𝑞, 𝑑reference frame. These signals are compared with the setpoints on the left and controlled by two PI controllers. These controllers determine the duty ratios resulting in the Pulse-Width Modulated (PWM) signals for the converter. In FOC, ̂𝑖d is set to 0. Eq. (7) shows that the machine torque is directly proportional to 𝑖q, resulting in a highly dynamic control. To achieve decoupled control of both currents, the coupling terms in Eq. (7) are used as a feedforward. Furthermore, a back-EMF estimator can be implemented in the 𝑞current control. Although FOC is highly dynamic and easy to implement, setting 𝑖d = 0 is not the most efficient way to reach a desired torque setpoint for a PMSM with saliency, like an interior magnet PMSM. Therefore, maximum torque per ampere (MTPA) control can reduce copper losses and increase overall efficiency in low-head hydropower applications. The MTPA accomplishes this by minimising 𝑖s = √ 𝑖2 q + 𝑖2 d for every torque setpoint [124]. 5.2. Torque and speed control Applications with interior magnet PMSMs in wind turbines found a reduction in Joule losses (up to 4.2%), while maintaining a dynamic response to changing torque setpoints [124, 125]. 5.3. Power control In the power control of low-head PHS, the goal is to reach a power setpoint as fast and efficiently as possible. Three main control parameters are determined: the pump/turbine rotational speed 𝛺; the inlet vane angle 𝛼; and the blade pitch 𝛽. An RPT with only 𝛺as control parameter is defined as a non-regulated RPT. A single- and double-regulated RPT further include, respectively inlet vane control, and inlet vane and blade pitch control. In low-head PHS, a regulated RPT is recommended, because it allows the RPT to be operated at high efficiency in a large operating range of heads, flow rate, and power setpoint. The position sensor plays a critical role in FOC. However, a po- sition sensor is costly and its signal can contain noise. Therefore, saliency-based sensorless rotor position estimators [88,89,126,127] are proposed for low-power systems, since they can increase reliability and reduce cost [89]. For low rotational speed runners, as in low-head PHS, the saliency-based approach is the most suitable [89]. Here, a high pulse frequency is injected, while the current response, which depends on the rotor magnetic flux position, is observed. 5.2. Torque and speed control In variable speed PHS, the machine speed is altered to reach a power setpoint as fast and precise as possible, both in pump and turbine mode. Therefore, the machine torque must be precisely controlled. Field oriented control (FOC) is a vector control method that has been widely used in low-head micro-hydropower installations [83–89]. The main advantage is an independent control of the machine torque, and thus, highly dynamic performance. This is necessary in PHS to quickly react to changes to the rapidly fluctuating grid frequency. The general principle of FOC is to regulate the 𝑖d and 𝑖q currents in the rotating reference frame. The electrical dynamics of a PMSM can be modelled by Eqs. (5) and (6). Here, 𝛺𝑒𝛹PM is the back-EMF of the permanent magnets. 𝑅is the stator resistance. 𝐿q and 𝐿d are the 𝑞and 𝑑axis inductances, respectively. 𝛺𝑒𝐿𝑖is the armature reaction EMF, through which the 𝑞and 𝑑schemes are coupled. Single-stator single-rotor topologies [101] are simple in design and compact. However, there is a strong imbalanced axial force between the stator and rotor. Therefore, the rotor disc width needs to be increased to avoid twisting [96]. Double-stator single-rotor topologies [102] are a valuable alternative to SSDR topologies. Double-stator single- rotor uses fewer PMs, but experiences more copper losses due to poor winding utilisation [96]. A DSSR machine with integrated permanent magnets has a high power-to-inertia ratio, since the rotor disc serves no magnetic purpose and is eliminated [100]. The reduced inertia is a significant benefit in a grid-supporting low-head PHS. In a slotted stator AF-PMSM, cogging torque results from the interaction between the PMs and the stator slots. This undesired torque can be significantly reduced by changing the angle between both stators [103]. However, this also reduces power output. Finally, SSDR is deemed the most favourable AF-PMSM topology. Next to decreased copper losses, the use 𝑣d = 𝑅𝑖d + 𝐿d d𝑖d d𝑡−𝛺e𝐿q𝑖q (5) 𝑣q = 𝑅𝑖q + 𝐿q d𝑖q d𝑡+ 𝛺e(𝐿d𝑖d + 𝛹PM) (6) (5) (6) By regulating the 𝑑and 𝑞axis currents, the torque can be regulated as shown in the general torque Eq. (7) of the PMSM. 𝑇= 𝑝3 2 [𝛹PM𝑖q + (𝐿d −𝐿q)𝑖d𝑖q ] (7) 𝑇= 𝑝3 2 [𝛹PM𝑖q + (𝐿d −𝐿q)𝑖d𝑖q ] (7) 8 Renewable and Sustainable Energy Reviews 158 (2022) 112119 J.P. Hoffstaedt et al. Fig. 6. Decoupled field oriented control of a PMSM with estimated back-EMF feedforward. Fig. 7. Control schematic of DTC. Fig. J.P. Hoffstaedt et al. Especially in a low-head high-power system, where the flow rate is high, these losses have a significant influence on the efficiency. Gradient descent control is a direct maximum efficiency point track- ing (MEPT) algorithm that allows multiple control variables, opposed to the perturb and observe algorithm. However, to derive the efficiency, an accurate flow sensor is necessary, which was discussed to be a challenge in low-head PHS systems [133]. On every operating point, the control variables are incremented with the direction of their partial derivatives of efficiency at the current operating point, multiplied by a step size 𝑘[138]. In Eq. (10), 𝛼is the vane opening and 𝛽is the blade pitch. 𝛺11 = 𝛺𝐷 √ 𝐻 , 𝑄11 = 𝑄 𝐷2√ 𝐻 (12) (12) 𝑄11 and 𝛺11 can also be compensated when the Reynolds number of the real system differs from the design [145]. Fraile-Ardanuy et al. [146] applied a hill chart to a control system in order to find the optimal efficiency speed for given 𝛼and measured 𝑄. For a reduced-scale RPT [147], a lookup table is trained based on measurements. The lookup table is used to find ̂𝛺for given measured 𝐻and ̂𝑃. However, the speed is controlled by 𝛼, while it was shown in the paper that 𝑄11 = 𝑓(𝛼, 𝛺). However, the proposed control system is promising for low-head hydropower if both 𝛺and 𝛼are controlled separately. Then, the RPT could be controlled to reach ̂𝑃at the highest efficiency for a certain measured 𝐻. 𝑄11 and 𝛺11 can also be compensated when the Reynolds number of the real system differs from the design [145]. Fraile-Ardanuy et al. [146] applied a hill chart to a control system in order to find the optimal efficiency speed for given 𝛼and measured 𝑄. For a reduced-scale RPT [147], a lookup table is trained based on measurements. The lookup table is used to find ̂𝛺for given measured 𝐻and ̂𝑃. However, the speed is controlled by 𝛼, while it was shown in the paper that 𝑄11 = 𝑓(𝛼, 𝛺). However, the proposed control system is promising for low-head hydropower if both 𝛺and 𝛼are controlled separately. Then, the RPT could be controlled to reach ̂𝑃at the highest efficiency for a certain measured 𝐻. 𝛥𝛼= 𝑘𝜕𝜂 𝜕𝛼 𝛥𝛽= 𝑘𝜕𝜂 𝜕𝛽 𝛥𝛺= 𝑘𝜕𝜂 𝜕𝛺 (10) (10) If the time constants of the control parameters are known, 𝑘can be chosen differently for each control parameter. J.P. Hoffstaedt et al. If 𝐾is too low after oscillations and the MPP shifts, the dynamic response is poor. To allow both dynamic response and minimal power fluctuation around the MPP, 𝐾can be taken proportional to the power gradient 𝛥𝑃 𝛥𝛺[134]. Due to the parabolic nature of the turbine characteristics, 𝐾is high when far away from the MPP and gradually decreases when the MPP is neared. Note that using this gradient-based step size cannot be used to reach a lower power setpoint ̂𝑃. However, the step size here can be proportional to | ̂𝑃−𝑃|. In wind turbine applications, fuzzy logic is recently used to find the value of 𝐾, where the perturbed variable is the generator voltage, which is proportional to the generator speed [135–137]. Inherent to this method is that the system will still perturb 𝛺when the MPP is reached, resulting in an oscillation around the MPP. Step size 𝐾is an adaptive value that increases when the power is continuously rising and decreases when the power is fluctuating [83]. However, the dynamic behaviour of this method is not optimal. If 𝐾is too low after oscillations and the MPP shifts, the dynamic response is poor. To allow both dynamic response and minimal power fluctuation around the MPP, 𝐾can be taken proportional to the power gradient 𝛥𝑃 𝛥𝛺[134]. Due to the parabolic nature of the turbine characteristics, 𝐾is high when far away from the MPP and gradually decreases when the MPP is neared. Note that using this gradient-based step size cannot be used to reach a lower power setpoint ̂𝑃. However, the step size here can be proportional to | ̂𝑃−𝑃|. In wind turbine applications, fuzzy logic is recently used to find the value of 𝐾, where the perturbed variable is the generator voltage, which is proportional to the generator speed [135–137]. Hill charts define the relation between flow rate 𝑄, rotational speed 𝛺, inlet vane angle 𝛼, and efficiency 𝜂for a constant head 𝐻. Therefore, if 𝛺and 𝛼are known, 𝑄and 𝜂can be read from the graph. Furthermore, 𝛼and 𝜂are plotted versus the unitary rotational speed 𝛺11 and unitary flow rate 𝑄11 in Eq. (12), making hill charts scalable for different heads. However, in a real system, the losses 𝐻L(𝑄) have to be taken into account, making it difficult to estimate the net head across the turbine without a flow rate sensor. J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Although this model can be used in low-head control models, it is important to note that flow rate Q is a function of head and rotational speed 𝑄= 𝑓(𝐻, 𝛺) for a non-regulated RPT. Therefore, changing 𝛺will affect flow rate 𝑄as well. Zhang et al. [142] proposes a polynomial empirical equation for the efficiency of a turbine 𝜂ℎ(𝛺, 𝑄), where the coefficients can be derived from experimental data. Borkowski and Dariusz [143] used an ANN to compose and validate an efficiency equation 𝜂ℎ(𝛺, 𝑄) together with a flow rate characteristic 𝑄(𝛺, 𝛼) for a regulated turbine. The control system based on these characteristics requires no flow rate sensing. However, the 𝑄characterisation is for a constant head and the flow rate is approximated by a linear function of speed. This limits its application under varying head at low rotational speeds. Among direct MPPT control methods, the perturb and observe algorithm has been investigated for non-regulated low-head turbines. The principle operation consists of altering or perturbing the rotational speed 𝛺, i.e. accelerating or decelerating, and analysing the change in output power 𝑃, measured at the electric machine or converter. If the power has increased, the sign of d𝛺is maintained and the procedure continues. Otherwise, d𝛺is reversed [83]. Eq. (8) shows how the speed setpoint ̂𝛺is altered after each iteration. Note that 𝛿(𝑡) implies the sign of d𝛺. ̂𝛺= ∫ 𝑡𝑘−1+𝑇𝑠 𝑡𝑘−1 𝐾𝛿(𝑡) d𝑡 (8) 𝛿(𝑡) = sgn(𝑃𝑘−𝑃𝑘−1) sgn(𝛺𝑘−𝛺𝑘−1) (9) (8) (9) Similarly, ANN has also been used to form lookup tables [144]. Lookup tables can be constructed over a large operating range during on-site measurements or by using an existing dataset. Pérez-Diaz and Fraile-Ardanuy [144] use two ANNs to train the head and efficiency for input parameters 𝑄, 𝛺, and 𝛼. A possible application of the result- ing lookup tables is to find reference ̂𝛺and ̂𝛼to reach the optimal efficiency for a given head. In this control system, no flow sensor is necessary, thus reducing cost and increasing reliability, especially for low-head systems. Inherent to this method is that the system will still perturb 𝛺when the MPP is reached, resulting in an oscillation around the MPP. Step size 𝐾is an adaptive value that increases when the power is continuously rising and decreases when the power is fluctuating [83]. However, the dynamic behaviour of this method is not optimal. J.P. Hoffstaedt et al. Furthermore, 𝑘can be adaptive and defined by a line search algorithm at every itera- tion [139]. Although this control algorithm shows great potential, any disturbances on the gradient estimation due to measurement error or mutual influence between control parameters has a great impact on the convergence [138]. Therefore, a moving average filter and a Kalman filter can, respectively, be used to increase robustness [90,140]. Furthermore, Borkowski [90] accounts for the time delay of flow rate settlement after a change in turbine control parameter further reduce risk of a non-converging control. One drawback of using turbine characteristics is that the electrical machine and converter losses are not included. Therefore, the overall MPP may differ from the turbine MPP [87]. De Kooning et al. [148] found that the MPP displacement in wind turbines was greater for low wind and thus lower rotational speeds. In direct MPPT methods, these losses are included if the power is measured on the converter side. An- other drawback of indirect MPPT methods is that they do not account for system performance deterioration over a long time period. However, reinforcement learning, as proposed for wind turbines [149,150], can solve this problem at the cost of a higher real computational intensity. Indirect MPPT algorithms rely on prior knowledge of the system in order to determine the optimal torque or speed reference to achieve a power setpoint. Recently, this system knowledge is mostly captured in the form of empirical equations, hill charts, or lookup tables, which are derived from measurements or numerical analysis like computational fluid dynamics. Márquez et al. [141] derived an empirical formula for a propeller turbine, which is a modified Kaplan turbine, designed for low-heads and low flow rates. Eq. (11) relates the non-regulated turbine efficiency to flow rate and turbine speed. 5.3.1. Maximum power point tracking algorithms the torque must be increased, a voltage vector is selected so that the angle between stator and rotor flux is increased. Fig. 7 visualises the control schematic. To find the torque and stator flux, an 9 6. Modelling of low-head systems The overall objective of developing a model of a given system is to have a representation of the real world. Since such a model will always be a simplified depiction, it is crucial to weigh which aspects are essential and what should be left out or simplified. In the case of numerical models, this also helps to improve performance and reduce the computational resources necessary. At the end, a well formulated model allows to predict the behaviour of a system to a greater extent and wider scenarios than experiments and interpolating empirical data. These predictions are crucial in developing such systems to understand performance and dynamics, aid optimisation, and can be required for accurate control during operation. The mathematical models are typi- cally derived from first principles, such as balance equations of mass, energy, or momentum, but can also be based on phenomenological or empirical observations or a mixture of both. 𝜕𝐻 𝜕𝑡= −𝑈𝜕𝐻 𝜕𝑥−𝑎2 𝑔 𝜕𝑈 𝜕𝑥 (14) 𝜕𝑈 𝜕𝑡= −𝑈𝜕𝑈 𝜕𝑥−𝑓𝑈|𝑈| 2𝐷 −𝑔𝜕𝐻 𝜕𝑥 (15) (14) (15) Here we have H as the pressure head, U as water velocity, a as the pressure wave velocity, g as gravitational acceleration, D as the conduit diameter, and f as friction factor. If appropriate, simplifications can be made neglecting velocity head or friction losses. The set of partial differential equations can be solved either directly or by transforming it first into a series of ordinary differential equations. Models for high-head PHS are comprehensive and well researched while attention to low-head PHS applications has been limited. Fun- damentally, the same approaches can be used. There are, however, differences in the relevance of individual model components. From a hydrodynamic point of view, the major difference is a shift towards higher flow and reduced head for a given power. The increase in the mass flow rate of water may cause the system to be more prone to water hammer effects. Cavitation is a further effect to consider when choosing model components for a low-head scenario. Reaction turbines, such as Francis or Kaplan turbines that are suitable for medium- and low-head applications, are considered more susceptible to the effect [157]. Comparisons of similar approaches have shown that treating the conduit as rigid results in a reduction in computational resources neces- sary and hence, decreased simulation time. However, if the underlying scenario requires, higher accuracy can be achieved when considering elasticity and compressibility effects [163,164]. 5.3.2. Model predictive control However, the benefits of adapting MPC for low-head PHS could be substantial, because of the potentially increased influence of transient effects, as described in Sec- tion 6.1. For a dynamic system providing frequency support, the MPC’s computational intensity increases even further, which might slow down the optimisation algorithm. However, with the recent advancements made in parallel computing with, e.g., multi-core processors (CPUs) and many-core processors such as graphical processing units (GPUs), the MPC process can be accelerated [156], making it suitable for complex dynamic systems like low-head PHS. of variable speed control which may cause even lower speeds at off- design operation points, this may, for example, not just affect the choice of motor-generator architecture but could also affect the drivetrain losses. When choosing modelling approaches for individual system components, considering these characteristics specific for low-head PHS helps to cover the relevant aspects while also optimising performance. 5.3.2. Model predictive control An important factor in RPT operation and control that is often overlooked in traditional MPPT strategies is the transient effect of the water supply system caused by a control action. The transient flow equations are described in Section 6.1. Fang et al. [151] stated that increasing the control action magnitude actually decreased the output power, while the settling and maximum turbine pressure deviation increased. Therefore, it can be seen how using an MPPT control that does not account for these effects can have poor dynamic behaviour 𝜂ℎ(𝛺, 𝑄) = 3.33 𝑄 [ 1 2 ( 90 𝜆𝑖 + 𝑄+ 0.78 ) 𝑒 −50 𝜆𝑖 ] (11) 𝜆𝑖= [ 1 (𝜆+ 0.089) −0.0035 ]−1 , 𝜆= 𝑅𝐴𝛺 𝑄 (11) 10 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 when applied to a real system. In some studies on MPPT control, the water inertia time 𝑇𝑤is incorporated as a time delay on the control action [90] or as an extra mechanical inertia on the RPT [152]. How- ever, this does not fully capture the transient effects. Therefore, model predictive control (MPC) is applied to PHS systems [153–155]. In MPC, a detailed model of the full PHS system, including hydraulic transients, losses, and an RPT model, are used. Based on a certain operating state setpoint, an internal optimisation algorithm simulates control actions and observes the predicted outcomes of the model. The outcomes are then given a cost value based on the power response. These predictions are made for multiple future time samples. Each time sample, this process is repeated. Therefore, MPC is an accurate control method that can work on complex systems, at the cost of a significantly high computational intensity. Furthermore, MPC can also incorporate system constraints, such as maximum pressure deviation and mechanical rate limits. Chaoshun et al. [153] proposed using a nonlinear MPC, which includes the elastic water hammer effect in a high-head PHS plant. Liang et al. [154] used MPC to define the optimal switching time between pump and turbine mode for a multi-RPT PHS plant. However, these studies did not include pressure constraints, which are especially important in systems with long pipelines. The MPC for a 40 metre PHS plant by Mennemann et al. [155] included this effect. MPC is currently mostly investigated for high-head PHS. 6.1. Hydrodynamics One of the most common approaches in PHS is to model the penstock as a rigid conduit in addition to the consideration of water being an incompressible fluid. A widespread approach making these assumptions is based on the net force on the body of water which can be given through both the rate of change of momentum and the differences in pressure head to obtain the change in time of the volumetric flow rate as shown in Eq. (13) [158–160]. 𝐿d𝑄 d𝑡= (𝐻−𝐻T −𝐻L ) 𝐴𝑔 𝐿d𝑄 d𝑡= (𝐻−𝐻T −𝐻L ) 𝐴𝑔 (13) (13) Here, Q is the volumetric flow rate through the conduit and turbine, H is the head over the body of water, 𝐻T is the turbine head, 𝐻L represents the head losses within the conduit, g is the gravitational acceleration, A is the conduit cross-sectional area, and L is the conduit length. The turbine head can be obtained either from empirical data or physical models as a function of both its rotational speed and flow rate, while the major and minor head losses are typically represented through well known hydraulic models, such as the Darcy–Weisbach formulation in combination with the Colebrook equation [50]. To complete the conduit component of the system model, these equations are typically coupled with equations relating to a governor, such as a gate or guide vanes and mechanical power of the turbine as given in Eq. (1). Using the above mentioned combination of ordinary differential and algebraic equations to model the water column as a rigid body is one the simplest methods to cover the system dynamics. However, to accu- rately depict transients in the system, such as travelling pressure waves, an approach using coupled partial differential equations considering compressibility and elasticity is required [161]. This could be partic- ularly relevant in applications where enlarged mass flow rates of water are subject to sudden changes of flow rates and heads, such as low-head PHS. A commonly used 1-D approach considering these effects uses the so-called, water hammer equations. These relate pressure head and water velocity as a function of position and time as shown in Eqs. (14) and (15) [162,163]. 6.2. Power take-off However, it is important to include the whole system with its losses to have precise control. Model predictive control is a computationally intense control method that can account for transient effects in complex systems, making it a valuable option for low-head PHS. 𝐽d𝜔 d𝑡= 𝜏h −𝜏g −𝐷f𝜔 (17) 𝐽d𝜔 d𝑡= 𝜏h −𝜏g −𝐷f𝜔 (17) This ordinary differential equation relates the change in angular ve- locity 𝜔and rotational mass moment of inertia of the system J to the balance of hydraulic torque 𝜏h, generator torque 𝜏g, as well as friction typically represented as a viscous damping torque 𝐷f𝜔. Typical implementations modelling the electrical dynamics of motor-generators use space-vector representation with the d-q frame of reference. An example for such an approach for a PMSM can be found in Eqs. (5) and (6) described in Section 5.2. This ordinary differential equation relates the change in angular ve- locity 𝜔and rotational mass moment of inertia of the system J to the balance of hydraulic torque 𝜏h, generator torque 𝜏g, as well as friction typically represented as a viscous damping torque 𝐷f𝜔. Typical implementations modelling the electrical dynamics of motor-generators use space-vector representation with the d-q frame of reference. An example for such an approach for a PMSM can be found in Eqs. (5) and (6) described in Section 5.2. • When modelling low-head PHS, the same fundamental approaches of traditional PHS can be used. However, the change in system characteristics being a shift to larger masses of water and reduced head requires more attention on certain model components. Mod- elling hydrodynamics in more detail allows to cover transients such as water hammer and estimate the risk of cavitation. Paying further attention to changing motor-generator dynamics can help to accurately predict performance and improve control. • When modelling low-head PHS, the same fundamental approaches of traditional PHS can be used. However, the change in system characteristics being a shift to larger masses of water and reduced head requires more attention on certain model components. Mod- elling hydrodynamics in more detail allows to cover transients such as water hammer and estimate the risk of cavitation. Paying further attention to changing motor-generator dynamics can help to accurately predict performance and improve control. A further point of interest to modelling the PTO of low-head PHS in combination with variable speed operation is a potential shift to higher Joule and reduced iron losses. 6.2. Power take-off Variable speed operation of PHS does not just enable to work at improved efficiency under varying conditions but also improves the ca- pability to provide AS. When modelling newly developed low-head PHS systems, this should be considered. For variable speed systems in low- head PHS as well as related and comparable wind power generation, a variety of power take-off architectures and controls are available. Com- monly used are models representing doubly-fed induction or permanent magnet synchronous motor-generators, respectively, in combination with drivetrain models of the change in angular velocity based on torque balance and inertia [161,170,171]. To model the drivetrain, the simplest model consider a lumped rigid body approach with a rigid shaft such that all rotating masses and hence rotational inertias can be added together, leading to Eq. (17). • For the power take-off, axial flux PMSMs are the most promising electric machines for low-head PHS due to their high efficiency, high power density, and suitability for high-torque-low-speed operation. The machine torque can be controlled to achieve a speed setpoint by either field oriented control or direct torque control, with the latter having a slightly better torque response if a position sensor is used, but increasing torque ripple. Active dis- tribution rejection control can be used to complement the torque and speed control, increasing performance and robustness. To derive the speed setpoint, MPPT algorithms based on RPT models are suitable for low-head PHS because of their short response time and steady power output. However, it is important to include the whole system with its losses to have precise control. Model predictive control is a computationally intense control method that can account for transient effects in complex systems, making it a valuable option for low-head PHS. • For the power take-off, axial flux PMSMs are the most promising electric machines for low-head PHS due to their high efficiency, high power density, and suitability for high-torque-low-speed operation. The machine torque can be controlled to achieve a speed setpoint by either field oriented control or direct torque control, with the latter having a slightly better torque response if a position sensor is used, but increasing torque ripple. Active dis- tribution rejection control can be used to complement the torque and speed control, increasing performance and robustness. To derive the speed setpoint, MPPT algorithms based on RPT models are suitable for low-head PHS because of their short response time and steady power output. (16) Such cavitation numbers characterise how close the local reference pressure is to the vapour pressure of the fluid. It is calculated from the reference pressure 𝑝r, the vapour pressure at the given temperature 𝑝v(𝑇), and the characteristic system pressure difference 𝛥𝑝. Approaches using various forms of the cavitation number have shown widespread use in a variety of hydrodynamic applications. It has been found, however, that studies giving too much emphasis to it, result in poor repeatability and inconsistency between them. This is due to a wide variety of definitions of the cavitation number as well as the neglect of other factors, such as the influence of geometry, flow velocity, or fluid temperature [167]. More accurate approaches typically include representations of the dynamics of the vapour bubble cluster. While computationally more intensive, these allow to model growth and collapse of the nuclei based on the Rayleigh–Plesset equation derived from the conservation of mass allowing to solve for the time-dependent vapour bubble radius [168,169]. • In a low-head context, the choice of pump-turbine design is highly dependent on the flow rate of the system. Axial flow pump- turbines, with variable speed drives, are the most suitable solution for high flow rates which leads to higher power outputs. On the other hand, other designs, such as Archimedes screw or rotary positive displacement configurations, can be beneficial at lower flow rates and micro- or small-scale locations. • The discussion on grid integration has shown that, to compensate for an increase in intermittent generation and a reduction in spin- ning reserves, a combination of grid-forming control alongside bulk energy storage is necessary. To ensure grid stability, such systems will need to provide synthetic inertia next to other an- cillary services, namely steady state voltage control, fast reactive current injections, short circuit currents, black start, and island operation capability. 6. Modelling of low-head systems Aside from travelling pressure waves, another effect that may be of higher relevance in a low-head high-flow system is cavitation. The inception of cavitation is dependent on the amount and type of particles in the water allowing for nuclei to sustain [165]. Low-head PHS is a promising candidate for seawater applications in coastal regions. In such, breaking waves mixing particles from the seabed with the salty water could increase the risk for cavitation to occur. Its likelihood is The different relation between flow and head also affects turbo- machinery and power take-off. The shift to a higher flow and lower pressure at the machine side closer to the upper reservoir typically results in lower angular velocity and higher torque at the runner and motor-generator. This is due to an increased runner tip speed as a consequence of enlarged machines. In combination with the likely use 11 Renewable and Sustainable Energy Reviews 158 (2022) 112119 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 also increased when pump-turbines work at off-design conditions as a consequence of variable speed operation. 7. Conclusion Due to the rapid rise of intermittent renewable energy sources, penetration levels will exceed what can be compensated for by alter- native stability measures, and large-scale integration of energy storage will become imperative. This increase in demand for short- and long- term balancing and the provision of ancillary services will contribute to novel systems turning into cost-effective solutions. While pumped hydro storage is a promising candidate to improve grid stability, its limitations in deployability call for a conceptual adaptation. Shift- ing the operating range from traditional high-head towards low-head applications could pave the way to utilise PHS in regions where so far it had not been feasible. Further technological advancements can significantly contribute to enhancing its capability to improve grid stability while also making it cost competitive. Based on the lack of research on low-head PHS, this review discusses challenges and the potential of low-head PHS while giving an overview of pumped storage technologies and their applicability to low-head applications. The main outcomes are the following: This formation of vapour bubbles occurs when the local refer- ence pressure in the fluid reaches its vapour pressure. Fundamental numerical estimates for the likelihood of cavitation occurrence use dif- ferent forms of a dimensionless cavitation parameter, such as Thoma‘s cavitation number shown in Eq. (16) [166]. 𝜎= 𝑝r −𝑝v(𝑇) 𝛥𝑝 (16) 𝜎= 𝑝r −𝑝v(𝑇) 𝛥𝑝 (16) References Speicherlösungen für den Energiemarkt der Zukunft. [13] Ren J, Ren X. Sustainability ranking of energy storage technologies under uncertainties. J Clean Prod 2018;170:1387–98. [44] Hogan TW, Cada GF, Amaral SV. The status of environmentally enhanced hydropower turbines. Fisheries 2014;39(4):164–72. [14] Stocks M, Stocks R, Lu B, Cheng C, Blakers A. Global atlas of closed-loop pumped hydro energy storage. Joule 2021;5(1):270–84. [45] Karassik IJ, Messina JP, Cooper P, Heald CC. Pump handbook, 4th ed.. New York: McGraw-Hill Education; 2008. [15] Blakers A, Stocks M, Lu B, Cheng C. A review of pumped hydro energy storage. Prog Energy 2021;3(2):022003. [46] Carravetta A, Derakhshan Houreh S, Ramos HM. Introduction. 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Deriaz pump-turbine for pumped hydro energy storage and micro applications. J Energy Storage 2019;24:100788. [50] White FM. Fluid mechanics, eighth edition. 2016. [49] Morabito A, de Oliveira e Silva G, Hendrick P. Deriaz pump-turbine for pumped hydro energy storage and micro applications. J Energy Storage 2019;24:100788. [19] Kougias I, Aggidis G, Avellan F, Deniz S, Lundin U, Moro A, et al. Analysis of emerging technologies in the hydropower sector. Renew Sustain Energy Rev 2019;113:109257. [50] White FM. Fluid mechanics, eighth edition. 2016. [51] Braitsch W, Haas H. 2.7 Turbines for hydroelectric power. In: Heinloth K, editor. Renewable energy, Vol. 3C. Springer Berlin Heidelberg; 2006, p. 197–222. [20] Javed MS, Zhong D, Ma T, Song A, Ahmed S. Hybrid pumped hydro and battery storage for renewable energy based power supply system. Appl Energy 2020;257(May 2019):114026. [52] Novara D, McNabola A. Declaration of competing interest [28] Nicolet C, Béguin A. Variable speed and ternary units to mitigate wind and solar intermittent production. 2014. The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper. [29] Sterner M, Stadler I. Handbook of energy storage. 2019. [30] European Environment Agency. EU Renewable electricity has reduced envi- ronmental pressures; targeted actions help further reduce impacts. 2021, p. 1–14. Acknowledgements [31] Foley AM, Leahy PG, Li K, McKeogh EJ, Morrison AP. A long-term analysis of pumped hydro storage to firm wind power. Appl Energy 2015;137(2015):638–48. This research is part of a project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 883553. The authors would like to thank the International Hydropower Association for providing us with Fig. 2. [32] Zakeri B, Syri S. 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Additionally, larger diameters of the rotating mass lead to increased rotational inertia. An increase here correlates with a reduction in speed ripple [173]. This becomes clear when including a cogging torque in Eq. (17). There are considerable opportunities for further research to improve low-head pumped storage technology and facilitate economic viability. Firstly, a detailed economic analysis incorporating a predicted increase in revenue from energy arbitrage and the provision of AS combined with improved control strategies optimised for these may serve as a further proof of concept. Additionally, modelling and simulation efforts integrating the proposed components and analysing performance on 12 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 a system level can help to estimate technical potential for round-trip efficiencies, mode-switching times, and power ramp rates. 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Análisis de publicaciones de los primeros 20 números de magis
Magis
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Análisis de publicaciones de los primeros 20 magis números de magis Analysis of publications for the first 20 editions of magis Fecha de recepción: DICIEMBRE 16 DE 2018 / Fecha de aceptación: FEBRERO 20 DE 2019 / Fecha de disponibilidad en línea: MARZO DE 2019 doi: 10.11144/Javeriana.m11-23.appm Christian Hederich-Martínez hederich@pedagogica.edu.co Universidad P edagógica N acional , C olombia https://orcid.org/0000-0003-1080-9973 C atalina Roa-C asas roa.a@javeriana.edu.co Pontificia Universidad Javeriana , C olombia https://orcid.org/0000-0002-8104-5782 Resumen Este artículo analiza el comportamiento de magis en sus 10 primeros años. Se analizan las investigaciones que se han publicado en 20 números, de 2008 a 2017, con una metodología que recoge estadísticas descriptivas y un análisis multivariante de redes de palabras claves. Los resultados presentan tipos de artículos publicados, enfoques de investigación, indicadores de colaboración. El análisis multivariante identifica siete diferentes conglomerados temáticos, definidos por coocurrencias de palabras clave, que resultan asociados con niveles educativos específicos y con momentos de aparición en la revista. Palabras claves Artículo científico; publicación científica; palabra clave; citación Abstract This article analyzes the behavior of magis in the first 10 years. The researches published in these first 20 editions, from 2008 to 2017, have been analyzed using a methodology that gathers descriptive statistical data and applies a multivariate analysis of keyword networks. The results indicate the types of articles published so far, their research approaches, collaboration indicators, quotes and downloads. Multivariate analysis identifies seven different clusters (groups) of topics defined by the concurrence of keywords associated to specific educative level and the number of times they have been included in the journal. Keywords Scientific article, scientific publication, keyword, citation Para citar este artículo / To cite this article Hederich-Martínez, C. & Roa-Casas, C. (2019). Análisis de publicaciones de los primeros 20 números de magis. magis, Revista Internacional de Investigación en Educación, 11 (23), 221-242. doi: 10.11144/Javeriana.m11-23.appm VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 13 magis VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 222 Descripción del artículo | Article description Este artículo se deriva de un proceso de investigación que analiza el comportamiento de magis en sus 10 primeros años. Las revistas científicas se han convertido en el escenario fundamental para la publicación de resultados de investigaciones en los diferentes campos del conocimiento. Conforman un espacio de intercambio activo y continuo, en la medida en que ponen a disposición de los investigadores un repositorio de los avances en su área. De este modo, posibilitan y promueven un intercambio permanente de información y conocimiento entre especialistas de diferentes instituciones y países, pues los resultados de investigaciones realizadas en diversos lugares del mundo coexisten en una publicación. Así, las revistas científicas ponen a disposición de los lectores artículos originales para ser leídos, valorados y difundidos entre quienes investigan un área. En este sentido, estas publicaciones constituyen la base de nuevos procesos que retoman y cuestionan los resultados publicados, para avanzar hacia la producción de otros nuevos. En línea con lo anterior, la valoración de los artículos que se publican en determinada área es un acercamiento a la investigación que una comunidad científica desarrolla. Así, valorar la producción de un campo es adentrarse en la dinámica de la investigación misma, con sus aportes, intereses, tensiones y cuestionamientos, para reconocerla como base de nuevas indagaciones. La continua publicación de artículos por parte de las revistas, y su disposición en bases de datos e índices internacionales, si bien no garantiza la lectura de los resultados de las investigaciones, sí hace posible su visibilidad en espacios a los cuales acudirán los interesados en su uso y divulgación. Desde sus planteamientos de origen, magis nace para ser un espacio académico que permita cualificar prácticas de investigación en el campo educativo en Colombia y la región (García, 2008). En su definición etimológica, magis significa más y mejor. En el marco de las definiciones de la revista, el término se refiere al compromiso, la actitud y el empeño de superación personal de maestros y alumnos, en busca de la realización del potencial de bondad que hay en cada ser humano. De este modo, su acercamiento al campo de conocimiento de la educación se postula desde su comprensión interdisciplinaria, en el marco de una perspectiva cultural, en un interés por la profesionalización de los agentes y las instituciones educativas, y un compromiso ético con la educación. En ese sentido se formula que magis promueva buenas prácticas pedagógicas, didácticas y de gestión educativa, en línea con la manera en que estas son manifestación de la formación integral de quienes las agencian, orientan y estudian, y cómo son apropiadas por los estudiantes cuando son acompañados por el maestro. En el marco de lo anterior, desde sus inicios, magis se dirige a investigadores de diferentes disciplinas que generan conocimiento sobre estas prácticas, y busca, también, llegar a maestros en formación y en ejercicio de los diferentes niveles de la educación que pretendan construir un panorama profundo de las prácticas educativas y su investigación, además de funcionarios de instancias gubernamentales que tienen a su cargo la formulación de políticas y directrices de gestión. magis cumple sus 10 primeros años y nos interesa ver las formas que han tomado esas apuestas iniciales que fundaron la revista. Así, este análisis se propone como marco de comprensión de su comportamiento en este tiempo. En este sentido, da cuenta de las investigaciones que se han publicado en 20 números, de 2008 a 2017, y hace un balance de los tipos de artículos, países, idiomas e instituciones presentados, el nivel de colaboración entre diferentes autores e instituciones, las metodologías seguidas, las descargas y citaciones, y un análisis de las redes y las tendencias temáticas que marcan las palabras clave de los artículos publicados. Se trata de Metodología PÁGINA Procedimiento Se calcularon las estadísticas descriptivas de la base total de artículos, comenzando con la distribución de frecuencias, continuando con el cálculo de medidas de tendencia central y concluyendo con un análisis multivariante de redes de palabras claves para determinar la coocurrencia de todas las palabras definidas por el autor e incluidas en el resumen. Estos datos fueron agrupados en tres grandes temas, que aparecen bajo estos títulos en el artículo: primero, aquellos que resultaban ser indicadores de colaboración; segundo, los relacionados con un alto indicador de uso, descarga y citaciones de los artículos publicados; y, tercero, los que describen asociaciones temáticas y de naturaleza semántica, que cierran el análisis. Resultados Descripción general Los textos incluyen reportes de investigación, reflexión y revisiones, presentaciones de la revista, artículos del cuerpo editorial (editoriales), ensayos, entrevistas, ponencias y reseñas. La tipología de artículos con la que se realizó el procesamiento no necesariamente corresponde a la que se indica en la descripción de cada artículo, dado que esta ha cambiado a lo largo de los números publicados. Para este análisis, se organizaron de acuerdo con las siguientes definiciones: Muestra Para el análisis se trabajó sobre la totalidad de los artículos publicados por magis : desde el volumen 1, número 1, publicado en 2008 hasta el volumen 10, número 20, publicado en 2017. En total, se consideraron 251 textos. La base de datos trabajada identifica títulos de los artículos, cada uno de sus autores, sus instituciones de afiliación y los países de su localización, tipología del artículo, metodología seguida —en el caso de los reportes de investigación—, número de descargas del archivo PDF en plataforma y de citaciones en Google magis Académico y Scopus, y palabras clave definidas por los autores y presentes en el abstract de los artículos. Es importante anotar que, en estos veinte números, magis ha editado tres números monográficos: el número 8, de 2011, dedicado al tema de violencia en las escuelas; el 12, publicado en 2013, enfocado en la enseñanza de las ciencias y la diversidad cultural; y el número 14, disponible en 2014, con foco en los dilemas y los desafíos de la investigación cualitativa en educación. Interesa, de manera especial, examinar el comportamiento de estos números en el contexto general de lo publicado, a fin de alimentar decisiones futuras al respecto con base en la evidencia disponible. • Presentación: textos elaborados por el editor o el director de la revista para anunciar los artículos que componen el número. • Editorial: textos de reflexión y/o análisis sobre procesos o productos de la revista magis que permiten analizar asuntos concretos de su trayectoria. Son elaborados por miembros del comité editorial y/o del equipo editorial. • Artículos de investigación: textos inéditos que presentan procesos y resultados de investigaciones terminadas. Análisis de publicaciones de los primeros 20 números de magis una mirada en doble sentido. Interna, en la medida en que permite ver el comportamiento de magis en sus 10 primeros años y su marco de relación con los planteamientos iniciales que le dan origen. Externa, pues da cuenta de un balance de la investigación en educación en esos años. Desde otro punto de vista, creemos que la experiencia acumulada en magis puede ser útil a otras revistas científicas en el campo de la educación y, en general, de las ciencias sociales. Desde sus inicios, magis se dispuso y se mantuvo en el índice bibliográfico nacional —Publindex—, y fue la primera revista científica nacional del ámbito de la educación en ser aceptada en la base de Scopus. En este sentido, su experiencia puede ser muy útil para otras revistas nacionales que empiezan a seguir sus pasos. Es importante anotar que no se pretende hacer un juicio de valor sobre la calidad de lo publicado en estos años. La revista ha seguido procesos editoriales que, en mucho, incluso han excedido los controles habitualmente establecidos por las revistas científicas en relación con el número, la diversidad y el nivel de formación de los revisores, la cantidad de instancias y los filtros utilizados. Al respecto, estamos bastante seguros de la calidad de lo publicado. Sin embargo, las reglas que rigen el uso, la circulación y la difusión de un artículo particular o de un número completo aún están bajo nuestra lupa, y nos interesa comprenderlas mucho mejor. Este análisis es, también, un intento para avanzar en ese sentido. A continuación, se analiza el comportamiento de magis en estos 10 años, con una metodología que recoge estadísticas descriptivas y un análisis multivariante de redes de palabras claves. Los resultados presentan tipos de artículos publicados, enfoques de investigación, indicadores de colaboración, citas y descargas, y asuntos temáticos como niveles educativos objeto de las investigaciones y coocurrencia de palabras clave para visibilizar temáticas preferentes que definen ciertas tendencias. 223 magis VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 224 • Artículos de reflexión derivados de investigación: textos de reflexión que presentan elaboraciones teóricas, interpretaciones críticas, conceptualizaciones relacionadas con aspectos epistemológicos o construcciones metodológicas de una o más investigaciones realizadas por los autores. • Artículos de revisión: revisiones exhaustivas de los avances y tendencias en un campo de conocimiento, una temática o una problemática particular. Se caracterizan por presentar una cuidadosa revisión bibliográfica de mínimo 50 referencias citadas. • Entrevistas: transcripción de una entrevista oral realizada a un investigador que se destaca por su trayectoria en el campo de la educación y la investigación en educación. • Reseñas: textos expositivos sobre el contenido de un libro en educación, con el interés de promover su lectura. • Ensayos: documentos de reflexión en educación, no derivados de procesos de investigación concretos. • Ponencias: textos que se han presentado en eventos académicos y que recogen reflexiones relevantes y vigentes sobre educación e investigación en los campos pertinentes. En este punto puede ser importante distinguir los textos que son propiamente artículos de otros tipos de documentos que no lo son. Entre los primeros están los ensayos, los reportes de investigación, las reflexiones derivadas de investigación y las revisiones. Estos son 203 artículos, que representan el 80,9% de los textos publicados por la revista. El porcentaje restante está representado por 48 textos, que corresponden al 19,1% del total, y agrupan presentaciones de los números, textos editoriales, entrevistas, ponencias y reseñas. Estos últimos no serán tenidos en cuenta en lo que sigue. La tabla 1 muestra la distribución de la totalidad de los textos publicados en los primeros veinte números de magis. Una simple división nos indica que, en promedio, cada número de magis ha sacado a la luz 12,5 textos, de los cuales 10 son artículos. Tabla 1 Distribución de textos publicados en magis por tipología Tipología de textos No. (%) % en artículos Ensayo 19 7,6 9,4 Investigación 107 42,6 52,7 Reflexión derivada de investigación 61 24,3 30,0 Revisión 16 6,4 7,9 Subtotal 203 80,9 100,0 Presentación 20 8,0 41,7 Editorial 4 1,6 8,3 Entrevista 4 1,6 8,3 Ponencia 4 1,6 8,3 Reseña 16 6,4 33,3 Subtotal 48 19,1 100,0 251 100,0 Artículos Otros documentos Total Fuente: elaboración propia En relación con los artículos que serán objeto de nuestro análisis, la gran mayoría de los textos corresponde a reportes de investigación (107 artículos, que representan el 52,2% del total), seguido por reflexiones derivadas de investigación (61, 30,5%). Los ensayos y las revisiones son los tipos de artículo con menor frecuencia de publicación en magis, con porcentajes menores a 10%. Ver gráfica 1. Revisión Reflexión derivado de investigación Investigación Fuente: elaboración propia De la totalidad de los reportes de investigación, el 53,3% corresponde a investigaciones de enfoque cualitativo (57 artículos), mientras que solo 35 casos son investigaciones de enfoque cuantitativo (32,7%). En 15 investigaciones (14,0%), el enfoque elegido es de tipo mixto. Tabla 2 Distribución de investigaciones por enfoque de investigación Enfoque No. (%) Cualitativo 57 53,3 Cuantitativo 35 32,7 Mixto 15 14,0 Total 107 100,0 Fuente: elaboración propia La mayoría de los textos publicados está en castellano (86,7%), solo 15 artículos están escritos en idioma inglés (7,4%), 11 en portugués (5,4%) y solo 1 en francés (0,4%). Indicadores de colaboración En general, los textos son elaborados por un número variable de autores, hasta un máximo de diez. En su mayor parte, los textos poseen un solo autor (88 artículos, que representan el 43,3% del total) o dos autores (67, 33,0%). El 92,1% de los artículos es escrito por tres autores o menos. Ver tabla 3. PÁGINA Análisis de publicaciones de los primeros 20 números de magis Ensayo magis Gráfica 1 Distribución de artículos por tipología 225 Tabla 3 Tabla 4 Distribución de artículos por número de autores Distribución de artículos por país de la institución magis de afiliación de los autores VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 226 Número de autores No. (%) 1 88 43,3 2 67 33,0 3 32 15,8 4 9 5 País No. (%) Alemania 1 0,5 Argentina 13 6,0 Australia 2 0,9 4,4 Brasil 13 6,0 3 1,5 Canadá 1 0,5 6 3 1,5 Chile 25 11,5 10 1 0,5 Colombia 80 36,9 Ecuador 2 0,9 Total 203 100,0 España 40 18,4 Estados Unidos 5 2,3 Francia 1 0,5 Grecia 1 0,5 Inglaterra 1 0,5 Israel 1 0,5 México 23 10,6 Perú 1 0,5 Portugal 3 1,4 Reino Unido 2 0,9 Uruguay 1 0,5 Venezuela 1 0,5 Total 217 100,0 Fuente: elaboración propia Los autores de los artículos publicados aparecen afiliados a un total de 155 instituciones. Aquellas de aparición más frecuente son, en su orden, la institución de casa, la Pontificia Universidad Javeriana con 22 apariciones (14,2%), la Universidad Distrital con 16 apariciones (10,3%) y la Universidad Pedagógica Nacional de Colombia con 14 apariciones (9,0%). Otras instituciones con menores niveles de aparición son la Universidad de Antioquia con 9 apariciones (5,8%) y, en este punto, aparecen dos instituciones extranjeras: la Universidad de Barcelona y CONICET, cada una con 5 apariciones (3,2%). Las demás instituciones muestran cuatro o menos apariciones. El número de instituciones diferentes que colaboran para producir un único artículo alcanza un máximo de siete. La mayoría de los artículos están firmados por uno o varios autores de una misma institución (148, 72,9%). En 45 casos (22,2%) figuran dos instituciones. En un caso bastante atípico, los diez autores que firman un artículo aparecen afiliados a siete instituciones diferentes. En consonancia con lo anterior, la colaboración internacional no es muy alta. Solo en 14 artículos (6,9%), los autores del texto trabajan en instituciones localizadas en países diferentes. Con respecto al país en el que están ubicadas las instituciones representadas por los diferentes autores, en Colombia está el 36,9%. Otros países que muestran buenos niveles de participación en la revista son España (18,4%), Chile (11,5%), México (10,6%), Argentina (6,0%) y Brasil (6,0%). Entre estos seis países se ubica casi el 90% de la participación. Las instituciones ubicadas en otros países muestran, cada una, menos del 2% de participación. Fuente: elaboración propia Citas y descargas magis es una revista de acceso abierto. Los datos de descarga de los documentos en formato PDF representan una buena aproximación al grado de circulación del conocimiento que difunde, aunque, debe mencionarse, no es el único ni el más preciso. Una vez descargado un archivo en formato PDF, este puede ser reproducido y enviado un número indefinido de veces, sin que quede registro alguno de ello. Aun así, el dato de descargas puede darnos una buena idea de la demanda y del interés generado por el artículo entre los lectores. La tabla 5 muestra las medidas de tendencia central de tres indicadores: el número de descargas de archivos en formato PDF en la web de la revista, el número de citas registradas en la plataforma de Google Académico y el número de citas registradas en revistas indexadas en la base de Scopus. Tabla 5 Mínimos, máximos, medias y desviaciones típicas de las descargas y citaciones de artículos de magis Mínimo Máximo Media Desviación estándar Citas Scopus 0 13 1,0 1,75 Citas Google Académico 0 231 11,3 21,80 Descargas PDF 143 47.524 1.313,8 3.493,48 Fuente: elaboración propia magis Los resultados muestran que los artículos son descargados en formato PDF en numerosas ocasiones. En promedio, cada artículo ha sido descargado 1.313,9 veces. Si bien hay enormes diferencias al respecto entre los artículos (D. E. = 3.493,5). Acoso escolar. De las causas, origen y manifestaciones a la pregunta por el sentido que le otorgan los actores es el artículo más descargado entre los publicados por magis ; esta revisión, publicada en 2011, en el volumen 4, número 8, de Luis Evelio Castillo-Pulido, tiene 47.524 descargas, desde su publicación hasta diciembre de 2018. Este dato, sin embargo, es atípico, por lo que será excluido de los siguientes análisis. A buena distancia del anterior, el segundo artículo con mayor frecuencia de descargas es El informe de investigación con estudio de casos, una reflexión derivada de investigación, de Froylán Antonio Ceballos-Herrera (2009), con 9.214 descargas. La tabla 6 muestra los 20 artículos más descargados. Análisis de publicaciones de los primeros 20 números de magis PÁGINA Tabla 6 Lista de los 20 artículos de magis más descargados Volumen 4 1 2 8 1 Número 8 2 3 17 2 Año 2011 2009 2009 2016 2009 Tipo Referencia Revisión Castillo-Pulido, L. (2011). El acoso escolar. De las causas, origen y manifestaciones a la pregunta por el sentido que le otorgan los actores. Magis, Revista Internacional de Investigación en Educación, 4 (8), 415-428. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/3572 Descargas 47.524 Reflexión Ceballos-Herrera, F. (2009). El informe de investigación con estudio de casos. Magis, Revista Internacional de Investigación en Educación, 1 (2), 413-423. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/3394 9.214 Investigación González-Moreno, C.; Solovieva, Y. & Quintanar-Rojas, L. (2009). La actividad de juego temático de roles en la formación del pensamiento reflexivo en preescolares. Magis, Revista Internacional de Investigación en Educación, 2 (3), 173-190. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/3407 6.561 Investigación Montanares-Vargas, E. & Llancavil-Llancavil, D. (2016). Uso de fuentes históricas en formación inicial de profesores. Magis, Revista Internacional de Investigación en Educación, 8 (17), 85-98. https://doi.org/10.11144/Javeriana.m8-17.ufhf 6.537 Ensayo Hoyos-Vásquez, G. (2009). Educación para un nuevo humanismo. Magis, Revista Internacional de Investigación en Educación, 1 (2), 425-433. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3395 5.821 Continúa 227 Volumen 1 magis 1 VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 9 Número 1 1 19 Año 2008 2008 2017 Tipo Referencia Reflexión Touriñan-López, J. (2008). Teoría de la educación: investigación disciplinar y retos epistemológicos. Magis, Revista Internacional de Investigación en Educación, 1 (1), 175-194. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/3363 5.588 Reflexión Murillo-Torrecilla, F. J. (2008). Los modelos multinivel como herramienta para la investigación educativa. Magis, Revista Internacional de Investigación en Educación, 1 (1), 45-62. Recuperado a partir de https://revistas.javeriana.edu.co/ index.php/MAGIS/article/view/3355 4.463 Investigación Duque-Gómez, L. (2017). Conflicto social colombiano: representación en textos escolares de ciencias sociales. Magis, Revista Internacional de Investigación en Educación, 9 (19), 49-68. https://doi.org/10.11144/Javeriana.m9-19.cscr 4.237 Investigación Orellana-García, P. & Melo-Hurtado, C. (2014). Ambiente letrado y estrategias didácticas en la educación preescolar chilena. Magis, Revista Internacional de Investigación en Educación, 6 (13), 113-128. https://doi.org/10.11144/ Javeriana.M6-13.ALED 4.199 Reflexión Mireles-Vargas, O. (2015). Metodología de la investigación: operaciones para develar representaciones sociales. Magis, Revista Internacional de Investigación en Educación, 8 (16), 149-166. https://doi.org/10.11144/Javeriana.m8-16.miop 4.050 Investigación Torres-Puentes, E. & Arias-Gómez, D. (2011). La pedagogía crítica en la experiencia carcelaria de presas políticas. Magis, Revista Internacional de Investigación en Educación, 4 (7), 27-43. Recuperado a partir de https://revistas.javeriana.edu. co/index.php/MAGIS/article/view/3554 3.871 Reflexión Herrera-González, J. D. (2010). La formación de docentes investigadores: el estatuto científico de la investigación pedagógica. Magis, Revista Internacional de Investigación en Educación, 3 (5), 53-62. Recuperado a partir de https:// revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3526 3.284 Revisión Uribe-Álvarez, G. & Camargo-Martínez, Z. (2011). Prácticas de lectura y escritura académicas en la universidad colombiana. Magis, Revista Internacional de Investigación en Educación, 3 (6), 317-341. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/3543 3.260 Revisión Palacios-Mena, N. & Herrera-González, J. (2013). Subjetividad, socialización política y derechos en la escuela. Magis, Revista Internacional de Investigación en Educación, 5(11), 413-437. Recuperado a partir de https://revistas. javeriana.edu.co/index.php/MAGIS/article/view/5710 3.129 Revisión Bolívar-Botía, A. (2010). ¿Cómo un liderazgo pedagógico y distribuido mejora los logros académicos? Revisión de la investigación y propuesta. Magis, Revista Internacional de Investigación en Educación, 3 (5), 79-106. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/3528 3.086 Revisión Andrade-Lotero, L. (2012). Teoría de la carga cognitiva, diseño multimedia y aprendizaje: un estado del arte. Magis, Revista Internacional de Investigación en Educación, 5 (10), 75-92. Recuperado a partir de https://revistas.javeriana.edu. co/index.php/MAGIS/article/view/4166 2.685 Investigación Estévez-Nenninger, E.; Valdés-Cuervo, Á.; Arreola-Olivarría, C. & Zavala-Escalante, M. (2014). Creencias sobre enseñanza y aprendizaje en docentes universitarios. Magis, Revista Internacional de Investigación en Educación, 6 (13), 49-64. https://doi.org/10.11144/Javeriana.M6-13.CSEA 2.670 228 6 8 4 3 3 5 3 5 6 13 16 7 5 6 11 5 10 13 2014 2015 2011 2010 2011 2013 2010 2012 2014 Descargas Continúa 7 2 2 14 3 Año 2009 2014 2009 Tipo Referencia Reflexión Bozu, Z. (2009). El profesorado universitario novel y su proceso de inducción profesional. Magis, Revista Internacional de Investigación en Educación, 1 (2), 317-328. Recuperado a partir de https://revistas.javeriana.edu.co/ index.php/MAGIS/article/view/3387 2.601 Reflexión Guzmán-Valenzuela, C. (2014). Polos epistemológicos: uso y construcción de teoría en investigación cualitativa en educación. Magis, Revista Internacional de Investigación en Educación, 7 (14), 15-28. https://doi.org/10.11144/Javeriana. M7-14.PEUC 2.539 Investigación Ruiz, M. & Mora, M. (2009). Una aproximación a las prácticas de enseñanza de la lectura y la escritura en una escuela colombiana en 1940. Magis, Revista Internacional de Investigación en Educación, 2 (3), 15-28. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/3400 2.431 Fuente: elaboración propia Vale la pena observar dos puntos de la tabla 6. Primero, en casi todas las ocasiones, los más descargados corresponden a artículos publicados en los primeros cinco años de magis. Esto es esperable pues esos artículos han estado disponibles más tiempo en la plataforma. Solo seis artículos relativamente recientes figuran en esa lista: los de Duque-Gómez (2017), Montanares-Vargas y Llancavil-Llancavil (2016), Estévez-Nenninger, Valdés-Cuervo, Arreola-Olivarría y Zavala-Escalante (2016). Segundo, es importante notar una clara presencia de las revisiones en esta lista. Cinco revisiones aparecen en los 20 artículos más descargados, aun cuando esta no es una tipología particularmente frecuente en magis. Tal como lo presentamos en la tabla 1, las revisiones son apenas el 7,9% del total de artículos publicados en estos 10 años. Mientras que el número de descargas en PDF puede ser un buen indicador de lectura de un artículo por parte de estudiantes, los registros de citaciones son un mejor indicador de su uso por parte de investigadores. En lo que sigue, examinaremos las citaciones a los artículos publicados en magis en Google Académico y Scopus. En promedio, cada artículo de magis ha sido citado 11,38 veces en Google Académico, con grandes diferencias entre los artículos (D. E. = 21,8). En Scopus, el promedio de citaciones es bastante menor (M = 1,0, D. E. = 1,7). La tabla 7 muestra los 20 artículos con mayor cantidad de citaciones en Google Académico. Los resultados revelan que el artículo más citado en esta plataforma alcanza las 231 citaciones y corresponde, de nuevo, al artículo de revisión ¿Cómo un liderazgo pedagógico y distribuido mejora los logros académicos? Revisión de la investigación y propuesta, de Antonio Bolívar-Botía (2010), que ya había aparecido en el puesto 16 de los artículos más descargados. A buena distancia del anterior, el segundo artículo más citado es El acoso escolar. De las causas, origen y manifestaciones a la pregunta por el sentido que le otorgan los actores, de Luis Evelio CastilloPulido (2011), otra revisión, que alcanza 121 citas. En la tabla 6, este artículo aparece como el más descargado. Al igual que en las descargas, las revisiones tienden también a ser los artículos más citados. En la lista de los 20 más citados, cinco son revisiones y cuatro de ellas ocupan los primeros cuatro puestos. Descargas magis 1 Número PÁGINA Análisis de publicaciones de los primeros 20 números de magis Volumen 229 Por otro lado, como ya lo habíamos notado en el caso de las descargas, los artículos más citados han sido publicados en los primeros números de magis, si bien en este caso no hay excepciones. Entre los 20 artículos más citados en Google Académico no aparece ninguno de los últimos cinco volúmenes. Ver tabla 7. magis Tabla 7 Lista de los 20 artículos más citados en Google Académico VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA Volumen Número Año Tipo Referencia Revisión Bolívar-Botía, A. (2010). ¿Cómo un liderazgo pedagógico y distribuido mejora los logros académicos? Revisión de la investigación y propuesta. Magis, Revista Internacional de Investigación en Educación, 3 (5), 79-106. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3528 231 Revisión Castillo-Pulido, L. (2011). El acoso escolar. De las causas, origen y manifestaciones a la pregunta por el sentido que le otorgan los actores. Magis, Revista Internacional de Investigación en Educación, 4 (8), 415-428. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3572 121 Reflexión Ceballos-Herrera, F. (2009). El informe de investigación con estudio de casos. Magis, Revista Internacional de Investigación en Educación, 1 (2), 413-423. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3394 87 Revisión Uribe-Álvarez, G. & Camargo-Martínez, Z. (2011). Prácticas de lectura y escritura académicas en la universidad colombiana. Magis, Revista Internacional de Investigación en Educación, 3 (6), 317-341. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3543 61 Reflexión Murillo-Torrecilla, F. J. (2008). Los modelos multinivel como herramienta para la investigación educativa. Magis, Revista Internacional de Investigación en Educación, 1 (1), 45-62. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/3355 57 Investigación González-Moreno, C.; Solovieva, Y. & Quintanar-Rojas, L. (2009). La actividad de juego temático de roles en la formación del pensamiento reflexivo en preescolares. Magis, Revista Internacional de Investigación en Educación, 2 (3). 173-190. Recuperado a partir de https:// revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3407 53 Ensayo Hoyos-Vásquez, G. (2009). Educación para un nuevo humanismo. Magis, Revista Internacional de Investigación en Educación, 1 (2), 425-433. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3395 48 Investigación Padilla, C. (2012). Escritura y argumentación académica: trayectorias estudiantiles, factores docentes y contextuales. Magis, Revista Internacional de Investigación en Educación, 5 (10), 31-57. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/4164 41 230 3 4 1 3 1 2 1 5 5 8 2 6 1 3 2 10 2010 2011 2009 2011 2008 2009 2009 2012 Citas Google Continúa 5 1 3 1 4 2 3 1 5 10 2 6 1 7 4 6 2 Año 2010 2012 2009 2011 2008 2011 2010 2011 2009 Tipo Referencia Citas Google Reflexión Herrera-González, J. D. (2010). La formación de docentes investigadores: el estatuto científico de la investigación pedagógica. Magis, Revista Internacional de Investigación en Educación, 3 (5), 53-62. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3526 40 Ensayo Soares-Leite, W. & Nascimento-Ribeiro, C. A. do. (2012). A inclusão das TICs na educação brasileira: problemas e desafios. Magis, Revista Internacional de Investigación en Educación, 5 (10), 173-187. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/4172 40 Investigación Santos-Rego, M.; Lorenzo-Moledo, M. & PriegueCaamaño, D. (2009). Aprendizaje cooperativo: práctica pedagógica para el desarrollo escolar y cultural. Magis, Revista Internacional de Investigación en Educación, 1 (2), 289-303. Recuperado a partir de https://revistas. javeriana.edu.co/index.php/MAGIS/article/view/3385 39 Investigación Valdés-Cuervo, A.; Arreola-Olivarría, C.; AnguloArmenta, J.; Carlos-Martínez, E. & García-López, R. (2011). Actitudes de docentes de educación básica hacia las TIC. Magis, Revista Internacional de Investigación en Educación, 3 (6), 379-392. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3546 37 Revisión Solaz-Portolés, J. & Sanjosé-López, V. (2008). Conocimientos y procesos cognitivos en la resolución de problemas de ciencias: consecuencias para la enseñanza. Magis, Revista Internacional de Investigación en Educación, 1 (1), 147-162. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3361 37 Investigación Cartolari, M. & Carlino, P. (2011). Leer y tomar apuntes para aprender en la formación docente: un estudio exploratorio. Magis, Revista Internacional de Investigación en Educación, 4 (7), 67-86. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3557 36 Investigación Flórez-Romero, R. & Arias-Velandia, N. (2010). Evaluación de conocimientos previos del aprendizaje inicial de lectura. Magis, Revista Internacional de Investigación en Educación, 2 (4), 329-344. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3516 33 Investigación Muñoz-González, J.; Ontoria-Peña, A. & MolinaRubio, A. (2011). El mapa mental, un organizador gráfico como estrategia didáctica para la construcción del conocimiento. Magis, Revista Internacional de Investigación en Educación, 3 (6), 343-361. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3544 32 Investigación Jarauta-Borrasca, B. & Medina-Moya, J. (2009). La formación pedagógica inicial del profesorado universitario: repercusión en las concepciones y prácticas docentes. Magis, Revista Internacional de Investigación en Educación, 1 (2), 357-370. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3390 32 magis 3 Número PÁGINA Continúa Análisis de publicaciones de los primeros 20 números de magis Volumen 231 Volumen 1 1 10 Año 2008 2012 Tipo Referencia Investigación Soriano de Alencar, E. & de Souza-Fleith, D. (2008). Criatividade Pessoal: Fatores Facilitadores e Inibidores Segundo Estudantes de Engenharia. Magis, Revista Internacional de Investigación en Educación, 1 (1), 113-126. Recuperado a partir de https://revistas. javeriana.edu.co/index.php/MAGIS/article/view/3359 32 Reflexión Molina-Natera, V. (2012). Escritura a través del currículo en Colombia: situación actual y desafíos. Magis, Revista Internacional de Investigación en Educación, 5 (10), 93-108. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/4167 30 Investigación Rodríguez-Jiménez, O. & Murillo-Torrecilla, F. (2011). Estimación del efecto escuela para Colombia. Magis, Revista Internacional de Investigación en Educación, 3 (6), 299-316. Recuperado a partir de https://revistas. javeriana.edu.co/index.php/MAGIS/article/view/3959 30 magis 5 Número VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA Citas Google 232 3 6 2011 Fuente: elaboración propia Hay algunas coincidencias entre los artículos de las dos listas que hemos expuesto. Primero, ambas listas comparten todas las revisiones. Segundo, Educación para un nuevo humanismo, el texto de Guillermo HoyosVásquez (2009), aparece entre los 5 más descargados en la plataforma y es el séptimo más citado en Google Académico, siendo el único ensayo que se incluye en estas tablas. Igual ocurre con los artículos de MurilloTorrecilla (2008), Los modelos multinivel como herramienta para la investigación educativa, y González-Moreno, Solovieva y Quintanar-Rojas (2012), La actividad de juego temático de roles en la formación del pensamiento reflexivo en preescolares, que aparecen entre los 10 más descargados y los 10 más citados en Google Académico. Otras coincidencias en las tablas son la presencia repetida de algunos autores con más de un artículo reflejado en las tablas. Francisco Javier Murillo, por ejemplo, aparece con dos artículos entre los 20 más citados en Google Académico, al igual que José Darío Herrera quien, en la lista de los 20 más descargados, aparece destacado con dos artículos diferentes. Los dos autores aparecen tanto en la lista de los más descargados como en la de los más citados. En este sentido, son autores que han aportado a magis más de un artículo en los 20 números y sus publicaciones han generado un número destacado de descargas y citaciones. Por último, vale la pena examinar las citas en la base de Scopus. En este caso, el promedio de citas es bastante menor que el mostrado en la base de Google (media = 1,03; D. E. = 1,75). De los artículos publicados por magis en estos 10 años, el artículo más citado en esta base alcanza apenas 13 citas y coincide con el más citado en Google Académico: ¿Cómo un liderazgo pedagógico y distribuido mejora los logros académicos? Revisión de la investigación y propuesta, de Bolívar-Botía (2012). A este le siguen dos artículos que alcanzan 8 citas. Solo dos artículos, que alcanzan 4 citas, han sido publicados en los últimos cinco años. En la tabla 8 se presentan los artículos que han alcanzado, al menos, cuatro citas en la base de Scopus. Tabla 8 Lista de artículos con cuatro o más citas en la base de Scopus 1 2 2 1 5 4 1 5 3 5 1 4 3 2 10 9 2 10 6 Año 2010 2008 2010 2009 2009 2012 2012 2009 2012 2011 Tipo Referencia Citas Scopus Revisión Bolívar-Botía, A. (2010). ¿Cómo un liderazgo pedagógico y distribuido mejora los logros académicos? Revisión de la investigación y propuesta. Magis, Revista Internacional de Investigación en Educación, 3 (5), 79-106. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3528 13 Reflexión Murillo-Torrecilla, F. J. (2008). Los modelos multinivel como herramienta para la investigación educativa. Magis, Revista Internacional de Investigación en Educación, 1 (1), 45-62. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/3355 8 Investigación Gurr-Mark, D.; Drysdale-George, L. & Mulford, B. (2010). Australian Principal Instructional Leadership: Direct and Indirect Influence. Magis, Revista Internacional de Investigación en Educación, 2 (4), 299-314. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3514 8 Investigación González-Moreno, C.; Solovieva, Y. & Quintanar-Rojas, L. (2009). La actividad de juego temático de roles en la formación del pensamiento reflexivo en preescolares. Magis, Revista Internacional de Investigación en Educación, 2 (3), 173-190. Recuperado a partir de https:// revistas.javeriana.edu.co/index.php/MAGIS/article/ view/3407 7 Investigación Santos-Rego, M.; Lorenzo-Moledo, M. & PriegueCaamaño, D. (2009). Aprendizaje cooperativo: práctica pedagógica para el desarrollo escolar y cultural. Magis, Revista Internacional de Investigación en Educación, 1 (2), 289-303. Recuperado a partir de https://revistas. javeriana.edu.co/index.php/MAGIS/article/view/3385 6 Reflexión Molina-Natera, V. (2012). Escritura a través del currículo en Colombia: situación actual y desafíos. Magis, Revista Internacional de Investigación en Educación, 5 (10), 93108. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/4167 5 Investigación González-Moreno, C. (2012). Formación del pensamiento reflexivo en estudiantes universitarios. Magis, Revista Internacional de Investigación en Educación, 4 (9), 595617. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/3578 5 Reflexión Ceballos-Herrera, F. (2009). El informe de investigación con estudio de casos. Magis, Revista Internacional de Investigación en Educación, 1 (2), 413-423. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/3394 4 Investigación Padilla, C. (2012). Escritura y argumentación académica: trayectorias estudiantiles, factores docentes y contextuales. Magis, Revista Internacional de Investigación en Educación, 5 (10), 31-57. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/4164 4 Investigación Valdés-Cuervo, A.; Arreola-Olivarría, C.; AnguloArmenta, J.; Carlos-Martínez, E. & García-López, R. (2011). Actitudes de docentes de educación básica hacia las TIC. Magis, Revista Internacional de Investigación en Educación, 3 (6), 379-392. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/3546 4 magis 3 Número PÁGINA Continúa Análisis de publicaciones de los primeros 20 números de magis Volumen 233 2 1 2010 2008 Investigación 4 Investigación Soriano de Alencar, E. & de Souza-Fleith, D. (2008). Criatividade Pessoal: Fatores Facilitadores e Inibidores Segundo Estudantes de Engenharia. Magis, Revista Internacional de Investigación en Educación, 1 (1), 113126. Recuperado a partir de https://revistas.javeriana. edu.co/index.php/MAGIS/article/view/3359 4 Investigación Rodríguez-Jiménez, O. & Murillo-Torrecilla, F. (2011). Estimación del efecto escuela para Colombia. Magis, Revista Internacional de Investigación en Educación, 3 (6). Recuperado a partir de https://revistas.javeriana.edu. co/index.php/MAGIS/article/view/3959 4 Investigación Conejeros-Solar, M.; Gómez-Arizaga, M. & DonosoOsorio, E. (2013). Perfil docente para alumnos/as con altas capacidades. Magis, Revista Internacional de Investigación en Educación, 5(11), 393-411. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/ MAGIS/article/view/5709 4 Reflexión Touriñan-López, J. (2008). Teoría de la educación: investigación disciplinar y retos epistemológicos. Magis, Revista Internacional de Investigación en Educación, 1 (1), 175-194. Recuperado a partir de https://revistas. javeriana.edu.co/index.php/MAGIS/article/view/3363 4 Investigación Salvà-Mut, F.; Oliver-Trobat, M. & Comas-Forgas, R. (2014). Abandono escolar y desvinculación de la escuela: perspectiva del alumnado. Magis, Revista Internacional de Investigación en Educación, 6 (13), 129-142. https:// doi.org/10.11144/Javeriana.M6-13.AEDE 4 Reflexión Tobin, K. (2013). A Sociocultural Approach to Science Education. Magis, Revista Internacional de Investigación en Educación, 6 (12), 19-35. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/7199 4 magis 1 4 Flórez-Romero, R. & Arias-Velandia, N. (2010). Evaluación de conocimientos previos del aprendizaje inicial de lectura. Magis, Revista Internacional de Investigación en Educación, 2 (4), 329-344. Recuperado a partir de https://revistas.javeriana.edu.co/index.php/MAGIS/ article/view/3516 VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 234 3 5 1 6 6 6 11 1 13 12 2011 2013 2008 2014 2013 Fuente: elaboración propia Varios puntos deben destacarse en esta lista, especialmente en contraste con las dos anteriores. Primero, aunque el artículo más citado es una revisión, es el único texto de esta tipología que aparece en la lista. Desaparece acá la preeminencia de las revisiones ya antes observada, para dar paso a las investigaciones: de los 17 artículos más citados en Scopus 11 son reportes de investigación. Segundo, sigue apareciendo una clara tendencia a la citación de algunos autores y algunos temas. Los trabajos de Bolívar-Botía (2010), González-Moreno, Solovieva y Quintanar-Rojas (2012), Murillo-Torrecilla (2008) y Ceballos-Herrera (2009), que ya habían aparecido en las listas anteriores, vuelven a aparecer en esta lista entre los más citados. En relación con el idioma, es importante anotar que entre las dos listas previas, los artículos más descargados y los más citados en Google Académico, no había presencia de artículos en idiomas diferentes al español. Ahora, en la lista de los más citados en Scopus hay un artículo en Gráfica 2 Promedio de descargas PDF por número (excluido caso atípico) 2000 1800 1600 1400 1200 1000 800 600 400 200 0 Vol. 1 Vol. 1 Vol. 2 Vol. 2 Vol. 3 Vol. 3 Vol. 4 Vol. 4 Vol. 4 Vol. 5 Vol. 5 Vol. 6 Vol. 6 Vol. 7 Vol. 7 Vol. 8 Vol. 8 Vol. 9 Vol. 9 Vol. 10 Núm. 1 Núm.  2 Núm. 3 Núm. 4 Núm. 5 Núm. 6 Núm. 7 Núm. 8 Núm. 9 Núm. 10 Núm. 11 Núm. 12 Núm. 13 Núm. 14 Núm. 15 Núm. 16 Núm. 17 Núm. 18 Núm. 19 Núm. 20 (2008) (2009) (2009) (2010) (2010) (2011) (2011) (2011) (2012) (2012) (2013) (2013) (2014) (2014) (2015) (2015) (2016) (2016) (2017) (2017) Fuente: elaboración propia magis PÁGINA Análisis de publicaciones de los primeros 20 números de magis portugués: el de Soriano de Alencar & Souza-Fleith (2008), titulado Criatividade Pessoal: Fatores Facilitadores e Inibidores Segundo Estudantes de Engenharia y dos artículos en inglés, el de Tobin (2013), A Sociocultural Approach to Science Education y el de Gurr-Mark, Drysdale-George & Mulford (2010), titulado Australian Principal Instructional Leadership: Direct and Indirect Influence. Esto parece señalar que la publicación en lengua extranjera, si bien no muestra una incidencia en los artículos más descargados y citados en Google Académico, sí marca una diferencia en la citación en Scopus. A fin de contrastar la hipótesis de que los artículos con mayor tiempo en la plataforma muestran más descargas y más citaciones, se examinan las medias de descargas y citaciones por número. Los resultados se presentan en las siguientes tres gráficas. En términos generales, el mayor volumen de descargas se presenta en el número 8 que casi alcanza 7.000 descargas. Esto se explica, sin embargo, por un solo artículo, al que ya nos habíamos referido. Excluyendo el artículo El acoso escolar. De las causas, origen y manifestaciones a la pregunta por el sentido que le otorgan los actores, de Luis Evelio CastilloPulido (2011), una mirada general sobre el promedio de descargas de los diferentes números muestra variaciones interesantes. En promedio, hay 1.313 descargas por número; se destacan los números 1, 2, 3, 16 y 17, que superan la media de 1.400 descargas, en especial los números 1, 2 y 17 que superan las 1.600 descargas. El número de descargas de los tres primeros números es esperable por el tiempo que han estado disponibles en línea. Sin embargo, resaltan los números 16 y 17 que, a pesar de haber sido publicados recientemente (menos de 4 años), tienen una media destacada de descargas en sus artículos. Igual tendencia muestra el número 19, de muy reciente aparición. Tal vez la única característica distintiva de estos números es que incluyen, en su contenido, artículos en idiomas diferentes del español. 235 Gráfica 3 Promedio de citas en Google Académico por número 35 30 25 20 magis 15 VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 236 10 5 0 Vol. 1 Vol. 1 Vol. 2 Vol. 2 Vol. 3 Vol. 3 Vol. 4 Vol. 4 Vol. 4 Vol. 5 Vol. 5 Vol. 6 Vol. 6 Vol. 7 Vol. 7 Vol. 8 Vol. 8 Vol. 9 Vol. 9 Vol. 10 Núm. 1 Núm.  2 Núm. 3 Núm. 4 Núm. 5 Núm. 6 Núm. 7 Núm. 8 Núm. 9 Núm. 10 Núm. 11 Núm. 12 Núm. 13 Núm. 14 Núm. 15 Núm. 16 Núm. 17 Núm. 18 Núm. 19 Núm. 20 (2008) (2009) (2009) (2010) (2010) (2011) (2011) (2011) (2012) (2012) (2013) (2013) (2014) (2014) (2015) (2015) (2016) (2016) (2017) (2017) Fuente: elaboración propia En relación con el promedio de citas de artículos de cada número en Google Académico, se destacan algunos con más de 20 citaciones (números 5, 8 y 6) que, si bien no son los publicados recientemente, tampoco corresponden a los más antiguos, de manera que presentan una tendencia destacada en citación en esta plataforma. Gráfica 4 Promedio de citas en Scopus por número 2.5 2,0 1.5 1,0 0.5 0,0 Vol. 1 Vol. 1 Vol. 2 Vol. 2 Vol. 3 Vol. 3 Vol. 4 Vol. 4 Vol. 4 Vol. 5 Vol. 5 Vol. 6 Vol. 6 Vol. 7 Vol. 7 Vol. 8 Vol. 8 Vol. 9 Vol. 9 Vol. 10 Núm. 1 Núm.  2 Núm. 3 Núm. 4 Núm. 5 Núm. 6 Núm. 7 Núm. 8 Núm. 9 Núm. 10 Núm. 11 Núm. 12 Núm. 13 Núm. 14 Núm. 15 Núm. 16 Núm. 17 Núm. 18 Núm. 19 Núm. 20 (2008) (2009) (2009) (2010) (2010) (2011) (2011) (2011) (2012) (2012) (2013) (2013) (2014) (2014) (2015) (2015) (2016) (2016) (2017) (2017) Fuente: elaboración propia Por su parte, la citación en Scopus muestra valores más bajos y tendencias diferentes a los más citados en Google académico. En esta base de datos, la tendencia es claramente decreciente, sin mayores excepciones. Los números más citados son, en su orden, los números 4 y 1, con una media de más de 2 citas por artículo, y el número 11, con poco más de 1,6 citas por artículo. El resto de los números publicados no supera una media de 1,3 citas por artículo. A partir del volumen 7, la media de citas Aspectos temáticos Nivel educativo En la medida en que magis es una publicación científica dedicada al ámbito de la educación, la gran mayoría de los artículos que publica se dirige a uno o varios niveles educativos específicos. Al respecto, la tabla 9 muestra la distribución de los niveles educativos. Tal como se observa, hay una buena cantidad de artículos, 55, que no se refieren a un nivel educativo específico (27,1%). El nivel educativo objeto más frecuente en los artículos examinados es el universitario, que es mencionado en 70 casos (34,4%). En 54 artículos se menciona el nivel de secundaria (26,6%) y, con proporciones similares, en 41 se menciona la primaria (20,2%). El preescolar es el nivel más desatendido en las publicaciones de magis, pues solo es mencionado en 25 casos (12,3%). La educación no formal, por otro lado, solo es objeto de trabajo en 6 textos (3,0%). Tabla 9 Distribución de artículos por el nivel educativo al que están dirigidos Nivel educativo No. (%) No refiere 55 27,1 Preescolar 25 12,3 Primaria 41 20,2 Secundaria 54 26,6 Universitario 70 34,4 Educación No formal 6 3,0 Fuente: elaboración propia Análisis de coocurrencia de palabras claves A fin de categorizar las temáticas preferentemente tratadas en los artículos, se corrió un análisis de conglomerados (cluster analysis) que identifica grupos de palabras claves, en lengua inglesa, que tienden a presentarse de forma simultánea en los artículos publicados. El análisis se hizo a través del software VOSviewer. Se consideraron las palabras clave que habían sido mencionadas en, al menos, tres ocasiones. magis PÁGINA Análisis de publicaciones de los primeros 20 números de magis cae de forma muy acentuada. Solo el volumen 8, número 16, muestra un mayor número de citas. En general, no se identifica un impacto destacable de los números monográficos (8, 12 y 14) en el panorama de las descargas por número. Sin embargo, en las citaciones en Google Académico y Scopus algunos de estos sobresalen. En Google Académico, el número 8, monográfico dedicado a la violencia en las escuelas, supera significativamente el promedio de citaciones de los números anterior y siguiente en este espacio; es el segundo número con promedio más alto de citaciones en Google académico. Por su parte, el número 14, dedicado al tema de dilemas y desafíos en investigación cualitativa en educación, muestra el promedio de citación más alto en Scopus entre los números publicados de 2013 a 2017. El número 12, sin embargo, dedicado a la educación en ciencias, presenta niveles relativamente bajos en descargas y citaciones, lo cual seguramente estará relacionado con una comunidad más pequeña y restringida de maestros, investigadores e interesados en el tema. Estos resultados nos pueden dar información importante para la elaboración de monográficos en el futuro. 237 magis VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 238 El procedimiento define siete conglomerados que agrupan 53 palabras clave. Cada palabra está representada por un nodo en la gráfica. Los tamaños de los nodos representan su peso relativo y el color de los nodos representa el conglomerado al que pertenece. El primer conglomerado, rojo en la gráfica 5, agrupa 11 palabras, a saber: bullying, violence, school violence, social representations, teacher development, teaching practice, school, primary education, youth, environmental education y knowledge. Por sus características, este conglomerado contiene artículos sobre agresión y violencia en la escuela primaria, en relación cercana con el rol del maestro frente a este fenómeno, en cuanto recoge elementos relacionados con su desempeño y sus prácticas. Vincula, además, las representaciones sociales y las condiciones del entorno que podrían influir en la presencia de fenómenos de violencia en la escuela. El segundo conglomerado de palabras clave, de color verde en la gráfica 5, agrupa 10 ítems. Por sus características, se incluyen los artículos relacionados con aspectos culturales: educación intercultural e indígena en la educación básica, en los que tiende a aparecer la metodología de enseñanza conocida como “aprendizaje cooperativo”, junto con el aprendizaje activo. Podría pensarse que refieren a investigaciones que se relacionan con procesos de intervención en comunidades específicas. Aparecen en este conglomerado referencias geográficas puntuales (México, Latinoamérica). El tercer conglomerado, azul oscuro en la gráfica 5, agrupa nueve ítems que se articulan alrededor de la enseñanza y el aprendizaje en la educación superior, en especial con objetos relacionados con la lectura, la escritura y el entrenamiento de habilidades en una disciplina concreta. La práctica reflexiva parece ser una metodología de aparición frecuente en este conglomerado. Dado que se trata de un tema de alta relevancia en la literatura en los últimos años, es posible pensar que se agrupan artículos que recogen experiencias sobre el tema del lenguaje en las universidades, desde la idea de repensarlas, tensionarlas y reconstruirlas, desde la práctica reflexiva. El cuarto conglomerado, amarillo en la gráfica 5, agrupa 8 ítems. Este parece ser de carácter bastante general, pues agrupa conceptos como educación, ciencia, comunicación y menciones a la metodología de la investigación en la universidad. Hay también referencia a la memoria y a las emociones, lo que hace pensar en reflexiones generales sobre el conocimiento pedagógico. El quinto conglomerado, de color violeta en la gráfica 5, contiene 7 ítems y parece girar en torno a temas de política e investigación educativa, mencionando las palabras epistemología, alfabetización, educación a través de los medios. Aparecen en este conglomerado referencias a la metodología de investigación y, específicamente, a metodologías de tipo cualitativo. El sexto conglomerado, azul claro en la gráfica 5, contiene cinco ítems y se refiere específicamente a temas de educación en ciencias y cultura científica en el nivel secundario. Los conceptos de argumentación y cultura escolar tienen una fuerte presencia en este grupo de artículos. Dado que en este conglomerado se ubica el nivel secundario, puede tener relación con los procesos escolares que buscan dar a los estudiantes las herramientas necesarias para afrontar la vida universitaria, pues es posible relacionar la cultura científica con procesos de argumentación académica. Finalmente, el séptimo conglomerado, de color naranja en el gráfico 5, contiene solo tres ítems y agrupa los trabajos sobre docentes y prácticas de enseñanza, en especial dirigidos a preescolar, que como vimos previamente es un nivel poco atendido en los artículos de magis. Gráfica 5 Red de coocurrencias de palabras clave magis En general, vale la pena anotar que ciertos objetos de investigación aparecen ligados a niveles educativos específicos. Los temas de maestros y prácticas de enseñanza están ligados a la educación preescolar. La educación básica primaria, por su parte, aparece en relación con temas de educación intercultural y acoso escolar. En la educación secundaria se han publicado temas de alfabetización y cultura científica. A su vez, la educación universitaria parece enfatizar en temas de la enseñanza y el aprendizaje de la lectura y la escritura. Lo anterior muestra una cercanía entre lo abordado en educación secundaria y universitaria. Análisis de publicaciones de los primeros 20 números de magis PÁGINA Fuente: elaboración propia Sobre la base de la red de palabras que se acaba de presentar, es posible superponer la información de años de publicación. El resultado se muestra en la gráfica 6. Como se observa, la tendencia de los artículos más recientemente publicados se relaciona con las temáticas de la educación intercultural (segundo conglomerado), los temas de juventud, educación primaria y secundaria, y los de alfabetización y desarrollo profesoral. Las temáticas relacionadas con educación en ciencias y las reflexiones sobre la naturaleza del saber educativo parecen concentrarse en los primeros números de magis. En relación con los cambios de esos asuntos que han sido objeto constante de los artículos publicados en magis en estos 10 años, los conglomerados muestran algunos temas que, más que cambios, evidencian transformaciones. Por ejemplo, las preocupaciones en torno al maestro han tomado formas diversas. Inicialmente, el foco estaba puesto en los 239 magis procesos de enseñanza que agenciaban, su formación y la práctica reflexiva como espacio de transformación. Estos temas se transforman en unos más específicos que, hacia los últimos números, abordan directamente el desarrollo de los profesores. Otro tópico constante que se va acotando es el relacionado con temas culturales. En los primeros números aparece como tema amplio “lo cultural”, mientras que, en los últimos números, aunque el interés permanece, se focaliza en asuntos concretos y poblaciones: aparecen con fuerza la educación intercultural y la educación con población indígena. 240 VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA Gráfica 6 Fecha de publicación en la red de coocurrencias de palabras clave Fuente: elaboración propia Discusión y conclusiones Los resultados indican una revista que se ha mantenido con buenos niveles de calidad y con una periodicidad regular. En su mayoría, lo publicado está conformado por reportes o reflexiones de investigación, mientras que los artículos de revisión bibliográfica y/o bibliométrica resultan ser minoritarios. Probablemente, este sea el momento para hacer un llamado a la comunidad científica —en general— a considerar los estudios históricobibliográficos como un área que se debe explorar, desarrollar y posicionar en la investigación en educación. magis PÁGINA Análisis de publicaciones de los primeros 20 números de magis En términos generales, magis ha publicado, principalmente, reportes de investigación, pues estos manuscritos representan más del 50% de los artículos de estos 20 números. En los artículos de investigación se identificó un predominio del enfoque cualitativo del 53,3%, frente a porcentajes más bajos del enfoque cuantitativo y mixto. Esto es consistente con la tendencia predominante en la investigación educativa, en especial la latinoamericana, que muestra cierto privilegio por este enfoque. Los indicadores de colaboración no son muy altos. El 43% de los artículos son escritos por un solo autor y el 72% de los artículos proviene de una institución. Solo en el 22% de los artículos aparecen dos instituciones y únicamente en el 7% de los casos, estas instituciones están localizadas en países diferentes. Esto puede estar en relación con una tendencia manifiesta a tratar problemas más bien locales que globales, lo cual es visible tanto en la dimensión de lo institucional como en la de lo nacional. Aun así, puede destacarse que en casi el 63% de los artículos aparecen autores afiliados a instituciones localizadas en países diferentes al de la revista, en especial España, Chile y México. En este sentido, en línea con su proyecto inicial, magis ha logrado llegar e impactar a lectores de la región. A partir de este panorama de los primeros 20 números de magis, se identifican oportunidades sobre asuntos y tipologías que podrían tener más fuerza en la revista. El análisis permite reconocer la potencialidad de los artículos de revisión. Este tipo de artículos se destaca en descargas y citaciones y, en este sentido, se reconoce como tipología apetecida por estudiantes e investigadores. Los idiomas diferentes al castellano (inglés y portugués, según las citaciones) son leídos y citados por quienes tienen acceso a la base Scopus. En este sentido, podría pensarse en aumentar la presentación de estos idiomas en los números de magis. En relación con los niveles educativos, se reconoció el preescolar como el más desatendido en los artículos de magis, de manera que podría potenciarse la publicación de artículos que aborden este nivel para presentar resultados más diversos y abarcadores. Al adentrarnos en aspectos temáticos, se destacan ciertos grupos en los artículos publicados en magis que aparecen, curiosamente, ligados a niveles educativos específicos. Las problemáticas ligadas a los docentes y las prácticas educativas han aparecido especialmente en relación con la educación preescolar. Los temas de acoso escolar y violencia en la escuela son de reciente aparición y se examinan en relación con la educación primaria. En la educación básica, primaria y secundaria aparecen también temas relacionados con interculturalidad en la escuela y, de igual forma, figuran entre los artículos de más reciente aparición. Se alcanzan a vislumbrar acá algunos temas que fueron apuestas iniciales de magis. La educación secundaria, por su parte, está ligada en especial a artículos sobre las temáticas de la educación en ciencias, que han sido publicados, principalmente, en los primeros volúmenes. La educación universitaria aparece, por su parte, asociada preponderantemente con investigaciones sobre lectura y escritura en los estudiantes. En este sentido, es posible evidenciar una transición tanto temática como en los niveles educativos, pues en los primeros números se focaliza sobre la enseñanza de las disciplinas y su abordaje desde la educación secundaria, mientras que, con el pasar de los años, la preocupación se concentra específicamente en la educación universitaria y el foco está puesto en las especificidades de la lectura y la escritura en los aprendizajes de cada disciplina. Un aspecto destacable es que, pese a que las revisiones, como tipo de artículo, representan menos del 10% de los artículos publicados en estos 10 años, tienen un alto impacto en las descargas y en las citaciones de 241 magis VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242 PÁGINA 242 Google Académico. De los 20 textos más citados, cinco son revisiones y cuatro están en los primeros puestos. En los más citados de Scopus, aunque solo aparece una revisión, es el primer artículo de la lista. Por otro lado, lo presentado da elementos para considerar la importancia y utilidad de los números monográficos. Al respecto, aunque positivos, los resultados pueden tener un dejo agridulce. Algunos de los monográficos muestran altos índices de citación y de descargas, y otros, no tanto. Esto nos lleva a considerar que las ventajas en la realización de este tipo de números estarán en relación directa con el interés que la temática despierte en una comunidad amplia de maestros y especialistas. Al plantear un monográfico, automáticamente se restringe de forma importante la cantidad de lectores del número a los directamente relacionados con —e interesados en— el tema específico. Esto, por otro lado, es también positivo pues este número puede convertirse en referencia obligada sobre el tema. Lo anterior permite sugerir la búsqueda de una solución intermedia que retome las ventajas de la restricción temática, sin que por ello enfrentemos una pérdida de lectores en el número. La solución, en ese sentido, puede estar dada por la publicación de dossiers especializados que no alcancen a cubrir la totalidad del número. Otras revistas, como la Revista Colombiana de Educación, han seguido antes, con bastante éxito, ese camino. En general, el balance muestra distancias significativas entre las descargas hechas sobre los artículos y la citación que se hace de ellos. Puede pensarse que quienes descargan son, en su mayoría, estudiantes universitarios de los diferentes niveles, que consultan los artículos como marcos para sus propios trabajos e investigaciones, o bien maestros en ejercicio, más que investigadores y autores que escriben y divulgan sus artículos en bases de alto reconocimiento como Scopus. Podría decirse que los artículos son mayoritariamente descargados por un público no especializado en publicaciones científicas, que no necesariamente investiga, pero que está altamente interesado y vinculado con la educación. Es decir, no solo estudiantes universitarios, sino también profesores de los diferentes niveles que, en el marco de estudios continuos o por interés propio, consultan determinados artículos de la revista que llaman su atención, de modo que la utilidad tiene qué ver más con la reflexión y la práctica propia que con la generación de conocimiento en una investigación formal. Ello respondería a una de las apuestas de magis desde sus orígenes: aportar a la consolidación de una comunidad académica que piensa la educación, no solo desde las investigaciones de alto nivel, sino en las reflexiones continuas de los maestros que ejercen cada día. Para magis, llegar a los 10 años ha implicado, además del presente análisis, una revisión interna de cara a las necesidades de cambio que se han evidenciado a lo largo de los años, así como externa en función de los nuevos requerimientos que ahora se hacen a las publicaciones seriadas. Después de un proceso de reflexión, desde el equipo editorial y de la mano con el comité editorial, hemos reformulado varios elementos para agilizar los tiempos de respuesta a los autores, ahorrar y focalizar esfuerzos en la evaluación, y ganar terreno en la visibilidad y la lectura de los artículos publicados. Esto nos llevó, entre otras decisiones, a reconsiderar la pertinencia del ejemplar impreso, que funcionaba más como tradición y colección que como lugar real de lectura. Hacia afuera, surgen cambios que representan aún retos por abordar, como el ajuste en la concepción del número publicado como unidad que nos ha organizado en este tiempo, para pensar el proceso de publicación del artículo a medida que este culmina su proceso editorial. En fin, seguimos aprendiendo y cambiando, para dar lo mejor a nuestros lectores, autores y revisores, en estos tiempos desafiantes. Sobre los autores Christian Hederich-Martínez es matemático. Magíster en Educación, Universidad Pedagógica Nacional, UPN, Colombia y Doctor en Psicología, Universidad Autónoma de Barcelona, UAB, España. Profesor, Universidad Pedagógica Nacional, UPN, Colombia. Editor de la Revista Colombiana de Educación. Miembro del comité editorial de magis. Catalina Roa-Casas es licenciada en Pedagogía Infantil, Pontificia Universidad Javeriana, Colombia. Magíster en Educación, Pontificia Universidad Javeriana, Colombia. Profesora, Pontificia Universidad Javeriana, Facultad de Educación y Departamento de Formación, Colombia. Editora de magis. Referencias García-Cepero, C. (2008). Documento maestro. Magis, Revista Internacional de Investigación en Educación. Inédito.
https://openalex.org/W2037443393
https://docs.lib.purdue.edu/cgi/viewcontent.cgi?article=1011&context=bmepubs
English
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Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons
PloS one
2,013
cc-by
10,752
Follow this and additional works at: http://docs.lib.purdue.edu/bmepub Recommended Citation Babbs, Charles F. and Shi, Riyi, "Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons." (2013). Weldon School of Biomedical Engineering Faculty Publications. Paper 14. http://dx.doi.org/10.1371/journal.pone.0067767 7-3-2013 Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons. Charles F. Babbs Purdue University, babbs@purdue.edu Riyi Shi Charles F. Babbs Purdue University, babbs@purdue.edu Follow this and additional works at: http://docs.lib.purdue.edu/bmepubs Purdue University Purdue e-Pubs Purdue University Purdue e-Pubs Weldon School of Biomedical Engineering Abstract This study explores in detail the functional consequences of subtle retraction and detachment of myelin around the nodes of Ranvier following mild-to-moderate crush or stretch mediated injury. An equivalent electrical circuit model for a series of equally spaced nodes of Ranvier was created incorporating extracellular and axonal resistances, paranodal resistances, nodal capacitances, time varying sodium and potassium currents, and realistic resting and threshold membrane potentials in a myelinated axon segment of 21 successive nodes. Differential equations describing membrane potentials at each nodal region were solved numerically. Subtle injury was simulated by increasing the width of exposed nodal membrane in nodes 8 through 20 of the model. Such injury diminishes action potential amplitude and slows conduction velocity from 19.1 m/ sec in the normal region to 7.8 m/sec in the crushed region. Detachment of paranodal myelin, exposing juxtaparanodal potassium channels, decreases conduction velocity further to 6.6 m/sec, an effect that is partially reversible with potassium ion channel blockade. Conduction velocity decreases as node width increases or as paranodal resistance falls. The calculated changes in conduction velocity with subtle paranodal injury agree with experimental observations. Nodes of Ranvier are highly effective but somewhat fragile devices for increasing nerve conduction velocity and decreasing reaction time in vertebrate animals. Their fundamental design limitation is that even small mechanical retractions of myelin from very narrow nodes or slight loosening of paranodal myelin, which are difficult to notice at the light microscopic level of observation, can cause large changes in myelinated nerve conduction velocity. R (2013) Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons. PLoS ONE 8(7): e67767 0067767 tation: Babbs CF, Shi R (2013) Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons. P i:10.1371/journal.pone.0067767 Received January 29, 2013; Accepted May 21, 2013; Published July 3, 2013 Copyright:  2013 Babbs, Shi. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: babbs@purdue.edu * E-mail: babbs@purdue.edu membrane. Charles F. Babbs*, Riyi Shi ences, Center for Paralysis Research, and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, Unite Department of Basic Medical Sciences, Center for Paralysis Research, and Weldon School of Biomedical Engineering, Purdue Unive States of America Department of Basic Medical Sciences, Center for Paralysis Research, and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States of America Recommended Citation This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact epubs@purdue.edu for additional information. Abstract Beyond the paranodal regions are the functionally important juxtaparanodal zones, where the density of transmem- brane channels carrying outward potassium current is high. Ring- like bands of paranodal cell-cell attachments separate and insulate the bare nodes from the juxtaparanodal regions. Since myelin inhibits the conduction of ionic current, the action potential tends to jump from one node to the next along the longitudinal axis of an axon. This process of jumping, or ‘‘saltatory conduction’’, boosts the speed of propagation of action potentials along myelinated axons to tens of meters per second, rather than tens of centimeters per second typical of unmyelinated axons [5]. Introduction Arrows indicate flow of positive ionic current during depolarization of Node 1 as conduction of the action potential moves toward Node 2. This sketch is foreshortened in the axial dimension. Anatomically L/s , 1000. (b) Schematic cross section of a myelinated nerve or fiber tract. Each axon (solid black) is surrounded by a sheath of myelin (white) and in turn surrounded by a sheath of non- myelinated tissue (shaded) known as endoneurium or neuropil. The mean radius of the sheath of endoneurium is denoted re and the thickness of the sheath is denoted h. The cross section of endoneurium, 2preh, is much greater than that of the axon. doi:10.1371/journal.pone.0067767.g001 Conduction in Injured Myelinated Axons Conduction in Injured Myelinated Axons Figure 1. Relevant anatomy. (a) Schematic longitudinal section of a myelinated axon. The width of each node of Ranvier is denoted s. The distance between nodes is denoted L. Arrows indicate flow of positive ionic current during depolarization of Node 1 as conduction of the action potential moves toward Node 2. This sketch is foreshortened in the axial dimension. Anatomically L/s , 1000. (b) Schematic cross section of a myelinated nerve or fiber tract. Each axon (solid black) is surrounded by a sheath of myelin (white) and in turn surrounded by a sheath of non- myelinated tissue (shaded) known as endoneurium or neuropil. The mean radius of the sheath of endoneurium is denoted re and the thickness of the sheath is denoted h. The cross section of endoneurium, 2preh, is much greater than that of the axon. doi:10.1371/journal.pone.0067767.g001 Figure 1. Relevant anatomy. (a) Schematic longitudinal section of a myelinated axon. The width of each node of Ranvier is denoted s. The distance between nodes is denoted L. Arrows indicate flow of positive ionic current during depolarization of Node 1 as conduction of the action potential moves toward Node 2. This sketch is foreshortened in the axial dimension. Anatomically L/s , 1000. (b) Schematic cross section of a myelinated nerve or fiber tract. Each axon (solid black) is surrounded by a sheath of myelin (white) and in turn surrounded by a sheath of non- myelinated tissue (shaded) known as endoneurium or neuropil. The mean radius of the sheath of endoneurium is denoted re and the thickness of the sheath is denoted h. The cross section of endoneurium, 2preh, is much greater than that of the axon. Introduction The majority of the axons in vertebrate nervous systems are wrapped with insulating layers of back-to-back cell membranes called myelin. The mechanisms by which myelin speeds axonal conduction in health and by which damage to myelin leads to loss of axonal conduction in disease have attracted much attention [1]. Still, the causes of myelin related functional deficits remain incompletely understood [2–4]. Continuing multidisciplinary investigation combining morphology, electrophysiology, and mathematical modeling may elucidate critical mechanisms and perhaps guide the development of effective treatments for medical conditions involving myelin damage, including traumatic injury to the brain or spinal cord and demyelinating diseases such as multiple sclerosis. Here we explore in detail the functional consequences of subtle injury to the well-known anatomic arrangement of the nodes of Ranvier, coupled with the more recently discovered segregation of nodal sodium channels from juxtaparanodal potassium channels [6–8]. Compared to the internodal regions, a node of Ranvier has a relatively high density of sodium channels, which enable the generation of action potential at the node. The virtual absence of sodium channels in the paranodal and juxtaparanodal regions, together with high electrical resistance of the paranodal region, blocks axial ionic current beneath the myelin sheath and favors saltatory conduction. Potassium channels, on the other hand, are segregated from sodium channels in nodes of Ranvier, residing predominantly beneath the myelin at the juxtaparanodal area, and separated from the node by the paranodal region [9]. There is now Myelin forming Schwann cells in the peripheral nervous system or oligodendrocytes in the central nervous system wrap around the axon multiple times to create laminated layers of insulating cell membrane, as shown schematically in Figure 1. Periodic short gaps in the myelin sheath along the axons having width, s, approximately 0.3 to 1 micrometer, are the Nodes of Ranvier, where the density of transmembrane channels carrying inward sodium current is high and where transmembrane action potentials are initiated. Immediately adjacent to the nodes themselves on either side in the axial dimension are the paranodal regions, where myelin is tightly attached to the underlying axonal July 2013 | Volume 8 | Issue 7 | e67767 1 PLOS ONE | www.plosone.org Figure 1. Relevant anatomy. (a) Schematic longitudinal section of a myelinated axon. The width of each node of Ranvier is denoted s. The distance between nodes is denoted L. Introduction doi:10.1371/journal.pone.0067767.g001 second order partial differential equation that describes the voltages at each node as functions of time and space. Such equations are known as cable equations, because of their similarity to cable or transmission line equations [25], and the corresponding models of nerve conduction are known as cable models. Subsequent, more detailed mathematical treatment of composite, myelinated axon models [23,24] leads to expressions essentially similar to the original solutions of FitzHugh. (Compare, for example, Basser Eq. (35) and Nygren and Halter Eq. (18) with FitzHugh Eq. (1)). general agreement that mechanical crush or stretch injury results in myelin damage, especially in the paranodal region [10,11]. This idea is consistent with biomechanical modeling showing significant stress in this area under mechanical insults [12], causing retraction of myelin away from the node [13,14]. With more severe injury there is also de-coupling and detachment of paranodal myelin, opening a low resistance gap or sleeve-like channel between the node and the juxtaparanodal region, through which increased potassium ion current may flow [10]. Subtle retraction and detachment of paranodal myelin can have two electrical effects. The first is increasing bare nodal area, which increases the electrical capacitance of the node. The second is loosening of paranodal cell-cell junctions, which reduces the normally high electrical resistance between the juxtaparanodal potassium channels and the nodal capacitance. In the present paper we consider the biophysics of nodal function before and after traumatic myelin retraction and partial detachment to explain and predict the effects of such subtle mechanical injury, which is sufficiently mild that it is clearly visible only at the electron microscopic level of observation [10]. Our approach is to employ a mathematical model of ionic current flow that reflects changes caused by such subtle injury to the nodal and paranodal regions. The goal of the present paper is to determine quantitatively how myelinated nerve conduction velocity depends upon the local integrity of the nodes and paranodal regions at the electron microscopic level of observation and how nerve conduction might be slowed or blocked by the known pathological changes to these structures [2]. We also explore how drug treatments that block potassium conductance in the juxtaparanodal region may act to restore conduction in subtly damaged regions. Toward this end it is insightful to derive from first principles a FitzHugh style cable model of myelinated axon conduction for the specific purpose of characterizing the parameters related to subtle injury. Methods The use of mathematical models to study mechanisms of saltatory conduction is well precedented [15–24]. Classical and more modern approaches almost universally involve a form of cable equation or cable model, as originally described by FitzHugh [15]. This approach regards successive nodes as a chain of leaky capacitors having voltage sensitive ion channels, and connected in parallel by extracellular and intracellular resistances between the nodes. Currents and voltages at each node are computed from a Model of a Myelinated Axon PLOS ONE | www.plosone.org for constants, a and b. Such functions nicely describe the classical waveforms of time- varying conductance determined experimentally in units of Seimens per square centimeter. It is easy to show using calculus that the values of a and b required to produce a peak conductance G* at time t* after onset of activation are given by the expressions a~G  (e=t  )2 and b~2=t, were e is the base of the natural logarithms <2.781. In turn, values of a and b describing textbook normal activation functions for sodium and potassium conduc- tance [5] can be computed as shown in Table 2. Specifically, we assume for the purpose of this study that the densities of sodium and potassium of ion channels per square centimeter of membrane in the regions of the nodes of Ranvier where such channels are concentrated is similar to that classically described for non- myelinated axons [6]. To specify extracellular and axonal resistances, Re and Ra, we note that the resistance of a volume conductor in terms of its length, cross-section, and resistivity is R~r L A , ð1aÞ ð1aÞ where r is resistivity, L is length, and A is cross sectional area [26]. Resistivity is the intrinsic property of a material that opposes the flow of steady electric current and is expressed in units of ohm-cm. From the geometry of Figure 1 we can write algebraic expressions for the internal resistance of an axon, Ra, between nodes as a function of the axon radius, ra, and also for the external resistance, Re, of the sleeve of tissue surrounding the myelin, through which current must flow between two adjacent nodes to complete the electrical circuit. For Ra and Re we have where r is resistivity, L is length, and A is cross sectional area [26]. Resistivity is the intrinsic property of a material that opposes the flow of steady electric current and is expressed in units of ohm-cm. From the geometry of Figure 1 we can write algebraic expressions for the internal resistance of an axon, Ra, between nodes as a function of the axon radius, ra, and also for the external resistance, Re, of the sleeve of tissue surrounding the myelin, through which current must flow between two adjacent nodes to complete the electrical circuit. Model of a Myelinated Axon A simplified equivalent electrical circuit for a series of equally spaced nodes of Ranvier, together with associated paranodal and juxtaparanodal regions, is shown in Figure 2. The intracellular resistance between nodes along the axons is denoted Ra and is much larger than electrical resistance of the extracellular current path between nodes, Re, (not show in Figure 2). The nodal July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org 2 Conduction in Injured Myelinated Axons Time-varying Sodium and Potassium Currents capacitances of the exposed, non-myelinated axon segments at each node are represented as C~2prasCm, where Cm is the axon membrane capacitance per unit area, s is the span of bare, unmyelinated axon in a node of Ranvier, and ra is the radius of the axon. Definitions of symbols and variables are provided for reference in Table 1. Local inward sodium currents, iNa0, iNa1, iNa2, … and outward potassium currents iK0, iK1, iK2, … for nodes 0, 1, 2, … and nearby juxtaparanodal regions are governed by time varying transmembrane conductances, GNa0, GNa1, GNa2 … for sodium and GK0, GK1, GK2 …. for potassium and the respective sodium and potassium equilibrium potentials [5]. Convenient descriptive formulas for ion specific conductances as functions of time, t’j, after onset of activation of node, j, (including both the bare nodal membrane and functional juxtaparanodal regions) have the form: Because of the relatively large distance between nodes compared to the node width, the segregation of ion channels for inward sodium current and outward potassium current in the nodal and juxtaparanodal regions does not change or invalidate the simplified equivalent circuit shown in Figure 2 as a realistic, model of a myelinated axon segment. However, the potassium current sources in the juxtaparanodal regions are a few micrometers distant from the sodium current sources in the bare nodal regions. Potassium current must flow between the axolem- ma and tightly adherent myelin in the paranodal regions in order to repolarize the nodal capacitance. This ultrastructural detail may add a substantial series resistance to the potassium ion ‘‘battery’’, which is omitted for simplicity in Figure 2 but described in detail in Appendix S1. G(t’j)~at’2 j e{bt’j ð2Þ ð2Þ for constants, a and b. for constants, a and b. For Ra and Re we have Using these descriptive functions for time-dependent sodium and potassium conductance per square centimeter of membrane area, the corresponding local nodal currents at node, j, and adjacent paranodal and juxtaparanodal membranes of the model become iNaj~2prasGNaj(ENa{Vj) ð3aÞ ð3aÞ and Ra~ raL pr2 a and Re~ reL 2preh , ð1bÞ ð1bÞ iKj~ Vj{EK 1 2pral GKj zRp ð3bÞ ð3bÞ where a sleeve of aqueous extracellular fluid of thickness h and mean radius re surrounds the myelin sheath over distance L and has extracellular fluid resistivity, re. Noting that extracellular resistance is about two orders of magnitude less than axonal resistance leads to a compact model shown in Figure 2. in terms of the axon radius, ra, unstretched node width, s, length of juxtaparanodal region on both sides of the node, l, local prevailing transmembrane potential, Vj, and the sodium and potassium Figure 2. Electrical model for multiple nodes of Ranvier. Arrows indicate directions of positive ionic current. Shading indicates foreshortened myelinated regions. In life the actual distance between nodes (,1000 mm) is much greater than the width of a single node (,1 mm). Current is denoted by i, capacitance by C, resistance by R, and voltage by V. Lumped ionic currents from sodium and potassium channels in and around each node are shown as a single current source. doi:10.1371/journal.pone.0067767.g002 Figure 2. Electrical model for multiple nodes of Ranvier. Arrows indicate directions of positive ionic current. Shading indicates foreshortened myelinated regions. In life the actual distance between nodes (,1000 mm) is much greater than the width of a single node (,1 mm). Current is denoted by i, capacitance by C, resistance by R, and voltage by V. Lumped ionic currents from sodium and potassium channels in and around each node are shown as a single current source. doi:10.1371/journal.pone.0067767.g002 July 2013 | Volume 8 | Issue 7 | e67767 3 PLOS ONE | www.plosone.org 3 Conduction in Injured Myelinated Axons Table 1. Nomenclature. for constants, a and b. Symbol Definition Units A Area cm2 C Capacitance of bare membrane in a node of Ranvier farads Cm Specific membrane capacitance per unit area farads/cm2 d Axon diameter cm e Base of natural logarithms (2.718…) G Membrane conductance per unit area ohm21 cm22 h Thickness of a conducting sheath surrounding a myelinated axon cm i, i0, i1 Positive ionic current during an action potential amperes l Length of juxtaparanodal region cm L Distance between nodes of Ranvier cm n Number of nodes in a discrete model p Circle ratio (3.1415 …) Ra Axonal electrical resistance between nodes of Ranvier ohms Re Extracellular electrical resistance between nodes of Ranvier ohms Rp Paranodal electrical resistance between a node and both juxtaparanodal regions of a myelinated axon ohms ra Radius of an axon cm re Radius of a conducting sheath surrounding a myelinated axon cm r, re, ra Resistivity of extracellular or axonal fluid ohm-cm s Span of bare axon in a node of Ranvier in the axial dimension cm t Clock time sec t Clock time of activation of a node sec t9 Time after node activation (t 2 t) sec V Transmembrane potential difference volts Vth Threshold potential for initiation of an action potential volts v Nerve conduction velocity m/sec doi:10.1371/journal.pone.0067767.t001 Table 1. Nomenclature. doi:10.1371/journal.pone.0067767.t001 Table 2. Constants for descriptive functions for membrane conductance per unit area, G(t’j)~at’2 j e{bt’j. Coordinates of peak conductance [5] Model function constants t* (sec) G* (ohm–1cm22) a (ohm–1cm–2sec–2) b (sec–1) Na+ 0.0001 0.028 2.16107 26104 K+ 0.0005 0.013 3.86105 46103 doi:10.1371/journal.pone.0067767.t002 Table 2. Constants for descriptive functions for membrane conductance per unit area, G(t’j)~at’2 j e{bt’j. Table 2. Constants for descriptive functions for membrane conductance per unit area, G(t’j)~at’2 j e{bt’j. equilibrium potentials ENa and EK, as described in [5]. In calculating potassium current, the juxtaparanodal length, l, including both sides of the node, is taken as approximately 10 times the normal nodal length based on cytochemical studies of potassium ion distribution [4,14]. Expression (3b) represents the current passing through the juxtaparanodal membrane conduc- tance in series with paranodal resistance, Rp. Rp is the resistance of the thin sleeve of extracellular matrix material through which potassium current must flow from the juxtaparanodal regions to the bare axonal membrane of the local node of Ranvier. Note for the case Rp = 0 the expression for potassium current is analogous to that for sodium current. for constants, a and b. If Rp were exceedingly large, then potassium current would be zero. Details of the treatment of potassium current in the presence of paranodal resistance and the estimation of a numerical value for paranodal resistance are provided in Appendix S1. equilibrium potentials ENa and EK, as described in [5]. In calculating potassium current, the juxtaparanodal length, l, including both sides of the node, is taken as approximately 10 times the normal nodal length based on cytochemical studies of potassium ion distribution [4,14]. Expression (3b) represents the current passing through the juxtaparanodal membrane conduc- tance in series with paranodal resistance, Rp. Rp is the resistance of the thin sleeve of extracellular matrix material through which potassium current must flow from the juxtaparanodal regions to the bare axonal membrane of the local node of Ranvier. Note for the case Rp = 0 the expression for potassium current is analogous to that for sodium current. If Rp were exceedingly large, then potassium current would be zero. Details of the treatment of potassium current in the presence of paranodal resistance and the estimation of a numerical value for paranodal resistance are provided in Appendix S1. doi:10.1371/journal.pone.0067767.t002 Conduction in Injured Myelinated Axons Conduction in Injured Myelinated Axons Estimation of other Parameters We consider a textbook normal case as a primary model. Membrane ionic conductances per square centimeter are as listed in Table 2. From anatomic sections such as those in [28] the radius of the sleeve of fibrous tissue is about 6 micrometers and the thickness of the sleeve is about 2 micrometers. Axon diameter averages 1.0 micrometer [29–31]. The width, s, of a normal node of Ranvier ranges from about 0.3 micrometer to about 1.0 micrometer, or 0.3 to 1.0 times the diameter of an axon, in various anatomic specimens [28]. A middle value for the width, s, of a node is about 0.65 micrometer [10,32]. The span of the juxtaparanodal region, l, on both sides of a normal node is taken as 10 times the axon radius [33]. The resistivity of intracellular fluid is about 200 ohm-cm [31]. The paranodal resistance, Rp, is estimated as 3.261010 ohms, as explained in Appendix S1. The specific membrane capacitance of nerve cell membranes is about 1 mircofarad per square centimeter [5]. Using these values to evaluate parameters, together with normal membrane potentials [5] (resting membrane potential –85 mV, threshold potential – 50 mV, sodium equilibrium potential +67 mV, potassium equi- librium potential –95 mV), one can specify the values of all parameters for a standard normal model. Numerical Integration Increasing or decreasing Dt without effect on the results confirmed that a sufficiently small value was chosen for Dt. Wave propagation typically stabilized over three successive nodes. For active nodes the net current flowing onto a nodal capacitance is the difference between inflow to and outflow from the node. Thus, the general structure of equations (5) and (6) below is: rate of voltage change = transmembrane sodium current inflow, minus transmembrane potassium current outflow, plus axon current inflow, minus axon current outflow, all divided by local capacitance. Ohm’s Law allows calculation of axon current inflow and outflow from voltages at adjacent nodes. For interior nodes j = 1, 2, … n –1 dVj dt ~ ij Cj ~ iNaj{iKjz Vj{1{Vj Ra { Vj{Vjz1 Ra   1 Cj : ð5Þ For left hand node 0 dV0 dt ~ i0 C0 ~ iNa0{iK0{ V1{V2 Ra   1 C0 : ð6Þ dVj dt ~ ij Cj ~ iNaj{iKjz Vj{1{Vj Ra { Vj{Vjz1 Ra   1 Cj : ð5Þ ð5Þ Simulation of Injury and Drug Treatment In models of localized crush injury of the spinal cord an impulse was initiated at node 0. Nodes 1 through 7 had normal parameters, representing undamaged tissue. A mild form of stretch or crush injury causing retraction of myelin around stretched nodes was simulated by increasing the width of exposed nodal membrane, s, in nodes 8 through 20. Normal nerve conduction velocity was taken as the inter-nodal distance, divided by the steady-state wave propagation time between nodes 4 and 5, namely (t5–t4)/L. Injured nerve conduction velocity was taken as the inter-nodal distance, divided by the steady-state wave propagation time between nodes 15 and 16, namely (t16–t15)/L. In some simulations a more severe form of stretch or crush injury was simulated that included both retraction and detachment of paranodal myelin. Paranodal myelin detachment, that is, radial separation of paranodal myelin from the underlying axon membrane, was simulated by decreasing paranodal resistance to one tenth or one hundredth of its normal value. In other simulations the effect of the potassium channel blocking drug, 4- aminopyridine, was simulated by decreasing peak potassium conductance to 20 percent of its normal value, representing mild-to-moderate inhibition that would be reasonable to achieve experimentally. For left hand node 0 dV0 dt ~ i0 C0 ~ iNa0{iK0{ V1{V2 Ra   1 C0 : ð6Þ ð6Þ Numerical Integration us [27], based upon the definition of capacitance and Ohm’s Law. The definition of capacitance is C~dQ=dV, where C is nodal capacitance and dV is the incremental change in electrical potential or voltage across the capacitance as charge dQ is introduced. For current i = dQ/dt flowing during time increment, dt, we must have dV/dt = i/C. Ohm’s Law, which relates current to voltage and resistance, is i~(DV)=R, where DV is the instantaneous difference in voltage across resistance R as current i occurs. To describe waveforms of membrane potential vs. time at each node j = 0, 1, 2, … n –1 equations (5) and (6) for dV/dt were integrated numerically using the simple Euler method, imple- mented in Visual Basic code within an Excel spreadsheet on an ordinary personal computer. For each node the membrane potential Vj at successive time steps Dt was calculated as Vj(tzDt)~Vj(t)z dVj dt Dt, ð7Þ ð7Þ Applying these basic concepts with reference to Figure 2 provides a set of governing differential equations that can be used to describe transmembrane potentials in the chain of nodes. Beginning with the most distant node, n, we set as a boundary condition using expressions (4), (5), and (6) for dVj/dt. Initial conditions at t = 0 describe the state of the axon segment at rest, with Vj set to the resting transmembrane potential for j = 1, 2, … n. Only node 0 is active at time zero (t0 = 0). Nodes j = 1, 2, … n –1 become active when t.tj. As a boundary condition, the rightmost node, n, in the chain is clamped at the resting potential and is never activated. dVn dt ~ 0: ð4Þ ð4Þ p g p A model of 21 nodes (including bare nodal axon and functional juxtaparanodal membrane) separated by a nominal average internodal distance, L, of 1 mm represented a hypothetical axon segment 2 cm in length. The combined bare nodal, paranodal, and juxtaparanodal regions of each node had a total axial length on the order of 0.01 mm or about 1 percent of the axial distance between nodes. Given initial conditions at t = 0, the evolution of potentials Vj(t) was computed as a ‘‘marching solution’’, for which stability and accuracy are ensured by using a sufficiently small value of Dt, such as 0.1 microsecond. Solving for Membrane Potentials at Successive Nodes Solving for Membrane Potentials at Successive Nodes The model of the myelinated axon includes an arbitrary number ,20 nodal capacitances, connected by equal axonal resistances, Ra~(raL=(pr2 a)), through which ionic current may flow, as shown in Figure 2. The capacitances, C~2prasCm, correspond to the bare membrane areas of successive nodes of Ranvier. Transmembrane potential is specified as the intracellular minus extracellular electrical potential at each node. To simulate myelinated nerve conduction node 0 at the left boundary is actively depolarized by a suprathreshold stimulus. The successive nodes 1, 2, … n become active if their membrane potential exceeds a threshold value, Vth, such as –50 mV. The same conductance functions GNa(t9j) and GK(t9j) are used for all nodes. However, the time variable, t9j, for each node differs among nodes, depending on the time of activation. Here we let tj represent the clock time of activation of nodes j = 0, 1, 2, … n in the model. In particular, time tj is defined as the clock time, t, when the transmembrane potential Vj(t) equals or exceeds the threshold potential Vth for each node j = 0, 1, 2, …. In turn, t9j = max (0, t2tj). Then the node-specific ionic conductance is computed for times t.tj as Gj(t’j)~a(t’j)2e{bt’j. For Node 0, the first node in the chain, t0 = 0, so that activation begins at node 0, proceeding from left to right in Figure 2. The computational approach used here is an adaptation of that previously published for modeling the circulatory system by one of July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 4 Normal Nerve Conduction Figure 3 shows successive action potentials in a normal myelinated axon model. Transmembrane potentials for nodes numbered 0 through 16 in the series are shown in successive curves from left to right. A propagated wave of excitation travels down the chain of nodes from left to right. Nerve conduction velocity is 19.1 m/sec. July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org 5 Conduction in Injured Myelinated Axons Figure 3. Modeled action potentials at successive nodes of Ranvier in a normal myelinated axon. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; node width 0.65 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential – 50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g003 Figure 3. Modeled action potentials at successive nodes of Ranvier in a normal myelinated axon. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; node width 0.65 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential – 50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g003 Figure 4 shows transmembrane sodium and potassium currents in this normal model as a function of time for node number 6 in the chain of normal nodes. The peak of potassium conductance is blunted somewhat, compared to that in an unmyelinated axon, because of the effect of paranodal resistance, Rp, the anatomic substrate for which is the thin sleeve of extracellular matrix material and tight junctions in the paranodal region between the axolemma and overlying myelin. Recharge current for the node must pass through this normally thin space to reach the nodal capacitance. Figure 5. Normal myelinated nerve conduction velocity as a function of the node width. Normal Nerve Conduction doi:10.1371/journal.pone.0067767.g005 Conduction velocity in otherwise normal myelinated axons depends on node width, s, measured along the axial dimension, as shown in Figure 5. Discussion Mathematical modeling can help to organize and distill knowledge about complex systems, highlight the most important variables that govern system performance, and suggest testable hypotheses for future research. The simple computational model of myelinated nerve conduction presented here provides several insights into the normal functioning and the failure of myelinated axons, which remain points of discussion in the 21st century [35,36]. One critical and underappreciated variable is the width of the nodes themselves, compared to the diameter of the axon. Another is the paranodal electrical resistance that separates the high density of sodium channels in the node itself from the juxtaparanodal potassium channels. The predicted results are similar to the diminished amplitude and increased latency of compound action potentials observed experimentally by Shi and Blighty [34]. Because node width, s, is on the order of only one micrometer normally, subtle damage, separation, or retraction of the myelin sheath at the nodes of Ranvier might go unnoticed at the light microscopic level of observation. Such damage, however, can lead to substantial degradation in myelinated nerve conduction. When decreased paranodal resistance is included in the model, such as would occur with decompaction or detachment of paranodal myelin [10], conduction is further degraded (Figure 7). When node width is stretched three-fold (similar to that observed in [10]), and paranodal resistance is reduced to one-tenth normal, the nerve conduction velocity is decreased from 17 m/sec in the uninjured region on the left to 6.6 m/sec in the injured region on the right. The amplitude of the action potential is also further reduced. Evidently, decreased paranodal resistance allows more outward potassium current to reach the node, diminishing the net depolarizing current, the rate of rise of the action potential, and the peak amplitude of the action potential. Note that the 90 percent reduction in Rp represents very subtle injury in this model since a retraction of the paranodal junction leaving a gap of only 0.1 micron around the circumference of the axon would reduce Rp by .99.9 percent. Narrower nodes increase conduction velocity (Figure 5) as previously pointed out in a semi-quantitative way by Giuliodori and DiCarlo [36]. Simulated Crush Injury Figure 6 illustrates axonal conduction in a model of simulated crush injury. The widths, s, of nodes 8 through 20 on the right have been stretched from 0.65 mm to 1.95 mm, simulating the degree of nodal stretching observed in experimental crush injury [10]. However, the length of axon over which sodium channels are Figure 5. Normal myelinated nerve conduction velocity as a function of the node width. doi:10.1371/journal.pone.0067767.g005 Figure 4. Normal ionic currents for node 6 in the myelinated axon model of Figure 3. doi:10.1371/journal.pone.0067767.g004 concentrated, s*, is not changed in keeping with the pre-injury segregation of sodium channels in the nodal region. Here s* is substituted for s in Equation (3a). In the crushed segment (right) the time between action potentials at successive nodes increases, indicating slowed impulse conduction. In this example crush injury diminishes action potential amplitude by about one third and also diminishes the slope of the rising phase of the action potential, slowing conduction velocity from 18.8 m/sec in the normal region on the left to 7.8 m/sec in the crushed region on the right. The longer discharge time required for stretched nodes allows for greater outward potassium current to develop (Figure 4), and in turn less net inward positive current inflow during the rising phase of the action potential. Figure 4. Normal ionic currents for node 6 in the myelinated axon model of Figure 3. doi:10.1371/journal.pone.0067767.g004 The reduced action potential amplitude after stretch injury appears to happen for the following reason. The currently PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 6 Conduction in Injured Myelinated Axons Figure 6. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the right. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms throughout the model; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g006 Conduction in Injured Myelinated Axons Figure 6. Simulated Crush Injury Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the right. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms throughout the model; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g006 width, there is complete conduction block in the region of simulated injury (Figure 8). depolarizing node of Ranvier acts as a constant current source in the presence of stretch injury. When mild stretch injury increases node width, the length of axon over which sodium channels are concentrated is not changed, in keeping with pre-injury segrega- tion of sodium channels in the nodal region. However, simple stretch injury increases the nodal surface area, and in turn increases the nodal capacitance. When the increased nodal capacitance of the next downstream node is discharged by a constant current source the rate of change in voltage is reduced (dV/dt = i/C, where current, i, is constant and capacitance, C, is increased). The reduced slope between the threshold potential and the peak of the action potential reflects the increased capacitance. The peak does not reach normal height, because a reduced slope over a constant duration results in a reduced peak amplitude. Blocked conduction in the simulation of Figure 8 can be restored by inhibiting peak potassium conductance in all nodes of the model by 80%, that is, by replacing GKmax = 0.013 S/cm2 with introducing GKmax = 0.2*0.013 S/cm2. The effect of simulated drug treatment with a potassium channel blocker is shown in Figure 9. Conduction is restored. However, conduction velocity remains low at 7.1 m/sec. Discussion The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26109 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g007 Figure 7. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the right. The dotted horizontal line represents threshold potential. In this simulation node width was increased three-fold and paranodal resistance was decreased to one tenth the normal value. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26109 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g007 myelinated nerve conduction velocity as the distance, L, between nodes, divided by the time, Dt, it takes for the next downstream node (e.g. Node 2 in Figure 1) to reach threshold after the transmembrane potential at a given node (Node 1 in Figure 1) reaches threshold and nodal sodium channels are fully open. (The value of Dt can be appreciated graphically with reference to Figure 3.) As soon as Node 1 becomes a low resistance pathway for ionic current, downstream Node 2 will depolarize to a value of about 1/e (37%) of its resting negative membrane potential in one time constant RaC. The time required to reach threshold is therefore Dt~aRaC, where a is a constant near 0.5. In turn, myelinated nerve conduction velocity v~ L Dt & L aRaC ~ 1 4raCm d s   : ð9Þ ð9Þ As node width, s, decreases, the myelinated nerve conduction velocity increases hyperbolically. Further, the node width and axon diameter are the major anatomic determinants of myelinated nerve conduction velocity. The direct dependence of conduction velocity on axon diameter is well established [16,18]. However, the importance of the node width or the nodal ratio is much less well appreciated. Discussion The discharge time constant, RaC, which governs the time required for downstream nodes to be depolarized from their resting membrane potential to the threshold potential, is simply RaC~ raL pr2 a   :2pras Cm~2 raCmL s ra ~4 raCmL s d , ð8Þ ð8Þ where s/d is the nodal ratio or node width divided by axon diameter. This expression includes relatively few variables and holds true as long as paranodal myelin remains tightly coupled to the underlying axonal membrane, so that paranodal resistance remains normal. When paranodal resistance if further reduced to 1 percent of the normal value, together with a three-fold increase in bare nodal The relationship of the discharge time constant (8) to nerve conduction velocity can be appreciated as follows. Consider PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 July 2013 | Volume 8 | Issue 7 | e67767 7 Conduction in Injured Myelinated Axons Figure 7. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the right. The dotted horizontal line represents threshold potential. In this simulation node width was increased three-fold and paranodal resistance was decreased to one tenth the normal value. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26109 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g007 Figure 7. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the right. The dotted horizontal line represents threshold potential. In this simulation node width was increased three-fold and paranodal resistance was decreased to one tenth the normal value. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. Discussion Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g009 Conduction in Injured Myelinated Axons Figure 9. Treatment of simulated crush injury to nodes on the right with a potassium channel blocker that reduces peak potassium conductance in all nodes by 80%. Parameters of injury: 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g009 and Pryor [37] revealed that the peak latency of action potentials increased by 35 percent after stretching guinea pig spinal cord segments. The stretched region in these preparations was about one third of the total distance over which latency was computed. The modified cable model derived here predicts that myelinated nerve conduction latency at node 10 in the model of Figure 7 would increase from a normal value of 0.57 sec (Figure 3) to 0.80 sec (Figure 7) a 40 percent increase in latency when the path from node 0 to node 10 in the model includes three injured nodes (only one third of the total). Shi and Prior also found that superfusion with 100 micromolar 4-aminopyridine (a potassium channel blocker) partially restored the loss in amplitude of the compound action potential 30 min after stretch injury. However, there was no significant change in conduction velocity following 4- aminopyridine treatment. Discussion Since the compound action potential represents a sum of signals from many axons, these experimental results are consistent with the model results in Figure 9, showing that an 80% blockade of potassium conductance restores conduction with slow velocity following severe stretch injury. The modeling results are also consistent with experimental studies in other animal models and with preliminary clinical trials of 4- aminopyridine as a therapeutic agent in spinal cord injury [4]. velocity is located near the elbow of the curve in Figure 5. This subtle feature hints that mammals are a highly evolved order of vertebrates. Natural selection would likely favor animals with higher nerve conduction velocity, quicker reaction times, and quicker thinking. However, as node spacing gets closer and closer, problems could arise with quality control in forming nodes to such close tolerances. For very small values of node span, s, small absolute change in the separation of myelinated segments on either side would cause a relatively large change in s and in the nodal ratio. (Indeed, the magnitude of the derivative dv/ds is proportional to 1/s2.) Simple movement of peripheral nerves or very mild concussions in the brain could cause relatively large changes in nerve conduction velocity, v, and hence in the stability and predictability of the nervous system. Coordinated complex movements would become more difficult if conduction time from brain to muscle, or within the central nervous system, varied unpredictably. Very narrow nodes would be fast, but they would also be delicate and perhaps too noisy. In this way the net survival advantages of quickness might plateau or even degrade as a function of decreasing node width. We may have reached a near optimal compromise through natural selection. The model also provides insights into the pathophysiology of crush injury. The subtle injury involving retraction and fraying of paranodal myelin increases the effective capacitance of the nodal membrane by perhaps an order of magnitude or more and also exposes paranodal and juxtaparanodal regions. Detachment of paranodal myelin, perhaps only visible at the electron microscopic level of observation [10], reduces by perhaps an order of magnitude or more the paranodal resistance through which repolarizing potassium current must flow to restore the resting membrane potential after an action potential. These small anatomical injuries increase early outward potassium current, opposing inward sodium current at the node. Discussion The sensitive dependence of myelinated nerve conduction velocity on the nodal width, s, of exposed axon membrane (or the ratio of nodal width to axon diameter, s/d) has important biological implications. Interestingly, normal nerve conduction Figure 8. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the right with 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g008 Figure 8. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the right with 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g008 July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org 8 Conduction in Injured Myelinated Axons Figure 9. Treatment of simulated crush injury to nodes on the right with a potassium channel blocker that reduces peak potassium conductance in all nodes by 80%. Parameters of injury: 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The dotted horizontal line represents threshold potential. Conclusions The straightforward cable model presented here recapitulates how the arrangement of myelin covered segments of axons, punctuated by narrow bare nodes of Ranvier, can produce saltatory conduction. The function of nodes of Ranvier in speeding impulse conduction is related to the ultrastructure of the nodes themselves. Normal myelinated nerve conduction velocity is inversely related to the node width, s, and to the nodal ratio, s/ d, in the presence of intact paranodal resistance, Rp. The narrow axial width of the nodes of Ranvier is the key to fast conduction. The biological tradeoff is that even a small mechanical retraction of myelin from very narrow nodes can cause large changes in conduction velocity. Very narrow nodes would be very fast, but exceedingly delicate. Hence evolution may have already found a near optimal tradeoff between quickness and toughness. Cable models are especially suited to predict outcomes of biological experiments that measure axonal conduction velocity because they can be used to simulate wave propagation through successive nodes. Goldman and Albus [16] described a cable model and focused on characterizing the relationship between conduction velocity and fiber diameter in myelinated axons, which was found to be nearly linear for realistic axon models. Over the next decade this model was further studied by several groups using alternative methods for numerical integration and slightly differing parameter values and to obtain myelinated conductions velocities between 12 and 22 m/sec and realistic dependence of conduction velocity upon fiber diameter [17,18]. Modeling also suggests how nodes can malfunction in disease. Subtle mechanical, chemical, or immunological injury to the nodes of Ranvier, causing small increases in effective node width, s, and nodal capacitance, C, which could be easily overlooked using ordinary light microscopes, can contribute to pathology of conditions such as spinal cord injury and multiple sclerosis that cause retraction of myelin. The effects of node stretching are magnified by detachment of paranodal myelin, a process that decreases paranodal resistance and increases repolarizing potas- sium current above the normal level. Thus the nodes of Ranvier are highly effective but somewhat fragile devices for increasing nerve conduction velocity and decreasing reaction time in vertebrate animals. Increased nodal capacitance or decreased paranodal resistance caused by subtle myelin retraction and detachment cause slowed saltatory conduction and ultimately conduction block. Conduction in Injured Myelinated Axons Conduction in Injured Myelinated Axons FitzHugh [15]. This approach modeled nodes as strings of leaky capacitors having Hodgkin-Huxley type ion channels, and connected in parallel by extracellular and intracellular internodal resistances. Currents and voltages at each node are computed. FitzHugh derived a second order partial differential equation for this system to describe voltages at each node as functions of time and space, and solved the equation numerically. (Much later Basser [23] and Nygren and Halter [24] used analytical methods to derive equivalent equations for a composite, myelinated axon, obtaining expressions essentially isomorphic with the original solutions of FitzHugh (compare Basser Eq. 35 and Nygren and Halter Eq. 18 with FitzHugh Eq. 1). Such equations are known as cable equations, because of their similarity to cable or transmission line equations [25], and the corresponding models of nerve conduction are known as cable models. idealized or typical myelinated axon. Noteworthy is the assump- tion that internodal myelin is an ideal insulator relative to uninsulated nodes. We ignore internodal leakage currents, which would likely change nodal currents and voltages by only a few percent. Supporting Information Figure S1 Electrical model for one side of one node of Ranvier with potassium conducting regions separated from the node by high resistance paranodal regions. (TIFF) Appendix S1 Electrical effects of segregated ion chan- nels. Detailed treatment of the additional complexity of ion channel segregation in the nodal, paranodal, and juxtaparanodal regions, leading to quantitative estimation of normal paranodal resistance Rp&3:2 : 1010 ohms. (DOC) Our model is obviously limited in being a theoretical rather than an experimental study in which only selected features of the complete complex biological system are included. Ours is a deterministic rather than a stochastic model, hence average or typical values of parameters are used and statistical variation in axon diameter, axon spacing or internodal width, density and distribution of ion channels, thickness of the myelin sheath, and so on are ignored for the sake of answering larger questions about an Conclusions Better understanding of the pathophysiology of nodal injury may lead to new treatments as well as heightened awareness of the dangers of subtle neurotrauma in closed head injury. Blight [19] studied a similar resistance-capacitance network representing a chain of 20 internodes. In the equivalent electrical circuit, internodes l-9 and 1 l-20 were each represented by a single segment, containing the resistance and capacitances of the internodal axolemma and myelin sheath, separated from the nodal circuits on either side by the resistance of the axon core. Internode number 10 was broken into 10 shorter sub-segments such that the importance of paranodal parameters could then be explored. The model quantitatively reproduced the voltage response of the axon to injected current pulses and to propagated action potentials, using Frankenhaeuser-Huxley kinetics. Blight highlighted the importance of the input resistance of the internode and the storage of charge at the axolemma. Later Stephanova and Bostock [20,21] created a distributed-parameter model of the myelinated human motor nerve fiber, and also highlighted the role of paranodal resistance. Similarly, McIntyre and coworkers [22] specifically included paranodal resistance in their model and studied the biophysical mechanisms underlying changes in excitability following an action potential. The re-derived cable equations of Nygren and Halter [24] have sodium channels that are localized at the node, whereas potassium channels and the transmembrane sodium/potassium pump are located predomi- nantly away from the node. In the present study we have retained the concept of separation of sodium and potassium channels, now well established as a constant feature of myelinated axons, and we have added the capability to systematically adjust parameters related to subtle injury during neurotrauma. Discussion As a result, the time required to discharge the next downstream node of Ranvier to the threshold level is increased. In turn, nerve conduction velocity falls, since it is related to the inter-nodal distance, divided by the discharge time. py p g p j y [ ] Retraction of myelin around nodes of Ranvier can be produced by chemical injury as well as by stretch injury. Fu and coworkers [14], using CARS imaging, reported paranodal myelin splitting and retraction in response to glutamate excitotoxicity in isolated rat spinal cord. The nodal ratio in these animals increased from a normal value near 1.0 to a pathological value near 3.0 after glutamate. Similarly, Shi and coworkers [32] found that nodal ratios increased from a control mean of 0.6 to 2.9 twelve hours after application of 500 micromolar acrolein to isolated guinea pig spinal cord. Subtle nodal injury, accompanied by slowed conduction velocity, is also recognized as an important mechanism in the pathology of multiple sclerosis [3,38]. Hence the mecha- nisms described in this paper may have relevance beyond the field of neurotrauma. The modeling approach used in the present study to explore the particular effects of node width and paranodal resistance builds on a rich intellectual history. The original model of saltatory conduction in myelinated nerve fibers was done by Richard The calculated changes in conduction velocity with subtle paranodal injury in the present paper agree with experimental observations. Experiments in isolated guinea pig spinal cord by Shi July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 9 References Roper J, Schwarz JR (1989) Heterogeneous distribution of fast and slow potassium channels in myelinated rat nerve fibres. J Physiol 416: 93–110. 27. Babbs CF (1999) CPR techniques that combine chest and abdominal compression and decompression: Hemodynamic insights from a spreadsheet model. Circulation 100: 2146–2152. 9. Waxman SG, Ritchie JM (1985) Organization of ion channels in the myelinated nerve fiber. Science 228: 1502–1507. 28. diFiori MSH (1981) Atlas of Human Histology. Philadelphia: Lea & Febiger. 267 p. 10. Sun W, Fu Y, Shi Y, Cheng J, Cao P, et al. 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3D subsurface characterisation of the Belgian Continental Shelf: a new voxel modelling approach
Geologie & mijnbouw/Geologie en mijnbouw
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Netherlands Journal of Geosciences www.cambridge.org/njg Netherlands Journal of Geosciences www.cambridge.org/njg Netherlands Journal of Geosciences 1. Introduction This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. To model aggregate resources in 3D, it is important to define deposits with uniform lithological properties. In a voxel model, 3D interpolation techniques can be implemented to estimate a representative lithological class (or another property) for each voxel based on the Keywords: 3D stochastic modelling; aggregate resource estimation; information entropy; North Sea; Quaternary Author for correspondence: Vasileios Hademenos, Email: Vasileios.Chademenos@UGent.be 3D subsurface characterisation of the Belgian Continental Shelf: a new voxel modelling approach Vasileios Hademenos1, Jan Stafleu2, Tine Missiaen1,3, Lars Kint4 and Vera R.M. Van Lancker1,4 Review 1Renard Centre of Marine Geology, Department of Geology, Ghent University, Krijgslaan 281 s8, 9000 Gent, Belgium; 2TNO – Geological Survey of the Netherlands, Princetonlaan 6, 3584 CB Utrecht, the Netherlands; 3Flanders Marine Institute, Wandelaarkaai 7, 8400 Ostend, Belgium and 4Operational Directorate Natural Environment (RBINS OD Nature), Royal Belgian Institute of Natural Sciences, Gulledelle 100, 1200 Brussels, Belgium Cite this article: Hademenos V, Stafleu J, Missiaen T, Kint L, and Van Lancker VRM. 3D subsurface characterisation of the Belgian Continental Shelf: a new voxel modelling approach. Netherlands Journal of Geosciences, Volume 98, e1. https://doi.org/10.1017/ njg.2018.18 Abstract Modelling of surface and shallow subsurface data is getting more and more advanced and is demonstrated mostly for onshore (hydro)geological applications. Three-dimensional (3D) modelling techniques are used increasingly, and now include voxel modelling that often employs stochastic or probabilistic methods to assess model uncertainty. This paper presents an adapted methodological workflow for the 3D modelling of offshore sand deposits and aims at demonstrating the improvement of the estimations of lithological properties after incorpora- tion of more geological layers in the modelling process. Importantly, this process is driven by new geological insight from the combined interpretation of seismic and borehole data. Applying 3D modelling techniques is challenging given that offshore environments may be heavily reworked through time, often leading to thin and discontinuous deposits. Since voxel and stochastic modelling allow in-depth analyses of a multitude of properties (and their associated uncertainties) that define a lithological layer, they are ideal for use in an aggregate resource exploitation context. The voxel model is now the backbone of a decision support system for long-term sand extraction on the Belgian Continental Shelf. Received: 13 September 2018 Accepted: 22 November 2018 Received: 13 September 2018 Accepted: 22 November 2018 1. Introduction The Belgian Continental Shelf (BCS) is bounded in the north and south by the Dutch and French parts of the North Sea, on the west by the British part and on the east by the Belgian coast (Fig. 1). The BCS is a sediment-depleted shallow shelf environment comprising a series of sandbanks. There is no distinct shelf break (De Batist,1989), so there isvery littleaccommodationspaceto accumulate younger and preserve older sediments. In turn this caused important recycling and redistribution of the sediment, creating a complex thin and discontinuous Quaternary sediment cover. There is a high demand to exploit the resources within this sedimentary cover (Van Lancker et al., 2010) and the demand is only increasing due to coastal nourishment projects and new visions for the development of the marine and coastal zone of Belgium. Availability of sand is critical to support these initiatives, and therefore the research project TILES was initiated to develop Transnational and Integrated Long-term Marine Exploitation Strategies (Van Lancker et al., 2017). Hitherto, no quantitative resource data were available, and also internationally such data remain scarce, apart from site-related datasets. In a marine aggregate context, it is also important to have information on admixtures that may adversely affect the quality of the resource (e.g. shells, mud and gravel content) and/or the environment (e.g. mud; Newell et al., 1998). Therefore, we opted to develop a three-dimensional (3D) voxel model allowing us to obtain a holistic view on resource quality and quantity of the Quaternary over wide areas and enabling the addition of any desired information relevant from a resource or environmental impact perspective. For this application, voxels are a regular grid of rectangular blocks with defined dimensions (x, y, z) in a Cartesian coordinate system. Each voxel in the model can contain multiple attributes describing, for example, the stratigraphy, or the spatial variation of lithology in geological units and other parameters such as uncertainty. Because of their structure, voxels can better define complex geology and heterogeneities within geological layers (Stafleu et al., 2011). In addition, voxel models can be created using stochastic techniques that allow the construction of multiple, equally probable 3D realisations. Furthermore, they facilitate easy querying and analysis: for example, volume calculations can be performed by selecting and counting the voxels that meet certain criteria. © Netherlands Journal of Geosciences Foundation 2019. © Netherlands Journal of Geosciences Foundation 2019. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms rg/10.1017/njg.2018.18 from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available a siteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2 2 Vasileios Hademenos et al. Fig. 1. (A) Map showing the depth of the Top-Palaeogene unconformity and the main geomorphological features: the Middle and Offshore Platform, separated by the Middle and Offshore scarp (De Clercq et al., 2016). These scarps were used to split up the model into regions with similar lithological characteristics. (B) Cross-section showing the extent and geometry of each stratigraphical unit subdividing the Cainozoic sediments of the BCS. Most of the sandbanks (e.g. Middelkerke, Hinder) have a characteristic internal architecture. Fig. 1. (A) Map showing the depth of the Top-Palaeogene unconformity and the main geomorphological features: the Middle and Offshore Platform, separated by the Middle and Offshore scarp (De Clercq et al., 2016). These scarps were used to split up the model into regions with similar lithological characteristics. (B) Cross-section showing the extent and geometry of each stratigraphical unit subdividing the Cainozoic sediments of the BCS. Most of the sandbanks (e.g. Middelkerke, Hinder) have a characteristic internal architecture. quality and reliability of the models becomes crucial. Using stochastic models, the probability of occurrences of each lithologi- cal class can be calculated as a first estimate of model uncertainty. To summarise the probabilities per voxel, Wellmann & Regenauer- Lieb (2012) suggested calculating information entropy as a mea- sure of model uncertainty. The quality measure thus obtained can be used to compare different versions of a model. lithological description of boreholes available in the model area (Van Haren et al., 2016). In many cases, modelling results can be greatly improved by subdividing the 3D volume into lithostrati- graphical units that have uniform sediment characteristics. For example, in the GeoTOP voxel model of the onshore part of the Netherlands (Stafleu et al., 2011, 2012) the borehole descriptions were first used to construct 2D bounding surfaces. These surfaces represented the top and base of each of the lithostratigraphical units and were used to place each voxel in the model within the correct unit. Next, the 3D interpolation of lithological class was performed for each lithostratigraphical unit separately. In this paper, a step-by-step modelling approach is described to depict the quality and quantity of the available geological resources in the BCS. For the first time, 3D stochastic modelling is applied to quantify sand resources in a marine setting making use of both borehole and seismic data. For this application, the approach is also new in the sense that it incorporates various levels of geological knowledge in the modelling process and this is shown to improve the characterisation of the subsurface. A more detailed case study is presented to demonstrate how the resolution of a model affects the depiction of the lithostratigraphy and the volume of the resource. Creating bounding surfaces from borehole data works well if the spatial data density of the boreholes is relatively high, as is the case in the GeoTOP model (~10 boreholes per km2 on average). In the BCS however, borehole density is only about 0.3 per km2. Such low bore- hole density necessitates the incorporation of other data sources. Most evident are geophysical line data such as shallow 2D/3D seismic profiles that allow the interpretation of ‘horizons’ to subsequently allow the generation of bounding surfaces to define the different lith- ostratigraphical units in 3D modelling (e.g. Bartakovics et al., 2013; Van Heteren et al., 2014; Jørgensen et al., 2015). https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.2. Quaternary Our core dataset contained a total of 1770 cores on the BCS extend- ing to 1 km beyond the border (Fig. 3A) provided by the Royal Belgian Institute of Natural Sciences (Kint & Van Lancker, 2016). Data originated from the public and private sector and span several decades (1900–2016). The majority of the cores were rela- tively shallow, with depths ranging from 0 to 5 m (Fig. 3A). The spatial distribution of the cores is denser close to the shore, and sparser further offshore and near the borders (especially towards France) (Fig. 3A). Metadata were all revised according to SeaDataNet standards (Schaap, 2017). Thin, discontinuous/heterogeneous Pleistocene and Holocene sedi- ments overlay the unconformity (Mathys, 2009). The variability of the geological formations of the Quaternary poses a major challenge when modelling; the lithostratigraphy content of each sandbank is unique and interpolation will cause generalisations that may lead to faulty assumptions about their geological content. To avoid this, the stratigraphical layers that will be used must be carefully defined. The Pleistocene sediments occur in two main regions of the BCS. On the Offshore Platform, they form the core of the sandbanks (e.g. Hinder Banks), and closer to the coast they are preserved in palaeovalleys such as the Ostend Valley (Fig. 1) (De Clercq et al., 2016, 2018). In the Middle Platform region they were mostly eroded down to the Palaeogene clays by both the Eemian and the Holocene transgressions (Mathys, 2009). The Pleistocene sediments originate mainly from the Eemian interglacial period and comprise mixed sediments spanning from gravel to clay. There is a lateral difference in lithology between the nearshore area, where clay to fine sands predominate (in the palaeovalleys), and the offshore area where coarser-grained sands with abundant shells are found. p Due to the diversity of core descriptions and different param- eterisation schemes dependent on the various project objectives, all of the descriptions were checked and encoded following European guidelines on geological data formats (Geo-Seas: Van Heteren, 2010). For lithology terms, the Wentworth (1922) classification was used to define sediment classes (e.g. clay, silt, fine sand, medium sand, coarse sand, gravel). In some cases, harmonisation of data across the original data sources was needed to resolve differences in assigning Wentworth classes to a given grain-size range. 2.1. Palaeogene The BCS is marked by two major geological units greatly varying in lithology and stratigraphy. These units, respectively the Palaeogene With the advent of 3D models, their increasing complexity and the variety of methods employed, finding better ways to assess the Netherlands Journal of Geosciences 3 et al. (2011) and expanded with seismic data. It comprises the fol- lowing steps (Fig. 2): and Quaternary, are bounded by the Top-Palaeogene unconform- ity (De Clercq et al., 2016). The Palaeogene is a polygenetic layer composed of compacted clays, sands and sandy clays that were deposited in a shallow-marine to outer-shelf environment (Le Bot et al., 2003). The geological units within the Palaeogene range in age from the upper Palaeogene to the upper Eocene. The layers dip towards the NE by approximately 1°. Their lithology varies from west to east, from consolidated clays (Ypresian) to alternating sequences of silt and clay, but also silty sand, muddy sand and even calcareous sandstone beds (Le Bot et al., 2005). (1a) Standardisation and lithological classification of borehole descriptions (1b, 3) Delineation of seismic acoustic facies and their seismostra- tigraphical interpretation (2) Stratigraphic interpretation of the boreholes (4) Construction of the 2D stratigraphical layer model (5) Assignment of lithostratigraphical units to the 3D voxel model (6) 3D interpolation of lithological class within each lithostra- tigraphical unit (7) Assessment of the information entropy of the model. The top of the Palaeogene is an angular unconformity (Fig. 1) representing a hiatus in time between the Lower and Middle Eocene formations (De Batist, 1989) and the overlying Quaternary deposits. The depth of the surface varies between 8 and 70 m below lowest astronomical tide (LAT), and its geomor- phology is characterised by a series of features ranging from planation surfaces, bounded by scarps and slope breaks, to palae- ovalleys and elongated depressions (Liu, 1990; Liu et al., 1992; Mathys, 2009; De Clercq et al., 2016). A detailed description of steps 1 to 7, following an iterative process as is indicated in Fig. 2, is given below. 2.2. Quaternary For interpolation purposes in the lithological description, six numerical classes were used, follow- ing the lithological classification of Vernes & Van Doorn (2005) ranging from gravel to clay (Table 1). Descriptions on sandy sediment layers without any further information on lithological content were characterised as sand and categorised in a separate numerical class. The Holocene sediments are diverse in origin and composition. They form the major part of the tidal sandbanks of the BCS (e.g. Trentesaux et al., 1999; Mathys 2009; Van Lancker et al., 2010). Two layers are distinguished, the Lower and Upper Holocene respectively. In the nearshore area, the Lower Holocene layer (LHL) is representative of a tidal flat environment (Mathys, 2009) which was formed around 10,950 cal BP (before present) until it was submerged around 7500 cal BP. This Lower Holocene layer was first defined in the Middelkerke Bank (Fig. 1); its sediments varied from coarse-grained to very-fine sand. The Upper Holocene layer (UHL) covers the total BCS and forms the most important sand resource. In the nearshore area, south of the middle scarp (Fig. 1), it predominantly consists of fine sands related to an estuarine–marine depositional environment. In the offshore area, north of the middle scarp (Fig. 1), the layer comprises coarser material, with medium to coarse sand being typical for the offshore marine depositional environment. 2.5. Step 1b and 3: Delineation of seismic acoustic facies and their seismostratigraphical interpretation The available seismic database (Renard Centre of Marine Geology, Ghent University) comprises over 12,000 km (Fig. 3B) of 2D seis- mic lines collected during a large number of scientific cruises from the late 1970s until today. The seismic sources used for the measurements mainly involved various types of sparkers and boomers, in combination with a single-channel streamer. The seismic dataset comprised both digitally recorded data (SEG-Y format) and older analogue data converted to SEG-Y from scanned paper rolls. The latter make up one-third of the total database to roughly 4000 km of seismic profiles (Mathys, 2009). https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. siteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.3. Methodology To model offshore aggregate resources, a methodological workflow was developed based on the voxel modelling approach of Stafleu The converted analogue data often caused serious problems related to the high uncertainty in geographical location (many lines Fig. 2. Modelling procedure flow chart. Fig. 3. (A). Map showing the seismic network on the BCS against a background of the bathymetry (Flanders Hydrography). (B) Map showing the depth (m) distribution of the co d t t th BCS Th t l i th iddl d fi th t t f th Hi d B k t d 4 Vasileios Hademenos et a Vasileios Hademenos et al. Fig. 2. Modelling procedure flow chart. Fig. 2. Modelling procedure flow chart. Fig. 3. (A). Map showing the seismic network on the BCS against a background of the bathymetry (Flanders Hydrography). (B) Map showing the depth (m) distribution of the core dataset on the BCS. The grey rectangle in the middle defines the extent of the Hinder Banks case study area. Fig. 3. (A). Map showing the seismic network on the BCS against a background of the bathymetry (Flanders Hydrography). (B) Map showing the depth (m) distribution of the core dataset on the BCS. The grey rectangle in the middle defines the extent of the Hinder Banks case study area. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/term 5 Netherlands Journal of Geosciences Table 1. Wentworth (1922) and the classification used in the voxel modelling Wentworth (1922) μm 2000 Gravel Gravel 1000 Very coarse sand Coarse sand 500 Coarse sand 250 Medium sand Medium sand 125 Fine sand Fine sand 62.5 Very fine sand 4 Silt Silt Clay Clay Table 1. Wentworth (1922) and the classification used in the voxel modelling depth of the stratigraphic layers. In order to perform this conver- sion, the sound velocity within each layer was calculated. To achieve this and to validate the seismic interpretations, borehole and bathymetric data were incorporated and each seismic reflector was compared to the lithostratigraphical information from the cores. This allowed the creation of a velocity model (Fig. 4B) assigning a laterally varying internal velocity to each layer, rather than simply providing a constant value for the velocity of each layer. 2.6. Step 2: Stratigraphic interpretation of the boreholes Only a limited number of boreholes were already assigned a stratigraphical interpretation, and if available, these interpretations were mostly made in different projects, each having different qual- ity requirements (Kint & Van Lancker, 2016). Thus the original borehole data were far from uniform with respect to stratigraphy, and therefore we decided to systematically reassign stratigraphical interpretations to all boreholes in the dataset using both sediment characteristics and seismic data. Based on geological knowledge and on an iterative process between step 2 and step 3, the acoustic facies from the interpreted seismic lines were assigned a strati- graphical unit exported to the borehole dataset for further use in the modelling process. For the stratigraphical description of the borehole intervals a lithostratigraphical unit was assigned based on the conceptual framework in Fig. 2. The labels in the core data were derived from the borehole stratigraphical descriptions or, when that information was unavailable, from the seismic interpretations. A constant cross-validation between two different types of data was needed. It is noteworthy that only a few cores on the BCS were dated, cre- ating uncertainty when assigning lithostratigraphical information. A final quality check included the identification of duplicate bore- holes, gaps or overlapping borehole intervals. Errors were sub- sequently corrected manually. The bounding surfaces were then combined to create the layer- based model defining the lithostratigraphical units (see step 5); cross-cutting between these surfaces has been resolved in seismic interpretation. The bathymetry (Flanders Hydrography) was used as the top surface. 2.8. Step 5: Assignment of lithostratigraphical units to the 3D voxel model Thenextstepistodefinethevolumeinwhichtheinterpolationoflitho- logical properties will take place. The highest point in the seafloor bathymetry was 4 m MSL (in the port of Zeebrugge), and the lowest point in the volume was −70 m MSL (corresponding to the bottom of the deepest borehole in the dataset). The grid resolution for modelling (i.e. the size of a single voxel) was set to 200 × 200 × 1 m (x, y, z), a choice based on data density and scale of the geological features that were described, while assuring a reasonable speed for 2.7.2. Geological layer creation Th d h f h h The depth of each seismic horizon was exported per line in a point format. Next, these points were interpolated by co-kriging using geostatistical software (ISATIS®), resulting in the creation of 2D bounding surfaces (grid size 200 × 200 m for the BCS and 100 m × 100 m for the Hinder Banks case study). All maps and models were vertically referenced to mean sea level (MSL). This reference level was chosen because it is calculated using onshore fixed points, while it also serves the need for a unified system between the Netherlands and Belgium in view of a future cross-border model- ling programme. Since the Belgian seismic dataset and bathymetry were referenced to the lowest astronomical tide (LAT) they were converted to MSL using a grid provided by Deltares. Standard deviation of the surfaces was estimated using the co-kriging function and was subsequently used as a measure of uncertainty in the modelled stratigraphy. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.3. Methodology The laterally varying velocity model was used to calculate more accurately the Top-Palaeogene surface, the Top-Pleistocene surface and the Upper/Lower Holocene boun- dary. If a constant velocity model (e.g. 1500 m s−1) had been used, the thickness of the Quaternary units would have been locally over- or underestimated. Additionally, errors in depth caused by local- ised velocity anomalies due to morphological features such as the pull-up effect of sandbanks (Fagin, 1996) were addressed using this dynamic velocity model. Locally, the laterally varying velocity model based on the core dataset introduces artefacts such as depressions (e.g. in the Zeebrugge valley) that are not visible in or not covered by the seismic dataset. Other artefacts include unrealistically low velocity values in the water body related to the outcrop of the Palaeogene layer in between the sand banks (Fig. 4). Nonetheless, the core-based approach leads to better results than the constant velocity model which generally overesti- mates the depth of the top Palaeogene surface. Furthermore the velocity model, due to artefacts inherent in the nature of the dataset (digitised from paper, seismic lines), does not always refer to a physical velocity but is often a conversion factor. showed a spatial misplacement). A second problem was related to the presence of shallow gas preventing seismic penetration (e.g. Missiaen et al., 2002). Other factors, such as bad weather condi- tions, also resulted in lower data quality. These problems were addressed either by excluding the problematic line from the dataset or ignoring the problematic segment. Facies with similar acoustic characteristics were delineated (e.g. Fig. 13 below). This was mostly a seismic interpretation revision of Mathys (2009). Boundaries were identified between the following stratigraphical units: Palaeogene, Quaternary, further divided into Pleistocene, Upper and Lower Holocene (Fig. 1). Acoustic facies were linked to stratigraphic boundaries. In this process, cross-veri- fication with the borehole data was essential. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.7. Step 4: Construction of 2D stratigraphical layer models 2.7.1. Time-to-depth conversion 2.7.1. Time-to-depth conversion 2.7.1. Time-to-depth conversion The horizons identified on seismic profiles were picked in time, because seismic traces are recorded in two-way travel time of the signal. A time-to-depth conversion is required to define the 6 Vasileios Hademenos et al. 6 Fig. 4. Map showing the laterally varying velocity model in m s−1 used to calculate the depth of (A) the picked seafloor horizon (water column) and (B) the picked Top-Palaeogene horizon (Quaternary layer). Fig. 4. Map showing the laterally varying velocity model in m s−1 used to calculate the depth of (A) the picked seafloor horizon (water column) and (B) the picked Top-Palaeogene horizon (Quaternary layer). the interpolation process. A higher-resolution model of 100× 100 × 0.5 m was tested at the Hinder Banks. taking into account the previous information, resulting in a prob- ability between 0 and 1 for each lithological class. The values are plotted in a cumulative distribution function. Then a random value between 0 and 1 is drawn and compared to the cumulative distribution function. The simulated lithological class at the target voxel corresponds to the rank of the interval to which the random value belongs. The bounding surfaces, as described in step 4, are now added to the volume of the model, and the space between them is filled with voxels. The centres of the voxels (whether above or below a surface) define the lithostratigraphical unit they belong to. The voxels are assigned a constant integer value that corresponds to the lithostratigraphy (e.g. 1 for Nearshore Upper Holocene, 2 for Offshore Upper Holocene, etc.). Especially in the deeper parts of the model, the neighbourhood search at a target voxel may end up with no data (neither hard data from boreholes nor already simulated voxels). The result is then drawn from proportions. These are the global proportions of each lithological class observed in the boreholes, which are assumed to be constant throughout the lithostratigraphical unit (Fig. 5). https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.9. Step 6: 3D interpolation of lithological class The next step in voxel modelling is to estimate a lithological class for each voxel, for which the Sequential Indicator Simulation (SIS) (Goovaerts, 1997; Chilès & Delfiner, 2012) technique was used (ISATIS®). SIS requires modest computation time and has been applied earlier in the creation of voxel models in similar geological settings (Stafleu et al., 2011; Maljers et al., 2015). The SIS method can be extremely useful in relatively homo- geneous geological units or in cases where good data coverage is available. However, on the BCS, and especially in the Holocene layer with its diverse sediment types, it may lead to errors in the form of so-called ‘flying’ voxels. These comprise voxels in regions of low data density that have been assigned a lithological class according to the global proportions (percentage of lithological class in each layer). This problem can be resolved by splitting the layer into smaller, better-defined sublayers. To allow a good comparison of the results, parameters such as the size of the neighbourhood (10 km) and the dimensions of the voxels (200 m × 200 m × 1 m) were kept constant as new lithostratigraphical layers were added. Borehole data were first migrated to the closest voxel and considered as hard data afterwards. All the remaining voxels were scanned using a random path. A neighbourhood is established, centred on the target voxel, and within this neighbourhood the procedure searches for the hard data from the boreholes and for voxels that are already simulated. The neighbourhood is examined using a variogram model which ensures that data most closely correlated with the target voxels are assigned the greatest weight. The data are then coded into a set of indicators; hence the name indicator simulation. For each lithological class, the indicator is set to 1 if the data belong to the lithological class and to 0 if not. The next step in SIS consists of a co-kriging phase (block kriging) The SIS resulted in 100, statistically equally probable, simulations of lithological class distributions. From these simulations probabil- ities of occurrence for each lithological class were calculated giving an indication of model uncertainty. In addition, the probabilities were used to compute a ‘most likely’ lithological class model using Netherlands Journal of Geosciences 7 Fig. 5. 2.9. Step 6: 3D interpolation of lithological class Chart representing the global proportions of each lithological class in each lithostratigraphical layer in the process of adding more lithostratigraphical divisions in each model run (NUH: Nearshore Upper Holocene; OUH: Offshore Upper Holocene). Fig. 5. Chart representing the global proportions of each lithological class in each lithostratigraphical layer in the process of adding m model run (NUH: Nearshore Upper Holocene; OUH: Offshore Upper Holocene). roportions of each lithological class in each lithostratigraphical layer in the process of adding more lithostratigraphical divisions in each ocene; OUH: Offshore Upper Holocene). Fig. 5. Chart representing the global proportions of each lithological class in each lithostratigraphical layer in the process of adding more lithostratigraphical divisions in each model run (NUH: Nearshore Upper Holocene; OUH: Offshore Upper Holocene). and the differences amongst the probabilities (the greater the differences, the lower the entropy). the averaging method for indicator datasets described by Soares (1992). However, the 100 individual simulation results remain available for further use. As suggested in Wellmann & Regenauer-Lieb (2012), informa- tion entropy can be used as a quality measure of 3D models. In our study, information entropy is used as a comparative measure between different runs of the model. This comparison between the distribution of the information entropy helps quantify the effect of each layer addition on the model. Moreover, it allows us to visualise the overall quality of the model and make compar- isons between the different interpolations. 2.10. Step 7: Assessment of the information entropy of the model The probabilities of occurrence provide a measure of model uncer- tainty. Probabilities of an individual voxel can be displayed in a single bar chart, thus showing its probability distribution and hence model uncertainty. Similar displays are possible in visualisa- tions of virtual boreholes (i.e. vertical stacks of voxels). However, in two- and three-dimensional visualisations (e.g. cross-sections or 3D views) it is not possible to show all probabilities for each voxel in a single view; the user will always be presented with one of the probabilities at a time. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core ter 4. Results Five different units were distinguished defining the lithostrati- graphical succession in the BCS: Palaeogene, Pleistocene, Lower and Upper Holocene, the latter with a subdivision into Nearshore and Offshore. Results are presented on the five model runs starting from the use of a uniform stratigraphy in the modelling process up to using all five units. This was done to compare the effects of the addition of each unit to the model. Additionally, the workflow was applied in a higher resolution in an area with higher data coverage. Results become progressively more detailed as the introduction of each new lithostratigraphical layer divides the model into smaller segments. 4.2.2. Two–layer model (Palaeogene–Quaternary) The first surface added to the model was the Top-Palaeogene unconformity (De Clercq et al., 2016). It is the bounding surface between the Palaeogene and the overlying Quaternary deposits and constrains the lower boundary of the resource units, and as such it has a significant impact on the resource calculations. The results of the two-layer model are shown in Fig. 8B. The Top-Palaeogene layer is now defined as consisting of 56% of clay (see Fig. 5, in green). Moreover, as the Top-Palaeogene surface comprises complex geomorphological features, abrupt lateral changes in the sediment composition are now much better con- strained. A good example is the Ostend Valley (Fig. 1) cutting into the underlying clay sediments (Fig. 15). 3.2. Hinder Banks case study: higher-resolution voxel modelling geological features are clearly visible in each unit, such as the sand- banks and the platform in the Holocene and the Ostend Valley in the Pleistocene. Following the steps described above, a case study has been con- ducted on a smaller area with better data coverage. The case study area (see Fig. 3A) is located in the Hinder Banks area and com- prises three major sandbanks (Noordhinder, Westhinder and Oosthinder). The dense bathymetric, seismic and borehole data coverage allowed the size of the voxels to be reduced to 100 × 100 × 0.5 m (x, y, z). The main reasons for choosing a higher voxel resolution were: (1) to test to what extent voxel size affects resource volume calculations; (2) to evaluate whether a higher resolution allows a better depiction of the different layers within the sand- banks; and (3) to compare the effects of different voxel sizes on the assigned lithological classes and probabilities of occurrence. The layers that are used for this test include the bathymetry, Top-Palaeogene and Top-Pleistocene; all three layers were interpo- lated at a resolution of 100 × 100 m, similar to the voxel size. The latter was achieved by re-interpolating the points from the seismic interpretation (cf. step 4). The Hinder Banks area is the main target for sand dredging in the years to come (Mathys et al., 2011); as such, an accurate estimate of the resource volume and lithology is crucial. 4.2. Stepwise incorporation of geological knowledge g y ( g p y) In the single-layer model, the volume in which the 3D interpola- tion of lithological class takes place is bounded only by the bathym- etry (in MSL) at the top and a horizontal boundary at −70 m MSL. The lithological classes of the borehole dataset were used without stratigraphical interpretation. The results for the single-layer model are shown in Fig. 8A. The model seems to work well in the area around Zeebrugge due to the good data coverage and where the model clearly shows the transition from Palaeogene clays to Quaternary sands. Although no strati- graphic information was added to the model, the model still correctly predicts clay in the depth intervals that contain Palaeogene layers, and sand in the depth intervals that contain Quaternary layers. In other parts of the BCS, with much lower data coverage, the litho- logical class ‘sand’ is wrongly propagated into the Palaeogene layer due to the global proportion of sands (see Fig. 5, in blue) in the bore- hole dataset. The model uncertainty map on the right shows high uncertainties (red voxels) in many areas. 3.1. Stepwise improvement by adding layers One of the aims of this study was to demonstrate how adding more geological information to the modelling process would improve the lithological characterisation of the lithostratigraph- ical units. As such, each borehole interval was attributed different levels of stratigraphy. For example, in the second run of the model when only two layers were used, the labels in the borehole descriptions are ‘Palaeogene’ and ‘Quaternary’, while in the last interpolation using five layers the core dataset contains the full set of relevant lithostratigraphical information (Nearshore Upper Holocene, Offshore Upper Holocene, Lower Holocene, Pleistocene, Palaeogene). To deal with this problem, Wellmann & Regenauer-Lieb (2012) proposed the use of information entropy as a measure of uncer- tainty in 3D models. The information entropy of a voxel is a single value ranging from 0 to 1 that can be calculated from each of the probabilities of lithological classes. An entropy value of 0 means that there is no uncertainty, whereas a value of 1 occurs when all lithological classes have the same probability. Values in between 0 and 1 account for both the number of lithological classes with a probability higher than 0 (the more classes, the higher the entropy) Vasileios Hademenos et al. 8 4.1. Lithological and stratigraphical framework The 2D bounding surfaces, that were created in step 4 of the meth- odology section, are shown in Fig. 6. When overlaid, they form the top and bottom of each unit that will be used in the different inter- polations. The space between them is filled with voxels. The vox- elised lithostratigraphical units and their extent can be seen in Fig. 7. 4.2.3. Three-layer model (Palaeogene–Pleistocene–Holocene) The second added surface is the Top-Pleistocene surface, allowing differentiation of Quaternary deposits in terms of their lithological composition. The patchy and lithologically mixed Quaternary layer comprises the infill of the palaeovalleys in the nearshore area of the BCS, and forms the core of the sandbanks in the offshore area (see Fig. 1). The results of the three-layer model for the BCS are shown in Fig. 8C. In the Ostend Valley, for example, fine sands can now be distinguished from overlying clay sediments. Results show that the Quaternary cover is very thin and its sedi- ments are accumulated mainly in the sandbanks. Each sandbank is unique in its stratigraphical and lithological content. This makes the modelling procedure more challenging because these geological features and their internal structure must be taken into consideration when splitting a lithostratigraphical unit. Fig. 7 shows the results of the lithostratigraphical characterisation of the borehole dataset, as discussed in step 5. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 4.2.4. Four-layer model (Palaeogene – Pleistocene – Lower Holocene – Upper Holocene) 4.2.4. Four-layer model (Palaeogene – Pleistocene – Lower Holocene – Upper Holocene) The level of detail even in the 200 × 200 m resolution of the surfaces allowed the robust modelling of the different geological units described previously, taking into account the stratigraphical boundaries and features (Fig. 6) of the Quaternary cover. For example the extent of the Ostend valley is 10 × 5 km; its internal features can be described well by a 200 × 200 m resolution model. Other resolutions have been tested for the BCP model, though based on the data density and calculations time the 200 × 200 m resolution was decided upon as the most effective. All of these In the nearshore, one extra layer was added to account for the litho- logical differentiation in the Lower Holocene which is here related to a fine-grained tidal-flat environment (Mathys, 2009). Further offshore, the Holocene deposits are coarser. The main purpose of adding this layer was to demonstrate the sensitivity of the model in describing a lithologically varying layer with very few cores crossing it. The results of the four-layer model are shown in Fig. 8D. 9 Netherlands Journal of Geosciences 9 Fig. 6. Views of the modelled bounding surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lower Holocene and (D) Top-Upper Holocene (bathymetry), the latter with a subdivision into Nearshore and Offshore defined by the Middle Scarp. Fig. 6. Views of the modelled bounding surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lowe (bathymetry), the latter with a subdivision into Nearshore and Offshore defined by the Middle Scarp. g surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lower Holocene and (D) Top-Upper Holocene ion into Nearshore and Offshore defined by the Middle Scarp. Fig. 6. Views of the modelled bounding surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lower Holocene and (D) Top-Upper Holocene (bathymetry), the latter with a subdivision into Nearshore and Offshore defined by the Middle Scarp. 4.2.5. Five-layer model (Palaeogene – Pleistocene – Lower Holocene – Nearshore Upper Holocene – Offshore Upper Holocene) the Holocene layer of the three-layer model (Top-Pleistocene surface). These ‘flying’ voxels were introduced by the SIS as the global proportions of the clay–silt percentages were forced through the entire model (see Fig. 5). https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. re. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/te 5. Case study with higher resolution of the voxel model The analysis of the results of the case study gives a detailed over- view of the lithological properties of the area. The 100 × 100 × 0.5 m voxel model shows a predominance of medium sand (Fig. 10). A major difference with the 200 × 200 × 1 m resolution five-layer model is the detailed lithological variation within the sandbanks. The 100 × 100 × 0.5 m was chosen based on the data density. The sandbanks are characterised by a Pleistocene core of coarse sand and gravel, mixed with clay and silts. The silt is mainly found in the upper part of the Pleistocene layer forming a transition boundary towards the Offshore Upper Holocene layer. Coarse sand and gravel populate the space between the sandbanks as well. Moreover, the Palaeogene layer is now composed almost completely of clay since the limited number of cores that penetrate the Palaeogene layer in the Hinder Banks have a global proportion of 92% clay. For the BCS, the Middelkerke Bank (localisation, see Fig. 1) was the first sandbank from which the succession of geological layers was defined (Trentesaux et al., 1999). Fig. 13 shows the original seismic line and its interpretation, based on the combination with the boreholes. The same information was now used in the voxel modelling. Compared to the previous interpretations, we are now able to model the lithological distribution, as well as the related information entropy of the model (Fig. 14). The higher val- ues of information entropy can be seen in the centre of the sand- bank associated with the uncertainty caused by the lack of cores. By visualising and querying the different models, the effects of the changes are discussed and evaluated. As a first example, the effect of a better parameterisation of the Top-Palaeogene is shown in Fig. 15. This was most striking for the delineation of the Ostend palaeovalley in the nearshore zone. In the single-layer model, fine sand voxels extended horizontally and masked the boundary of the valley. With the addition of the Top- Palaeogene surface, a distinct V-shape of the palaeovalley became apparent and allowed showing an infill of the valley with fine-sand (yellow) voxels above the Palaeogene clay layers. A second example illustrates the dramatic change that the addition of layers can make to the distribution of the lithological classes (Fig. 16). This is most evident on the level of the sandbanks (e.g. 4.2.4. Four-layer model (Palaeogene – Pleistocene – Lower Holocene – Upper Holocene) Clay–silt percentages are significantly higher in the nearshore area, because of the nearby estuary of the Scheldt river. In order to separate the two regions (with and without Scheldt influence), the Middle Scarp (see Fig. 1) was used ) In the final model, the Upper Holocene layer was further split (laterally) into two smaller areas. This was done primarily because of the increasing presence of medium sand further offshore as well as to reduce the presence of ‘flying’ clay voxels that were present in ) In the final model, the Upper Holocene layer was further split (laterally) into two smaller areas. This was done primarily because of the increasing presence of medium sand further offshore as well as to reduce the presence of ‘flying’ clay voxels that were present in 10 Vasileios Hademenos et al. Fig. 7. Fence diagram of voxelized lithostratigraphical units in the BCS. The borehole dataset is colour-coded following their lithostratigraphical interpretation. The blue line represents the extent of the modelled area. ostratigraphical units in the BCS. The borehole dataset is colour-coded following their lithostratigraphical interpretation. The blue line Fig. 7. Fence diagram of voxelized lithostratigraphical units in the BCS. The borehole dataset is colour-coded following their lithostra represents the extent of the modelled area. of the higher-resolution model is the fact that the lithostratigraph- ical units are much better defined, as shown by the Pleistocene inner core of the sandbanks. Additionally, some features were bet- ter delineated, e.g. the fine sand in the top zone of one of the sand- banks which is present in both models. as proxy where the depositional environment changes from a near- shore estuarine–marine to an offshore marine environment. The results of the five-layer model are shown in Fig. 8E. Another way of querying the model is the creation of cross- sections (Fig. 9). This type of data visualisation allows inspection of the in-depth distribution of the different assigned variables. Following the geological cross-section of Fig. 1, we can see all five lithostratigraphical units with the assigned lithology and informa- tion entropy. Cross-sections like these give a first glimpse of the areas where we are confident of the assigned lithology (blue colour in bottom figure), e.g. the fine sand above the Ostend Valley and the coarse sand tips off the Hinder Banks. 6.1. Importance of geological knowledge in voxel modelling Modelling the subsurface geology of shallow marine environments is highly challenging because of the complex depositional environ- ments that often vary on short spatial scales. Detailed interpolation of lithological information is hence critical and requires the best available knowledge on the geological layers constraining different resource qualities. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 5. Case study with higher resolution of the voxel model Hinder Banks) where in the second run of the model the base of the sandbank is better defined because of the addition of the Top-Palaeogene To compare the resource volumes between the different resolu- tion models of the Hinder Banks, resource quantity was queried in the first 2 m below seafloor (Fig. 11). The different resolutions do not affect the total volume of the queried voxels. However, there is a difference when comparing the lithologies of the two models (Fig. 12). This difference is due to the fact that in the 100 × 100 × 0.5 m resolution model the core dataset used is confined to a buffer zone around the modelled area, while in the 200 × 200 × 1 m resolution model the core dataset of the entire BCS (Fig. 3) is used. This clearly influences the voxels assigned with gravel, since the percentage of gravel is much higher in the cores around the Hinder Banks compared to the BCS dataset. Another advantage Netherlands Journal of Geosciences 11 Fig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy) B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the Lower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining ig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy). B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the ower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining ve lithostratigraphical layers of which the Nearshore (bottom) and Offshore Upper Holocene (top) are shown here. Fig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy). (B) One bounding surface (Top-Palaeogene). 5. Case study with higher resolution of the voxel model (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the Lower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining five lithostratigraphical layers of which the Nearshore (bottom) and Offshore Upper Holocene (top) are shown here. Fig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the Lower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining five lithostratigraphical layers of which the Nearshore (bottom) and Offshore Upper Holocene (top) are shown here. 12 Vasileios Hademenos et al. Fig. 9. Cross-section of the final model (for location, see Fig. 1). Top: lithostratigraphical units. Middle: lithological class. Bottom: model entropy on the lithological class. 0 indicates low and 1 high uncertainty. Fig. 9. Cross-section of the final model (for location, see Fig. 1). Top: lithostratigraphical units. Middle: lithological class. Bottom: mo indicates low and 1 high uncertainty. Fig. 9. Cross-section of the final model (for location, see Fig. 1). Top: lithostratigraphical units. Middle: lithological class. Bottom: model entropy on the lithological class. 0 indicates low and 1 high uncertainty. Fig. 10. High-resolution voxel model (100 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithological class, shown only for the Quaternary. Fig. 10. High-resolution voxel model (100 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithological class, shown only for the Quaternary. 00 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithological class, shown only for the Quaternary. Fig. 10. High-resolution voxel model (100 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithologi surface. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. rg/10.1017/njg.2018.18 from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available a 5. Case study with higher resolution of the voxel model In the third run of the model the introduction of the Pleistocene unit allowed us to define a core of lithologically mixed sediments. Finally, the fifth run of the model allowed us to differ- entiate layers of different lithological classes in the main body of the sandbanks once the offshore Upper Holocene unit was defined. The final figures illustrate that more data allow the creation of higher-resolution models that constrain better the stratigraphical layers and their lithological properties. Offshore Upper Holocene layer, as depicted in Fig. 15B, there is sig- nificantly less clay–silt in the global proportions (Fig. 5) when split from the Upper Holocene. As a result of the lower percentages, the majority of ‘flying voxels’ (Fig. 15A) are no longer present. To quantify the added value of incorporating geological knowl- edge in the voxel modelling procedure, the model uncertainty cal- culations, performed on each of the model runs, are now discussed and compared (Schweizer et al., 2017). Fig. 17 shows distribution curves of the model uncertainty with each new addition of a geo- logical layer. The addition of geological layers also allowed the reduction of the number of ‘flying voxels’, that were introduced as a consequence of the SIS method. In areas with low data density, SIS draws the litho- logical class from the global proportions per lithostratigraphical unit. By splitting the model volume into separate lithostratigraphical units, these percentages change according to the lithological content of the boreholes belonging to the lithostratigraphical units. In the In the single-layer model (first run – blue line) the mean of the model uncertainty distribution is close to 0.7 (Fig. 17A). Adding the Quaternary layer (second run – red line), the mean is close to 0.5, indicating an improvement of the modelling of the Palaeogene layer. The different runs of the model did not have 13 Netherlands Journal of Geosciences Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. the same pronounced effect on the model uncertainty distribution of the Pleistocene layer (Fig. 17B). Also, the addition of the Lower Holocene layer did not show any improvement in the model uncertainty distribution of the model (Fig. 17C). 5. Case study with higher resolution of the voxel model This can be attributed to the fact that this layer is poorly describe and that there are no distinct differences in th content that the addition of the lithostratigraphi describe. Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). ps://doi.org/10.1017/njg.2018.18 wnloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cam Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). attributed to the fact that this layer is poorly described in the cores and that there are no distinct differences in the lithological content that the addition of the lithostratigraphical layer can describe. the same pronounced effect on the model uncertainty distribution of the Pleistocene layer (Fig. 17B). Also, the addition of the Lower Holocene layer did not show any improvement in the model uncertainty distribution of the model (Fig. 17C). This can be 14 Vasileios Hademenos et al. Fig. 14. Left: lithostratigraphical units. Middle: lithological class. 5. Case study with higher resolution of the voxel model Right: model uncertainty, as queried from the voxel model along the same cross-section of Trentesaux et al. (1999; see Fig. 13). For values see legend of Fig. 9. Fig. 14. Left: lithostratigraphical units. Middle: lithological class. Right: model uncertainty, as queried from the voxel model along the same cross-section of Trentesaux et al. (1999; see Fig. 13). For values see legend of Fig. 9. Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One- layer model. Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. layer model. Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One- layer model. laeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One- Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One- layer model Fig. 16. Cross-section in the area of the Hinder Banks showing the distribution of lithoclasses of the different runs of the 200 × 200 × 1 m model. (A) One-layer model (no bound surfaces). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene). (D) Four bounding surfaces (Top-Palaeogene, T Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene). Followed by the final results from the 100 × 100 × 0.5 m resolution model. (E) litholog class, and (F) lithostratigraphical units. Fig. 16. Cross-section in the area of the Hinder Banks showing the distribution of lithoclasses of the different runs of the 200 × 200 × 1 m model. (A) One-layer model (no bounding surfaces). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene). (D) Four bounding surfaces (Top-Palaeogene, Top- Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene). Followed by the final results from the 100 × 100 × 0.5 m resolution model. (E) lithological class, and (F) lithostratigraphical units. Fig. 16. 5. Case study with higher resolution of the voxel model Cross-section in the area of the Hinder Banks showing the distribution of lithoclasses of the different runs of the 200 × 200 × 1 m model. (A) One-layer model (no bounding surfaces). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene). (D) Four bounding surfaces (Top-Palaeogene, Top- Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene). Followed by the final results from the 100 × 100 × 0.5 m resolution model. (E) lithological class, and (F) lithostratigraphical units. 15 Netherlands Journal of Geosciences Fig. 17. Distribution of the model entropy on the lithological class for each layer queried for different runs of the model. 0 indicates low and 1 high uncertainty. Fig. 17. Distribution of the model entropy on the lithological class for each layer queried for different runs of the model. 0 indicates low and 1 high uncertainty. he model entropy on the lithological class for each layer queried for different runs of the model. 0 indicates low and 1 high uncertaint Fig. 17. Distribution of the model entropy on the lithological class for each layer queried for different runs of the model. 0 indic with high uncertainty help us to understand the limitations of the datasets and will guide the planning of new data acquisition surveys. Adding the Nearshore Upper Holocene layer resulted in a more distinctive peak in the model uncertainty spectrum (Fig. 17D – green line). The high values of model uncertainty (0.6–0.8) are caused by the complex geology of the Scheldt estuary. The presence of laminated layers consisting of clay and fine sand makes it diffi- cult for the method to predict the lithology of the voxels in the region, at least at the scale of the present model. With increasing use of the BCS, both by the public and private sector, there are ample opportunities to validate and apply the model for different purposes, ideally by incorporating new data. Some of these applications may require a higher vertical resolution, which will require more tests. With the 1 m vertical scale resolu- tion, certain features such as the clay–silt–fine-sand laminated layering in the estuarine deposits are easily overlooked, especially in regions with high lithological heterogeneity. A case study close to the port of Zeebrugge has been planned where the resolution will be decreased to 0.7–0.4 m. 5. Case study with higher resolution of the voxel model In this area, it is also planned to incor- porate additional data, such as cone penetration tests, making the model more valuable for geotechnical applications. The Offshore Upper Holocene layer shows two distinct peaks (0.5 and 0.65) in the model uncertainty spectrum in the second and third runs of the model (Fig. 17E – red, light blue and purple lines). When the Holocene layer is split in the fifth run of the model (green line), the model uncertainty improves (peak around 0.4). This is because the offshore sandbanks have a better data coverage than the areas in the gullies between the sandbanks, where the information entropy is higher (0.5–0.7). Last but not least, it needs emphasising that the model is the backbone of a resource decision support system (Van Lancker et al., 2017). As such, users have direct access through the TILES website to the available geological information and can query resource quality and quantity, in combination with environ- mental and socio-economic datasets, in view of different applica- tions, now and in the future. 6.2. Future perspectives The model provides the uncertainty (information entropy) of the lithological class in each voxel based on the calculations of the SIS method. However, this uncertainty is now solely related to statis- tical calculations, whilst there is also uncertainty imposed by the dataset itself. Data-related uncertainties include those originating from the seismic lines (bad quality, misplacement) and borehole descriptions (mislabelling, insufficient interpretations, inadequate metadata) datasets and may have an adverse effect on the confi- dence we have in the resulting model. These uncertainties have already been quantified and incorporated in the model as flags, and procedures are now evaluated on how to propagate those uncertainties in the voxel models (De Tré et al., 2018). The areas https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 7. Conclusions A 3D voxel model was created depicting in detail stratigraphical and lithological information of the subsurface of the BCS. The 3D environment allows easy viewing of geological properties providing spatial context to certain features or heterogeneities in the subsurface, which is highly valuable in resource management. Vasileios Hademenos et al. 16 Geological knowledge was incorporated in a stepwise approach and gave a level of detail in the model that would not have been achieved by only using available coring data. Moreover, the step- wise approach allowed us to monitor the effect of each layer addi- tion on the model and its effectiveness in describing the geological features. In addition, model uncertainty, calculated as information entropy, was added to the voxels, providing insight into how suc- cessful the model is in unambiguously estimating lithological classes. Because of the automated workflow, new data can be incor- porated easily and results can be compared and evaluated against previous versions of the model. Goovaerts, P., 1997. Geostatistics for natural resources evaluation. Oxford University Press (New York): 483 pp. Goovaerts, P., 1997. Geostatistics for natural resources evaluation. Oxford University Press (New York): 483 pp. Jørgensen, F., Høyer, A., Sandersen, P., He, X. & Foged, N., 2015. Combining 3D geological modelling techniques to address variations in geology, data type and density – an example from Southern Denmark. Journal of Computers & Geosciences 35: 1311–1321. Kint, L. & Van Lancker, V., 2016. SediLITHO@SEA v2 (06/10/2016). Database lithological descriptions, with relevance to Belgian part of the North Sea. Royal Belgian Institute of Natural Sciences (Brussels). Le Bot, S., Van Lancker, V., Deleu, S., De Batist, M. & Henriet, J.P., 2003. Tertiary and Quaternary geology of the Belgian Continental Shelf. Scientific support plan for a sustainable development policy, SPSD II. PPS Science Policy (Brussels): 75 pp. Available at: http://www.vliz.be/imisdocs/ publications/215061.pdf. The new voxel model forms the backbone of a resource decision support system allowing structured querying of extractable aggre- gate resource volumes combining the geology-related information (stratigraphy, lithological class, model uncertainty) and any third party data that may constrain exploitation (e.g. shipping lanes, cable lines, port locations, ecologically valuable areas). Le Bot, S., Van Lancker, V., Deleu, S., De Batist, M., Henriet, J.P. & Haegeman, W., 2005. Geological characteristics and geotechnical properties of Eocene and Quaternary deposits on the Belgian continental shelf: synthe- sis in the context of offshore wind farming. Netherlands Journal of Geosciences 84: 147–160. References Bartakovics, E., Koma, Z., Székely, B., Kovács, G., András Zámolyi, A. & Tímár, G., 2013. Considerations on voxelization techniques for 3D geological and geophysical modeling using borehole data and seismic section interpretation– examples from Little Hungarian Plain, Pannonian Basin. Geophysical Research Abstracts 15, EGU2013-904-1, 2013 EGU General Assembly. Schaap, D., 2017. SeaDataNet: towards a pan-European infrastructure for marine and ocean data management. In: Diviacco, P., Leadbetter, A., &Glaves, H. (eds): Oceanographic and marine cross-domain data manage- ment for sustainable development. IGI Global (Hershey, PA): 155–177. Chilès, J.-P. & Delfiner, P., 2012. Geostatistics– modeling spatial uncertainty. Wiley (Hoboken): 699 pp. Schweizer, D., Blum, P. & Butscher, C., 2017. Uncertainty assessment in 3-D geological models of increasing complexity. Solid Earth 8: 515–530. De Batist, M., 1989. Seismo-stratigrafie en Struktuur van het Paleogeen in de Zuidelijke Noordzee, PhD thesis Ghent University (Ghent): 107 pp. Soares, A., 1992. Geostatistical estimation of multi-phase structures. Mathematical Geology 24: 149–160. De Clercq, M., Chademenos, V., Van Lancker, V. & Missiaen, T., 2016. A high-resolution DEM for the Top-Palaeogene surface of the Belgian Continental Shelf. Journal of Maps 12: 47–1054. Stafleu, J., Maljers, D., Gunnink, J.L., Menkovic, A. & Busschers, F.S., 2011. 3-D modelling of the shallow subsurface of Zeeland, the Netherlands. Netherlands Journal of Geosciences / Geologie en Mijnbouw 90: 293–310. De Clercq, M., Missiaen, T., Wallinga, J., Hurtado, Z.O., Versendaal, A., Mathys, M. & De Batist, M., 2018. A well-preserved Eemian incised-valley fill in the southern North Sea Basin, Belgian Continental Shelf-Coastal Plain: implications for northwest European landscape evolution. Earth Surface Processes and Landforms. 43: 1913–1942. Stafleu, J., Maljers, D., Busschers, F.S., Gunnink, J.L., Schokker, J., Dambrink, R.M., Hummelman, H.J. & Schijf, M.L., 2012. GeoTOP mod- ellering. TNO Rapport 2012 R10991 TNO – Geological Survey of the Netherlands (Utrecht): 216 pp. De Tré, G., De Mol, R., Van Heteren, S., Stafleu, J., Chademenos, V., Missiaen, T., Kint, L., Terseleer, N. & Van Lancker, V., 2018. Data quality assessment in volunteered geographic decision support. In: Bordogna, G. & Carrara, P. (eds): Mobile information systems leveraging volunteered geo- graphic information for Earth observation. Springer (Cham): 173–192. Trentesaux, A., Stolk, A. & Berne, S., 1999. Sedimentology and stratigraphy of a tidal sand bank in the southern North Sea. Marine Geology 159: 253–272. Van Haren, T., Dirix, K., De Koninck, R, De Groot, C. & De Nil, K., 2016. Author ORCIDs. Vera Van Lancker 0000-0002-8088-9713 Author ORCIDs. Vera Van Lancker 0000-0002-8088-9713 Author ORCIDs. Vera Van Lancker 0000-0002-8088-9713 Acknowledgements. This paper is a contribution to the Brain-be project TILES (Transnational and Integrated Long-term marine Exploitation Strategies), funded by Belgian Science Policy (Belspo) under contract BR/121/A2/TILES, and the Flemish research project SeArch (IWT SBO, contract nr. 120003). The research is fully supported by the ZAGRI project, a federal Belgian programme for con- tinuous monitoring of sand and gravel extraction, paid from private revenues. Contributing EU projects have been EMODnet Geology (MARE/2008/03; MARE/2012/10; EASME/EMFF/2016/1.3.1.2 – Lot 1/SI2.750862)), Geo-Seas (FP7, Grant 238952) and ODIP (FP7, Grant 312492). Underlying data have been acquired during numerous campaigns on RV Belgica, RV Simon Stevin and many Dutch research and monitoring vessels. For Belgian waters, ship-time was granted by Belspo / RBINS ODNature and Flanders Marine Institute. Nikki Trabucho, TNO, is acknowledged for the graphical design of Figures 2 and 5. The Federal Public Service Economy, Continental Shelf department is thanked for continuous support. Results, datasets, findings and the final version of the models are available through https://odnature.naturalsciences.be/tiles/. Dr P. Kruiver and an anonymous reviewer are thanked for very constructive comments. Liu, A.C., Missiaen, T. & Henriet, J.P., 1992. The morphology of the top- Tertiary erosion surface in the Belgian sector of the North Sea. Marine Geology 105: 275–284. Maljers, D., Stafleu, J., Van der Meulen, M.J. & Dambrink, R.M., 2015. Advances in constructing regional geological voxel models, illustrated by their application in aggregate resource assessments. Netherlands Journal of Geosciences / Geologie en Mijnbouw 94: 257–270. Mathys, M., 2009. The Quaternary geological evolution of the Belgian Continental Shelf, Southern North Sea, PhD thesis. Ghent University (Ghent). Available at http://hdl.handle.net/1854/LU-716421. Mathys, M., Van Lancker, V., De Backer, A., Hostens, K., Degrendele, K. & Roche, M., 2011. Application for a sand extraction concession in exploration Zone 4: baseline studies on the Hinderbanks and future impact monitoring. In: Study day: marine aggregate extraction: needs, guidelines and future pros- pects. Bredene: 113–124. Missiaen, T., Murphy, S., Loncke, L. & Henriet, J.P., 2002. Very high- resolution seismic mapping of shallow gas in the Belgian coastal zone. Continental Shelf Research 22: 2291–2301. Newell, R.C., Seiderer, L.J. & Hitchcock, D.R., 1998. The impact of dredging works in coastal waters: a review of the sensitivity to disturbance and sub- sequent recovery of biological resources on the sea bed. Oceanography and Marine Biology: An Annual Review 36: 127–178. 7. Conclusions Liu, A.C., 1990. A seismic and geomorphological study of the erosion surface at the top of the Tertiary in the Southern North Sea (Belgian and Northern French sectors), PhD thesis Gent University (Gent): vol. 1: 119 pp. þ vol. 2: 97 figs. Author ORCIDs. Vera Van Lancker 0000-0002-8088-9713 References An interactive voxel model for mineral resources: loess deposits in Flanders (Belgium). Zeitschrift der Deutschen Gesellschaft für Geowissenschaften 167: 363–376. Fagin, S., 1996. The fault shadow problem: its nature and elimination. The Leading Edge 15: 1005–1013. 1017/njg 2018 18 m https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at Netherlands Journal of Geosciences 17 H. (eds): Oceanographic and marine cross-domain data management for sustainable development. IGI Global (Hershey, PA): 224–252. Van Heteren, S., 2010. Standards for formats for transport of geological data. WP4– Development of content standards, profiles, common transport and ser- vices protocols. EU ProjectGeo-Seas: Pan-Europeaninfrastructure for manage- ment of marine and ocean geological and geophysical data: 28 pp. Available at: http://www.geo-seas.eu/content/content.asp?menu=0330000_000000. Vernes, R.W. & Van Doorn, Th.H.M., 2005. Van Gidslaag naar Hydrogeologische Eenheid– Toelichting op de totstandkoming van de dataset REGIS II. Report 05-038-B Netherlands Institute of Applied Geosciences TNO (Utrecht): 105 pp. Van Heteren, S., Meekes, J.A.C., Bakker, M.A.J., Gaffney, V., Fitch, S., Gearey, B.R. & Paap, B.F., 2014. Reconstructing North Sea palaeoland- scapes from 3D and high-density 2D seismic data: an overview. Netherlands Journal of Geosciences / Geologie en Mijnbouw 93: 31–42. Wellmann, J.F. & Regenauer-Lieb, K., 2012. Uncertainties have a meaning: information entropy as a quality measure for 3-D geological models. Tectonophysics 526–529: 207–216. Wentworth, C.K., 1922. A scale of grade and class terms for clastic sediments. The Journal of Geology 30: 377–392. Van Lancker, V.R.M., Bonne, W., Garel, E., Degrendele, K., Roche, M., Van den Eynde, D., Bellec, V., Brière, C., Collins, M.B., & Velegrakis, A.F., 2010. Recommendations for the sustainable exploitation of tidal sandbanks. In: Van Lancker, V., Bonne, W., Uriarte, A., and Collins, M.B, (eds): EUMARSAND: European Marine Sand and Gravel Resources: 151–164. Journal of Coastal Research, Special Issue 51. Coastal Education and Research Foundation, Inc. (West Palm Beach, FL). https://doi.org/10.1017/njg.2018.18 Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. Flanders Hydrography: http://www.vlaamsehydrografie.be (accessed on May 2016) Geo-Seas: http://www.geo-seas.eu/ (accessed on June 2015) ISATIS® Geostatistical Software: https://www.geovariances.com/en/software/ isatis-geostatistics-software (accessed on April 2015) SeaDataNet: https://www.seadatanet.org/ (accessed on June 2015) TILES: http://www.odnature.naturalsciences.be/tiles/ (accessed on July 2018) Websites Flanders Hydrography: http://www.vlaamsehydrografie.be (accessed on May 2016) Geo-Seas: http://www.geo-seas.eu/ (accessed on June 2015) ISATIS® Geostatistical Software: https://www.geovariances.com/en/software/ isatis-geostatistics-software (accessed on April 2015) SeaDataNet: https://www.seadatanet.org/ (accessed on June 2015) TILES: http://www.odnature.naturalsciences.be/tiles/ (accessed on July 2018) Flanders Hydrography: http://www.vlaamsehydrografie.be (accessed on May 2016) Van Lancker, V., Francken, F., Kint, L., Terseleer, N., Van den Eynde, D., De Mol, L., De Tré, G., De Mol, R., Missiaen, T., Hademenos, V., Bakker, M., Busschers, F., Maljers, D., Stafleu, J. & Van Heteren, S., 2017. 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O filme de busca e a construção ensaística de uma memória familiar emoldurada pelas montanhas mineiras
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* Adriano Medeiros da Rocha: Universidade Federal de Ouro Preto, Instituto de Ci- ências Sociais Aplicadas, curso de Comunicação Social-Jornalismo. 35420000 - Ma- riana, Minas Gerais, Brasil. E-mail: adrianomedeiros.audiovisual@gmail.com Eduardo Henrique Moreira: Mestrando. Universidade Federal de Ouro Preto, Instituto de Ciências Sociais Aplicadas (ICSA), Programa de Pós-Graduaçao em Comunicação. 35420-000, Mariana, Minas Gerais, Brasil. E-mail: eduardomoreira.op@gmail.com O filme de busca e a construção ensaística de uma memória familiar emoldurada pelas montanhas mineiras Adriano Medeiros da Rocha & Eduardo Henrique Moreira* Seu Carlito, narrativas sobre um comerciante da roça (Brasil, 20 70min30seg) Direção e Montagem: Eduardo Moreira Produção: Eduardo Moreira e José Carlos Moreira Fotografia e Som Direto: Eduardo Moreira e Thiago Novais Trilha Sonara: Matheus Ferro Seu Carlito, narrativas sobre um comerciante da roça (Brasil, 2016 70min30seg) Direção e Montagem: Eduardo Moreira Produção: Eduardo Moreira e José Carlos Moreira Fotografia e Som Direto: Eduardo Moreira e Thiago Novais Trilha Sonara: Matheus Ferro O início, o fim, e o meio... Doc On-line, n. 24, setembro de 2018, www.doc.ubi.pt, pp. 294-306. DOI: 10.20287/doc.d24.ac05 DOI: 10.20287/doc.d24.ac05 O início, o fim, e o meio... A produção de um documentário envolve a captação de algum tipo matéria prima temática. Nesse processo, como também na edição, o cineasta expõe sua visão do tema abordado em seu filme, ele enquadra sua história. Na concep- ção de Sheila Bernard (2008), os filmes documentários se baseiam em fatos, e é disso que vem o seu poder e sua diferenciação do cinema de ficção. Par- tindo desse princípio, podemos entender os documentários como uma forma subjetiva de representação do mundo derivada das decisões do cineasta. Esses filmes são, portanto, carregados de sentidos próprios de quem os faz. De acordo com Bill Nichols (2005: 47), os documentários não são uma re- produção da realidade, mas são representações do mundo que vivemos. Assim como fazem os outros meios de comunicação, no processo de produção de um documentário, os fatos são representados através da abordagem escolhida pelo diretor e sua equipe. Assim, essa abordagem emerge de uma série de escolhas. Consuelo Lins e Cláudia Mesquita (2008: 58) consideram que a rede de escolhas que envolvem a produção documental é o “mais puro” artifício que gera o filme. Para os autores, os documentários “não brotam do coração do real, espontâneos, naturais, recheados de pessoas e situações autênticas”, eles dependem da dedicação e envolvimento de seus realizadores Sheila Curran Bernard (2008: 47) afirma que as redes de escolhas definem como as histórias são contadas nos documentários. Para ela, essa rede envolve O filme de busca e a construção ensaística de uma memória familiar... 295 escolhas criativas acerca da estrutura do filme, de perspectiva, de equilíbrio, de estilo, de elenco, entre outras. A autora entende que as escolhas caracterizam a abordagem do filme. Dê a um grupo de cineastas alguma aparelhagem e a mesma história a ser contada em linhas gerais, e você acabará tendo em mãos filmes bastante di- ferentes em estilos, ponto de vista, enfoque e, na verdade, diversos em muito mais do que isso. Essas diferenças caracterizam a abordagem: como apresen- tar a história na tela. (Bernard, 2008: 47) Cada filme documentário tem sua abordagem, ou seja, a rede de escolhas criativas feitas para sua realização. Portanto, como afirma Nichols (2005: 48), são todos diferentes, pois “não adotam um conjunto fixo de técnicas, não tra- tam apenas de um conjunto de questões, não apresentam apenas um conjunto de formas ou estilos”. O início, o fim, e o meio... Bernard considera a maior parte dos documentários como sendo também narrativas e, em sua opinião, isso significa, pura e sim- plesmente, que eles contam histórias. Para a autora, a forma como estas obras contam as histórias e quais histórias contam são os elementos que separa esses filmes em subcategorias de gêneros ou estilo (Bernard, 2008:15). Mesmo com as subcategorias que caracterizam alguma(s) abordagem(s), a prática está sujeita a constantes mudanças. Neste universo, há uma constante busca por abordagens alternativas e transformações no uso da linguagem. Re- fletindo sobre essa questão, Bernard ressalta que: Ao discutir abordagem, tenha consciência de que os cineastas, conforme se mencionou, não raro combinam diferentes abordagens. Alguns usam narra- ção (ou texto na tela, que desempenhe o mesmo papel), mas a usam com parcimônia. Outros combinam voice-over com narração, ou o roteiro com voice-over em lugar da narração. Alguns filmes fabricam situações que então se desdobram na tela, tornando-se, portanto, observacionais. (Bernard, 2008: 51) Bill Nichols (2005) argumenta que os documentários representam ques- tões, aspectos, características e problemas encontrados no mundo histórico por meio de sons e imagens que compões seus discursos, o que suscita a questão da voz que cada filme tem. O fato de documentários não serem uma reprodução da realidade da a eles uma voz própria. Eles são uma reprodução do mundo, e essa representação significa uma visão singular do mundo. A voz do documentário é, portanto, o meio pelo qual esse ponto de vista ou essa perspectiva singular se dá a conhecer. (Nichols, 2005: 73) O fato de documentários não serem uma reprodução da realidade da a eles uma voz própria. Eles são uma reprodução do mundo, e essa representação significa uma visão singular do mundo. A voz do documentário é, portanto, o meio pelo qual esse ponto de vista ou essa perspectiva singular se dá a conhecer. (Nichols, 2005: 73) Cada documentário tem sua voz fílmica, ou seja, uma natureza própria. Essa voz também pode ser entendida como uma impressão digital que atesta a individualidade do cineasta, ou até mesmo o poder de decisão de seus financia- 296 Adriano Medeiros da Rocha & Eduardo Henrique Moreira dores. Sendo a maneira como o cineasta traduz seu ponto de vista e estabelece uma relação com mundo histórico, a voz fílmica remete ao estilo do filme. O início, o fim, e o meio... Quando assistimos a documentários participativos, esperamos testemunhar o mundo histórico da maneira pela qual ele é representado por alguém que nele se engaje ativamente, e não por alguém que observa discretamente, recon- figura poeticamente ou monta argumentativamente esse mundo. O cineasta despe o manto do comentário com voz-over, afasta-se da meditação poética, desce do lugar onde pousa a mosquinha na parede e torna-se um ator social (quase) como qualquer outro. (Nichols, 2005: 154) Quando assistimos a documentários participativos, esperamos testemunhar o mundo histórico da maneira pela qual ele é representado por alguém que nele se engaje ativamente, e não por alguém que observa discretamente, recon- figura poeticamente ou monta argumentativamente esse mundo. O cineasta despe o manto do comentário com voz-over, afasta-se da meditação poética, desce do lugar onde pousa a mosquinha na parede e torna-se um ator social (quase) como qualquer outro. (Nichols, 2005: 154) Documentários participativos simulam o que é, para o cineasta, estar numa determinada situação e como ela consequentemente se altera. A experiência extraída do encontro entre o cineasta e o tema será o conteúdo do filme. O cineasta e tem a possibilidade de se posicionar como mentor, crítico, interro- gador, colaborador ou provocador, ou mesmo como fio condutor da história. (Nichols, 2005 :155). Silvio Da-Rin (2006) acredita que, nesse modo ensaístico e interativo, “o cineasta dispõe de novos recursos para recusar o papel de agenciador oculto de imagens sonoras e visuais, podendo exibir-se como um ser humano impli- cado” (Da-Rin, 2006: 155 e 156). Além dessas características, Dan-Rin aponta também para o antiilusionismo que se manifestou de forma colateral no estilo interativo que permeia o filme ensaio. No modo interativo, o antiilusionismo se manifesta de forma colateral, apesar da contribuição inaugural de ChroniquedùnÉté. Um dos fatores que marcou a novidade radical foi a liberdade com que os equipamentos de filmagem e os membros da equipe eram exibidos. (...) Fundava-se ali a tendência de deslocar o documentarista dos bastidores para a superfície do filme, substituindo a voz off incorpórea por um corpo humano visível que interage com os atores sociais. (Da-Rin, 2006: 183) No modo interativo, o antiilusionismo se manifesta de forma colateral, apesar da contribuição inaugural de ChroniquedùnÉté. Um dos fatores que marcou a novidade radical foi a liberdade com que os equipamentos de filmagem e os membros da equipe eram exibidos. O início, o fim, e o meio... Para Bill Nichols (2005: 135), ao definir traços característicos semelhantes nas vozes individuais de filmes e cineastas, podemos estabelecer grupos que compartilham vozes semelhantes. Essas vozes compartilhadas referem-se aos gêneros de documentário. Nichols divide os gêneros do documentário em seis modos: poético, expo- sitivo, observativo, participativo, reflexivo e performático. Apesar de determi- narem uma estrutura de afiliação frouxa, eles “estabelecem as convenções que um determinado filme pode adotar e propiciam expectativas específicas que os espectadores esperam ver satisfeitas” (Nichols, 2005: 135). Por meio desta pesquisa, buscar-se-á investigar e compreender o tipo ou modo de documentário que Consuelo Lins e Cláudia Mesquita (2008: 55) con- sideram como ensaio fílmico, ou seja, aquele formato que remete a uma forma híbrida, sem regras nem definição exata, mas que articula modos de abordagem e composições variados, objetos e discursos heterogêneos. Para Bill Nichols (2005: 170) essas características mencionadas pelas autoras acima se aproxi- mam ao modo que ele denomina performático. Este último teórico do cinema entende que filmes desse estilo dão mais ênfase às características subjetivas da experiência e da memória, se afastando do relato objetivo. Para ele, o signifi- cado é claramente um fenômeno subjetivo, carregado de afetos. Um carro, um revólver, um hospital ou uma pessoa terão significados diferen- tes para pessoas diferentes. Experiência e memória, envolvimento emocional, questões de valor e crença, compromisso e princípio, tudo isso faz parte de nossa compreensão dos aspectos do mundo que mais são explorados pelo do- cumentário (...). O Documentário performático sublinha a complexidade de nosso conhecimento do mundo ao enfatizar suas dimensões subjetivas e afe- tivas. (Nichols, 2005: 169) Já Sílvio Da-Rin (2006: 183) aponta como elemento principal desse sub- gênero a auto-reflexidade, que constitui uma busca de alternativas às insufici- ências e às limitações identificadas nos diversos modos de representação em lidar criticamente com o ilusionismo cinematográfico. Os conceitos sobre este tipo de filme são variados e nem sempre conduzem a um consenso. Contudo, para esta pesquisa, adotaremos a perspectiva de que o ensaio fílmico possui sua hibridez justificada em suas variações demasiadas - transições e transformações nas quais gêneros e estilos não se limitam, mas se complementam. A participação também acontece nesse tipo de filme, e é uti- lizada de diversas maneiras. Esses traços, segundo Nichols, são características que passam a aparecer nos documentários que ele denomina participativos. 297 O filme de busca e a construção ensaística de uma memória familiar... (...) é o desempenho de Emiko Omori na reconstituição da história reprimida da experiência de sua própria família, nos campos de confinamento de nipo- americanos durante a Segunda guerra Mundial, que dá forma a Coelho na lua (1999). Em Diário inconcluso (1983), Marilu Mallet propõe uma estru- tura ainda mais explícita, na forma de diário, ao fazer o retrato de sua vida de exilada chilena em Montreal, casada com o cineasta canadense Michael Rubbo. (...) Esses filmes fazem do cineasta uma pernona tão nítida quanto O início, o fim, e o meio... (...) Fundava-se ali a tendência de deslocar o documentarista dos bastidores para a superfície do filme, substituindo a voz off incorpórea por um corpo humano visível que interage com os atores sociais. (Da-Rin, 2006: 183) Conforme Nichols (2005: 158), há casos em que o cineasta se distancia da postura investigativa para adotar uma relação mais receptiva e reflexiva frente aos acontecimentos que o filme deseja contar ou que envolvem este profis- sional e seu tema. Essa escolha leva o filme a se aproximar do diário e do testemunho pessoal. Nesse tipo de narrativa é comum encontrarmos a voz na primeira pessoa predominando na estrutura global do filme. Seria o engaja- mento participativo do cineasta, agora também como sujeito registrado, um dos principais elos para se conseguir a atenção do público. 298 Adriano Medeiros da Rocha & Eduardo Henrique Moreira qualquer outra de seus filmes. Como testemunho ou confissão, muitas vezes, estes manifestam um poder revelador. (Nichols, 2005: 158 e 159) O crítico de cinema afirma que a intensidade emocional e a expressividade subjetiva do modo performático tomaram forma nos anos 80 e 90 e foram desenvolvidos principalmente por grupos que possuíam forte atenção para a esfera social e as questões comunitárias. De acordo com Cláudia Mesquita e Consuelo Lins (2008), essas características ensaísticas no Brasil são notáveis em filmes dos anos 80, como Mato eles? (1982), de Sergio Bianchi e Ilha das Flores (1989), de Jorge Furtado. Os filmes desse estilo são carregados de emoção e significados que dão a eles características expressivas de uma dada perspectiva. Desse modo, os docu- mentários performáticos dirigem aos espectadores uma representação pessoal e pontual de sujeitos específicos, incluindo o cineasta. (Nichols, 2005: 43). Dialogando com Nichols, Silvio Da-Rin entende que o cineasta assimila os re- cursos retóricos desenvolvidos ao longo da história do documentário e produz uma inflexão deles sobre si mesmos. Não satisfeito em simplesmente expor argumentos sobre seu objeto, o cine- asta passa a engajar-se em um metacomentário sobre os mecanismos que dão forma a este argumento. No lugar da ênfase absoluta sobre os personagens e os fatos do mundo histórico, o próprio filme afirma-se como fato no domínio da linguagem. O início, o fim, e o meio... (Da-Rin, 2006: 170) Para Nichols, o estilo performático, ou o ensaio-fílmico, mistura livre- mente as técnicas expressivas que dão textura e densidade àquela ficção que ainda era presente nos modos poético e expositivo, mas que foi sendo deixada de lado quando o modo observacional priorizou a filmagem direta do encon- tro social, ou seja, possibilidades audiovisuais como planos de ponto de vista, números musicais, representações de estado subjetivo da mente, retrocessos, fotogramas congelados. Nichols (2005: 171) ainda ressalta que os filmes desse estilo "nos envol- vem menos com ordens ou imperativos retóricos do que com a sensação re- lacionada com sua nítida sensibilidade”. Em sua opinião, a sensibilidade do cineasta busca estimular a do espectador, ou seja, é a carga afetiva aplicada no filme que indiretamente nos envolve na sua representação do mundo histórico. Um dos métodos utilizados para a realização dos documentários de ensaio é o que o crítico cineasta Jean-Louis Comolli (2008) chama de dispositivos de escritura. Consuelo Lins e Cláudia Mesquita entendem que o dispositivo de escritura está relacionado à criação, pelo realizador, de um artifício ou proto- colo produtor de situações a serem filmadas ou a um tipo de maquinação de uma lógica, de um pensamento, que atribui às condições, regras e/ou limites O filme de busca e a construção ensaística de uma memória familiar... 299 para que esses filmes aconteçam. Na opinião das autoras, a adoção de um dis- positivo nega diretamente a ideia de documentário como obra que aprende a essência de uma temática ou realidade fixa e preexistente. Não se trata, porém, de um procedimento produtor que gera efeitos seme- lhantes em todo o filme, mas que é criado a cada obra, imanente, contingente às circunstâncias de filmagem, e submetido às pressões do real. A simples adoção de um dispositivo não garante, em suma, o sucesso de um filme; tudo depende da sua adequação ao assunto eleito, mas sobretudo do trabalho con- creto de filmagem, que a maquinação anterior não dispensa. (Lins & Mes- quita, 2008: 57) O documentarista Eduardo Coutinho, ao refletir sobre dispositivo fílmico, utilizava o termo prisão para caracterizar o conjunto de regras auto impostas que delimitam o processo de realização de seus documentários. O dispositivo utilizado pelo cineasta, pelo menos até Edifício Master (2002), era de ordem espacial: filmar numa única locação (Lins e Mesquita, 2008). O início, o fim, e o meio... Porém, esse tipo de abordagem só foi evidenciado no decorrer dos anos 2000 pelos documentá- rios Um passaporte húngaro (2002), de Sandra Kougut, e 33 (2003), de Kiko Goffman, os quais Consuelo Lins e Cláudia Mesquita denominam filmes de busca. Neles, o motivo da realização do documentário deixa de ser a alteridades clás- sica para se relacionar a aspectos da experiência pessoal e da subjetividade dos próprios realizadores. Nos dois filmes, os diretores interagem com persona- gens e situações como sujeitos interessados, protagonistas de um processo de busca pessoal. (Lins & Mesquita, 2008: 51) Lins e Mesquita notam que as histórias registradas não preexistem à fil- magem, mas são produzidas por um agir do documentarista, portanto, eles são também o fio condutor de seus filmes. Segundo Lins e Mesquita, o crítico Bernadet aposta no híbrido pessoa-personagem: Como notou Jean Claude Bernadet: “O documentarista determina um pro- jeto, sabe de onde parte, sabe o que gostaria de alcançar, mas não pode prever os resultados a que chegará nem o percurso que terá de cumprir. (...) Essas pessoas-personagens têm objetivos, enfrentam obstáculos, alcançam seus ob- jetivos ou não, exatamente como nos filmes de ficção”. (Lins & Mesquita, 2008: 52) A partir do diálogo teórico apresentado anteriormente, pode-se entender que os filmes de busca são documentários dos modos participativo e perfor- mático, ou então são filmes de ensaio, que possuem um dispositivo que faz o cineasta sair em busca de um objetivo. Nesse tipo de documentário, é es- tabelecida uma conexão entre o domínio privado e o domínio público, como acontece em Um passaporte húngaro (2002). Dessa forma, o cineasta faz sua representação do mundo histórico de maneira subjetiva através da exibição de 300 Adriano Medeiros da Rocha & Eduardo Henrique Moreira uma busca íntima, marcando a diferença desse documentário em relação à ex- posição da vida privada a que se assiste diariamente na televisão. Lins e Mesquita apontam também como traço marcante dos documentários de busca a utilização da entrevista com uso deslocado, sem reproduzir a tradi- cional dicotomia sujeito-objeto. Para elas, as entrevistas aparecem nos filmes de busca como “instrumento para obtenção de informações no processo con- creto de pesquisa e busca empreendido pelos realizadores.” (Lins & Mesquita, 2008: 52). O início, o fim, e o meio... Inspirados em conceitos encontrados nos filmes de busca e no mecanismo da pessoa-personagem, os realizadores Eduardo Moreira e Thiago Novais tam- bém decidiram ir a campo para desenvolver o primeiro média metragem da dupla, em 2016. O documentário, ou ensaio fílmico, que realizaram também pode ser identificado como um filme de busca. Um dos realizadores da obra – Eduardo Moreira – é o personagem condutor da história numa busca pela memória do seu avô, Carlos Moreira. O dispositivo escolhido para a gravação é a trajetória da equipe saindo da cidade de Ouro Preto com destino ao sub- distrito de Piranga onde viveu Carlos, o Manja Léguas. Durante o percurso, a dupla passou pelos distritos ouropretanos: Santa Rita de Ouro Preto, Ban- deiras e Santo Antônio. Nas várias paradas a dupla entrevistou filhos e netos do patriarca da família Moreira. Também nessas localidades foi feito o regis- tro em vídeos das paisagens, situações cotidianas e objetos que representam a cultura e o estilo de vida da zona rural mineira. Da mesma maneira, foram registradas cenas do cotidiano dos entrevistados que remetem, ou representam, elos de relacionamento com Carlos Moreira. Segundo Bernard (2008: 185) é preciso haver imagens de cobertura suficientes para dar opções para a edição. Pense em rodar o seu documentário como você faria com um drama: no âm- bito de determinada cena você desejará tomadas amplas, tomadas médias, close-ups e cortes, para garantir que as tomadas sejam suficientemente longas e estáveis para serem usadas. Você quer ser capaz de criar cenas visuais que proporcionem contexto e alguma outra informação à história. (Bernard, 2008: 185) O percurso regido pelo dispositivo adotado representa um mergulho feito pelo personagem central na história do avô de Eduardo. Através da vontade de resgatar parte significativa de uma memória de sua família, o neto de Car- los, que só o conheceu pelas histórias que ouvia do seu pai, dos tios e da falecida avó, decidiu embarcar em uma viagem para o Manja Léguas e do- cumentar essa busca. A cada parada, um ou mais encontros com os tios e primos, que contaram e demonstraram, através das suas narrativas, quem foi Carlos dentro daquela memória coletiva. As interações tiveram um caráter mais íntimo/pessoal por se tratar de um encontro em família para compartilhar O filme de busca e a construção ensaística de uma memória familiar... 301 a memória da mesma. O início, o fim, e o meio... Esse tipo de encontro para conversas sobre temas fa- miliares, normalmente, é uma situação rotineira no cotidiano dos habitantes da zona rural. A partir do conteúdo das entrevistas houve a tentativa de apresentar os aspectos imagéticos e também emocionais desses encontros. Para que os entrevistados não se sentissem muito incomodados com a pre- sença da(s) câmera(s) durante os diálogos, primeiramente a equipe buscou constituir um tempo de interação com cada um deles. Logo no momento da chegada ao ambiente de moradia ou trabalho de cada entrevistado o registro já começava a ser feito, a câmera era logo apresentada ao entrevistado e o equipa- mento registrador seguia o caminho. Na sequência, o entrevistado apresentava a sua casa/trabalho e, aos poucos, era estabelecida uma relação mais amena com a câmera. Após esse período de aproximação, a entrevista em profundi- dade era realizada. Ao se aproximar e estabelecer um tempo de convívio com seus parentes do interior, o ator social central se aprofundou cada vez mais da história de sua família. A cada encontro era promovida também uma nova descoberta que remetia ao passado da família e atestava contestações de seu presente. O uni- verso de expectativas e impressões geradas pelos encontros foi ilustrado pelas imagens de uma segunda câmera, que colocava o pesquisador, literalmente, como agente participativo. Esse registro foi tentado no calor do momento em cada uma das chegadas e partidas. As experiências obtidas durante o trajeto pelas estradas repletas de pai- sagens diferenciadas e localidades típicas da zona rural mineira e também de cada um dos encontros com os familiares do personagem que buscava a his- tória do seu avô foram registrados em duas perspectivas: em primeira pessoa, pelo próprio personagem e sua câmera, e em terceira pessoa por um com- panheiro de viagem – Thiago Novais. O objetivo foi ter um ponto de vista além do que era registrado pelo personagem central que fez a busca. Essas imagens de cobertura explicitam detalhes dos cenários, dos personagens e da natureza dos encontros, na tentativa de ilustrar o universo cultural e simbólico que os envolve. Conforme o entendimento de Bernard (2008: 185), é impor- tante garantir as informações visuais necessárias para o registro da busca do personagem central, a personalidade dos entrevistados e os encontros. O início, o fim, e o meio... Note que você está filmando casualmente tudo que for possível: está asse- gurando que possui informação visual passível de transmitir a informação narrativa básica: o que, onde, como. Quer estabelecer tempo, lugar e pessoas, buscando imagens que possam lhe fazer cortar informações verbais. Atente nos detalhes narrativos que revelam personagens: pode ser um cigarro es- quecido enquanto queima ou a pilha de garrafas de licor no lixo reciclável. Busque tomadas que mostrem como as pessoas se comportam umas em rela- ção às outras e com que habilidade manuseiam suas ferramentas de trabalho. 302 Adriano Medeiros da Rocha & Eduardo Henrique Moreira Você pode querer humor. E conforme foi mencionado, precisa estar certo de ter uma série suficiente de ângulos, tomadas e cortes que possibilitarão ao editor condensar horas de material em um filme final que conte uma história coerente e visualmente satisfatória. (Bernard, 2008: 185) A dupla partiu da hipótese de que a identidade de Carlos Moreira, presente na memória daqueles que o conheceram, é resultado dos seus feitos em vida. Histórias sobre o patriarca da família Moreira são contadas e recontadas há cerca de meio século. Na família do caixeiro viajante as lembranças da sua trajetória já são repassadas para a geração dos seus bisnetos. Nas comunidades e distritos próximos ao Manja Léguas, até mesmo os filhos e netos dos que conviveram com Carlos em vida reconhecem sua importância na história da região. Inicialmente, a vontade da dupla foi entender como se dá a construção da identidade do indivíduo. Em seguida também houve a necessidade de com- preender o funcionamento da memória coletiva das famílias e da identidade familiar. Como o filme dialoga sobre memórias de uma família do interior, questões ligadas à complexidade do ambiente e das relações sociais encontra- das na zona rural mineira também permeiam a obra. No caminho teórico que embasou a pesquisa, a dupla de realizadores uti- lizou alguns conceitos chave, tais como identidade e família. Com base em Anthony Giddens (1938), pode-se afirmar que as ações do indivíduo são obser- vadas e compreendidas por ele próprio e que são sustentadas por suas convec- ções e estados emocionais. O meio que cerca o indivíduo, a época e sociedade em que vive, os atributos culturais que lhe são dados, tudo isso se relaciona diretamente com as referidas questões que relacionam sua existência. O início, o fim, e o meio... Esses fatores envolvem também o processo de construção de significados baseados nos conjuntos de atributos culturais inter-relacionados que levam a definição da(s) identidade(s) do indivíduo (Castells, 1942: 22). Nesse sentido, Manuel Castells afirma que para um determinado indivíduo, ou ainda um ator coletivo, pode haver identidades múltiplas. Porém essa pluralidade é apontada pelo au- tor como uma “[...] fonte de tensão e contradição tanto na auto-representação quanto na ação social” devido à confusão existente entre o que é a identidade “[...] e o que tradicionalmente os sociólogos têm chamado de papéis, e con- juntos de papéis”. (Castells, 1942: 22). Do ponto de vista sociológico, toda identidade é construída. As experi- ências adquiridas pelo indivíduo ao longo de sua vida são processadas pelas instituições sociais nas quais ele está inserido e assim sua identidade é esta- belecida. Nessa linha de raciocínio, Castells lança a hipótese de que quem constrói as identidades são os determinantes do conteúdo simbólico das iden- O filme de busca e a construção ensaística de uma memória familiar... 303 tidades coletivas e seu significado para aqueles que com ela se identificam ou dela se excluem. A construção de identidades vale-se da matéria prima fornecida pela histó- ria, geografia, biologia, instituições produtivas e reprodutivas, pela memória coletiva e por fantasias pessoais, pelos aparatos de poder e revelações de cu- nho religioso. Porém, todos esses materiais são processados pelos indivíduos, grupos sociais e sociedades, que reorganizam seu significado em função de tendências sociais e projetos culturais enraizados em sua estrutura social, bem como em sua visão de tempo/espaço. Havendo aqui a hipótese de que, em li- nhas gerais, quem constrói a identidade coletiva, e para quê essa identidade é construída, são em grande medida os determinantes do conteúdo simbólico dessa identidade, bem como de seu significado para aqueles que com ela se identificam ou dela se excluem. (Castells, 1942: 23) Castells (1942: 26) considera que a construção da identidade do indivíduo consiste no seu projeto de vida. Nesse ponto, o indivíduo que assume um pa- pel dentro da sociedade é apenas um sujeito. O autor entende que sujeitos não são indivíduos, mesmo considerando que são constituídos a partir de indiví- duos. Quando o indivíduo tem um projeto de vida diferente “talvez com base em uma identidade oprimida, porém expandindo-se no sentido da transforma- ção da sociedade como prolongamento desse projeto” ele atesta sua identidade (Castells, 1942: 26). O início, o fim, e o meio... Em um recorte espacial mais micro, Ana Rojas Acosta e Maria Amália Faller Vitale (2008) sugerem que as famílias sejam abordadas como “algo que se define por uma história que se conta aos indivíduos, ao longo do tempo, desde que nascem, por palavras, gestos, atitudes ou silêncios, e que será por eles reproduzida e ressignificada [...]” (Acosta & Vitale, 2008: 26 e 27). Cada família faz isso de maneira particular, dados os seus distintos lugares e mo- mentos. Ao construir a sua própria história, ou o seu próprio mito, a família ex- pressa o significado e a explicação da realidade que viveu. Ela se utiliza dos elementos objetivos e subjetivos da cultura em que vivem seus indivíduos. A delimitação simbólica da família acontece por meio dos referenciais sociais e culturais da determinada época e sociedade. Sendo assim, para Acosta e Vitale, trabalhar com famílias requer a abertura para uma escuta, a fim de localizar os pontos de vulnerabilidade, e também os recursos disponíveis. Pensar a família como uma realidade que se constitui pelo discurso sobre si própria, internalizado pelos sujeitos, é uma forma de buscar uma definição que não se antecipe à sua própria realidade, mas que nos permita pensar como ela se constrói, constrói sua noção de si, supondo evidentemente que isto se faz em cultura, dentro, portanto, dos parâmetros coletivos do tempo e do espaço em que vivemos, que ordenam as relações de parentesco (entre irmãos, entre pais e filhos, entre marido e mulher. (Acosta & Vitale, 2008: 26 e 27) 304 Adriano Medeiros da Rocha & Eduardo Henrique Moreira Considerando que o ser humano se constitui simbolicamente, ou seja, se constitui a partir dos elementos culturais, pode-se entender que não há reali- dade humana exterior à cultura. Dessa mesma forma, a construção simbólica da família é feita com a partir da sua memória coletiva. O discurso social que a define reflete nas diferentes famílias como um espelho. Cada família faz sua própria tradução desse discurso e o reflete com a sua imagem filtrada pela singularidade das experiências vividas. Partindo da noção de que a identidade da família é definida pela sua própria história, é possível considerar que a memória coletiva familiar está muito atre- lada ao papel dos avós. Esses atores sociais representam um elemento-chave nos processos de identificação e, portanto, na construção do sentido de perten- cimento entre os membros de uma família. O início, o fim, e o meio... Acosta e Vitale (2008) afirmam que, quando se pesquisa ao menos três gerações de uma família, a figura dos avós revela bem essa dimensão. Através deles é possível recuperar a história familiar, social, atingindo várias gerações. Tanto aqueles que conheceram os avós pessoalmente, como os que só os conheceram por fotografias e histórias, tem neles a representação de figuras presentes no imaginário. As inserções sociais, culturais e as relações de gê- nero definem a trajetória desses personagens. Recordamos deles através da posição que ocuparam na família relacionada ao nosso próprio lugar (Acosta & Vitale, 2008). Porém essas posições podem ir se modificando ao longo da vida. Acosta e Vitale defendem que a herança simbólica transmitida pelos avós, entendida também como os seus legados geracionais, compõe nossa me- mória coletiva familiar. Afinal, eles contribuem ou contribuíram de diversas formas na vida cotidiana das suas famílias. A importância dos avós para a família é inegável. Com base em Kaës (2001), as autoras Acosta e Vitale (2008) afirmam que “o pensamento psica- nalítico tem tradicionalmente apontado o papel simbólico da figura dos avós – vivos ou mortos – na função de assegurar aos netos uma identidade enraizada no tempo imemorial”. Segundo elas, os avós proporcionam uma identidade genealógica para a família. Nesse caso, a criança está no centro das trans- missões inconscientes dessa “vida psíquica” que transpassam as gerações. De acordo com Acosta e Vitale, eles são os personagens que se movimentam de acordo com as gerações, mas que permanecem em nossa memória “como figu- ras cristalizadas” em determinado momento do percurso. A herança simbólica que é transmitida por eles se mantém ao longo de nossas vidas pode ser recri- ada no processo de continuidade e descontinuidade dos bens simbólicos que recebemos. O filme de busca e a construção ensaística de uma memória familiar... 305 No filme Seu Carlito: narrativas sobre um comerciante da roça a dupla de realizadores promove a busca pela identidade de Carlos Moreira e acaba por encontrar elementos significativos para entender o processo de construção da identidade do indivíduo, mas também da memória coletiva da família e da própria comunidade, sobretudo ambientada na zona rural de Ouro Preto. Para explorar a trajetória de um personagem desse meio foi necessário ir atrás da- queles que ainda reproduzem as narrativas sobre ele. O início, o fim, e o meio... Como a identidade do indivíduo é moldada por suas ações em vida, é possível acessar as informa- ções presentes na memória coletiva e resgatar os elementos que estruturam a identidade do personagem pesquisado. da identidade do indivíduo, mas também da memória coletiva da família e da própria comunidade, sobretudo ambientada na zona rural de Ouro Preto. Para explorar a trajetória de um personagem desse meio foi necessário ir atrás da- queles que ainda reproduzem as narrativas sobre ele. Como a identidade do indivíduo é moldada por suas ações em vida, é possível acessar as informa- ções presentes na memória coletiva e resgatar os elementos que estruturam a identidade do personagem pesquisado. As narrativas da época em que o comerciante viveu no Manja Léguas fo- ram cuidadosamente mapeadas pelos realizadores. Cada um dos atores sociais apresentou fragmentos e versões das várias histórias sobre o avô de Eduardo. O realizador, e também personagem do filme, Eduardo Moreira, teve a opor- tunidade de imergir nos resquícios da realidade rural em que seu avô viveu e, através dos encontros e conversas com seus tios e primos mais velhos, consegui acessar e registrar as narrativas sobre seu avô. Em sua viagem para o Manja, passou pelos cenários onde aconteceu boa parte das histórias que ouviu. A experiência proporcionou ao realizador conhecer a trajetória do seu avô e as narrativas inspiradoras daqueles que ainda guardam suas lembranças. A cada encontro recebeu de seus familiares as lições, lembranças e memórias que o patriarca deixou como herança, traduzindo-a, cuidadosamente, neste filme do- cumentário de buscas e virtudes. Giddens, A. (2002). Modernidade e identidade. Rio de Janeiro: Zahar. Acosta, A. R. & Vitale, M. A. F. (2008). Famílias: redes, laços e políticas públicas. 4 ed. São Paulo: Cortez/Instituto de Estudos Especiais/PUC-SP. Bernard, S. C. (2008). Documentário: Técnicas para uma produção de alto impacto. Rio de Janeiro: Elsevier. Castells, M. (2001). O Poder da Identidade: Paraísos comunais: Identidade e significado na sociedade em rede, vol. II. São Paulo: Paz e Terra. Comerford, J. C. (2003). Como uma família: Sociabilidade, territórios de parentesco e sindicalismo rural. Rio de Janeiro. RelumeDumará. Comolli, J.-L. (2008). Ver e poder: A inocência perdida – Cinema, Televisão, Ficção, documentário. Minas Gerais: Editora UFMG. Da-Rin, S. (2004). Espelho Partido: Tradição e Transformação do Documen- tário. Rio de Janeiro: Azougue Editorial. Giddens, A. (2002). Modernidade e identidade. Rio de Janeiro: Zahar. Referências bibliográficas Acosta, A. R. & Vitale, M. A. F. (2008). Famílias: redes, laços e políticas públicas. 4 ed. São Paulo: Cortez/Instituto de Estudos Especiais/PUC-SP. Bernard, S. C. (2008). Documentário: Técnicas para uma produção de alto impacto. Rio de Janeiro: Elsevier. Acosta, A. R. & Vitale, M. A. F. (2008). Famílias: redes, laços e políticas públicas. 4 ed. São Paulo: Cortez/Instituto de Estudos Especiais/PUC-SP. Bernard, S. C. (2008). Documentário: Técnicas para uma produção de alto impacto. Rio de Janeiro: Elsevier. Castells, M. (2001). O Poder da Identidade: Paraísos comunais: Identidade e significado na sociedade em rede, vol. II. São Paulo: Paz e Terra. Comerford, J. C. (2003). Como uma família: Sociabilidade, territórios de parentesco e sindicalismo rural. Rio de Janeiro. RelumeDumará. Comolli, J.-L. (2008). Ver e poder: A inocência perdida – Cinema, Televisão, Ficção, documentário. Minas Gerais: Editora UFMG. Comolli, J.-L. (2008). Ver e poder: A inocência perdida – Cinema, Televisão, Ficção, documentário. Minas Gerais: Editora UFMG. Da-Rin, S. (2004). Espelho Partido: Tradição e Transformação do Documen- tário. Rio de Janeiro: Azougue Editorial. Da-Rin, S. (2004). Espelho Partido: Tradição e Transformação do Documen- tário. Rio de Janeiro: Azougue Editorial. Giddens, A. (2002). Modernidade e identidade. Rio de Janeiro: Zahar. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro contemporâneo. Rio de Janeiro: Jorge Zahar. Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro contemporâneo. Rio de Janeiro: Jorge Zahar. Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro contemporâneo. Rio de Janeiro: Jorge Zahar. Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro contemporâneo. Rio de Janeiro: Jorge Zahar. Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. 306
https://openalex.org/W2763167556
https://dergipark.org.tr/tr/download/article-file/519256
English
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Design and Acquisition of EOG Based Interactive Communications for ALS Patients
International journal of applied mathematics, electronics and computers
2,017
cc-by-sa
2,775
Accepted : 12/05/2017 Published: 21/08/2017 DOI: 10.18100/ijamec.2017Special Issue30460 Abstract: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease caused by loss of function of spinal cord and brain stem nerve cells. Loss of function in nerve cells leads to weakness and explosion (atrophy) in the muscles. The outcome of the weakness in the muscles needs the help of someone. Despite the limitations of the movement, studies on methods of increasing the daily quality of life ALS patients are continuing. Electrooculogram (EOG) signals were taken from an instrumentation amplifier with 48dB gain and 107dB CMMR ratio. A 16Hz Low Pass Filter and a 50Hz Notch Filter were used to increase the signal to noise ratio. EOG signals are digitized with 10bit resolution ADC and applied to ATmega328 microcontroller. So, the software of microcontroller determines the horizontal and vertical movements of the eye. In this way, the interactive PC software was controlled by the EOG signals. EOG-based interactive software which was developed using the C # programming language has provided to patients' daily requirements, social media accounts etc. The system has been tested on healthy subjects and it has been seen that people can control the software by eye movements. As a result, system will be useful not only for ALS patients, but also for permanent or partially bedridden patient (MS, Hemiplegia, etc.) groups. And also, while the quality of life in the patients' own habitats is being raised, they will also be able to benefit from health services within the scope of home health services. Keywords: Amyotrophic Lateral Sclerosis (ALS), Atrophy, Electrooculogram (EOG), Home Health Care, Interactive movements are recorded with the EMG signal [3]. Bulling (2009) and the working group have designed EOG eyewear which provide daily environmental awareness and sensitivity. Real-time EOG signals can be received with the specially designed glasses and transmitted to the computer interface via Bluetooth. 8 games designed in the computer interface were requested to play with 11 eye movements and at the same time the EOG signals obtained were analyzed by signal processing techniques [4]. In 2012, Mingmin Yan and others developed a mouse-controlled system using EOG signals for ALS patients. The position of the eye was determined using signal processing techniques on the received EOG signals [5]. Ayvaz and Gürkan (2014) have designed an EOG circuit with 3 electrodes in order to control the actuators in their work [6]. Advanced Technology and Science Original Research Paper Advanced Technology and Science Original Research Paper International Journal of Applied Mathematics, Electronics and Computers ISSN:2147-82282147-6799 www.atscience.o International Journal of Applied Mathematics, Electronics and Computers ISSN:2147-82282147-6799 www.atscience.o www.atscience.org/IJAMEC Original Research Paper Accepted : 12/05/2017 Published: 21/08/2017 In the other work they did in 2015, they developed the EOG based Human Computer Interface application. The system was tested on 10 people, the results of performing the desired eye movement commands were recorded and processed in MATLAB environment. [7-8]. Mondal et al (2015) have implemented EOG based prototype data system design. The signals from this low-cost EOG prototype demonstrated the success of the signal they received compared to the EOG module in the BIOPAC MP36 system [9]. In 2016, Tamura and others created EOG and EMG based computer interfaces. By applying signal processing techniques on the signals they have acquired with the interface they have created they have distinguished the right-left commands on the EOG sign [10]. In this study, it is aimed to realize a system that will enable EOG based interactive software to control many commands from daily necessities of ALS patients to social media accounts. In this way communication between the patient and the companion is ensured 1,2,3 Biomedical Engg., Afyon Kocatepe University, Afyonkarahisar – 03200, TURKEY * Corresponding Author: Email: nozkan@aku.edu.tr Note: This paper has been presented at the 5th International Conference on Advanced Technology & Sciences (ICAT'17) held in İstanbul (Turkey), May 09-12, 2017. Design and Acquisition of EOG Based Interactive Communications for ALS Patients Neşe ÖZKAN*1, Ali IŞIK2, Uğur FİDAN3 IJAMEC, 2017, Special Issue , 1–4 | 1 2. Materials and Methods In general, home care services are offered at the professional level or by family members in the home or living environment of the individual, in order to maintain, raise and rehabilitate the health of the individual. 29280 dated February 27, 2015 by the Ministry of Health of the Republic of Turkey, "Health Services at Home by the Ministry of Health and its Affiliates" regulates the scope and content of home health services [11]. These services aim to protect the quality of life and the social dignity of the individual in a wide range of needs, including both health and social services. Home care services are generally expressed as services that take place in institutional care and reduce or delay the need to stay in institutions [12]. ALS and paralyzed patients are often in need of treatment within the scope of this regulation. Fig. 2. EOG measuring circuit block diagram. When the eyes move in different directions, the reciprocating eye muscles are stimulated. Thus, horizontal and vertical eye movements can be observed simultaneously. In Figure 3, EOG signs appear in the muscle groups surrounding the eye during vertical movements. Fig. 3. Vertical Eye Movements and EOG Sign. 2.2. EOG Based Interactive Communication System Fig. 3. Vertical Eye Movements and EOG Sign. ALS disease is known to Aran Duchenne until 1840 and is known in many countries by the name Charcot. The first clinical and pathological description of ALS was made by Jean Martin Charcot (1874). In a case study of 25 patients, they defined ALS on the basis of various clinical findings such as weakness - melting and pathological reflexes in the muscles [13]. ALS disease is a central nervous system disease caused by loss of function of motor cells (neurons) in the spinal cord and brain Fig. 3. Vertical Eye Movements and EOG Sign. 2.2. EOG Based Interactive Communication System Figure 4 shows the block diagram of the EOG based interactive communication system design. The system consists of 3 basic parts. The first part forms the EOG measuring circuit, which translates eye movements into electrical action. It constitutes the second part of the digital process controller (DSP) system which converts from electrical signals to position information according to 360° and converts the position information into mouse control signals and transfers the information to the PC via telemetry method. 1. Introduction ALS disease is a central nervous system disease caused by loss of function of motor cells (neurons) in the spinal cord and brain stem. Loss of function in the nerve cells leads to weakness and eruption (atrophy) in the muscles. This situation removes the movement functions of ALS patients and leaves them in need of someone's help. Although there are limitations of the movement, studies on the methods that increase the daily life quality of ALS patients who are not different from healthy individuals as consciousness continue. In the literature, Barea et al. (2002) developed the human- machine interface to control the wheelchair with EOG signals [1]. Kherlopian et al. (2006) developed a computer-based system capable of automatic analysis and processing of EOG signals. Signal processing techniques have been used to reduce noise in the EOG signal during work and to eliminate unwanted signals caused by muscle movements around the eye. The right, left, and center positions of the eye were determined by asking them to print on the screen by eye movements with 13 words and 5 words with 6 letters [2]. In another work, they created a computer keyboard interface and implemented a virtual keyboard that can be controlled with EOG and EMG marks. When the eye gaze is obtained with the EOG signal during operation, the user's muscle IJAMEC, 2017, Special Issue , 1–4 | 1 This journal is © Advanced Technology & Science the S / N ratio of the measuring system, the EOG signals are passed through the Notch and band pass filter. and interactive communication with the patient's social environment will be strengthened. While the quality of life is being upgraded within the patients' own living spaces, technological infrastructure will be provided for health services within the scope of home health services. Fig. 2. EOG measuring circuit block diagram. Fig. 2. EOG measuring circuit block diagram. 2. Materials and Methods The last part is interactive PC software that will enable communication between the patient and the environment. Developed using the C # programming language on the Visual Studio .NET platform, EOG based interactive software enables patients to control many commands from everyday needs to social media accounts. stem. Figure 1 shows how the muscular movements due to deformation in the nerve cells in ALS disease are restricted. Fig. 4. EOG Based Interactive Communication System stem. Figure 1 shows how the muscular movements due to deformation in the nerve cells in ALS disease are restricted. This journal is © Advanced Technology & Science 3. Results In order to ergonomically place the EOG electrodes on the measurement surface, the eyewear frame was designed in the Solid Works program and was manufactured using the MassPortal Pharaoh ED model 3D printer (Fig. 5). Fig. 5. Produced Eyewear model Fig. 7. Eye Movements Fig. 7. Eye Movements Fig. 5. Produced Eyewear model EOG-based interactive software developed using the C # programming language The Lenovo Idea center model all in one is run on the Lenovo PC (Figure 8) with i5 processor with 21.5" 1920 *1080 resolution monitor capability. In order to enable interactive communication with the external environment of the patient, the software includes modules for Patient related, Conversation, Communication, Swift messages, Media and Social media. The distance between the monitor and the EOG measurement system was set to 80 cm. It was desirable for the person to control the modules on the software with eye movements. Table 1 shows the functions to be controlled and the control periods. During the test, the person controlled all modules in the software with eye movements. The average duration of access to the modules is 3.24 seconds and the module selection time is 4.46 seconds. The realized system open circuit diagram is shown in Figure 6. Ag / AgCl electrodes were placed around the orbicularis oculi muscles. Electrooculogram (EOG) signals were taken from an instrumentation amplifier with 48dB gain and 107dB CMMR ratio. A 16Hz Low Pass Filter and a 50Hz Notch Filter were used to increase the signal to noise ratio. EOG signals are digitized with 10bit resolution ADC and applied to ATmega328 microcontroller. So, the software of microcontroller determines the transvers and sagittal movements of the eye. The movement information of the eye was sent to the computer with NRF24L01 transceiver at 2.4GHz operating frequency with 9.6Kb/s data transmission speed. This way, the interactive PC software was controlled by the EOG signals. Fig. 6. Circuit diagram of the system Fig. 8. Implemented Interface Program Fig. 6. Circuit diagram of the system Fig. 8. Implemented Interface Program Fig. 8. Implemented Interface Program Fig. 6. Circuit diagram of the system Table 1. Accessing and Controlling Modules with EOG Table 1. Accessing and Controlling Modules with EOG Table 1. 2.1. Measurement of Electrooculogram (EOG) Signals Eye movements are provided by three pairs of muscles, mainly medial-lateral recti muscle, superior-inferior recti muscle and superior-inferior muscular muscle. The stimulus from the cerebral nerves induces potential tension in the ocular muscles with frequency components between 0.5uV and 0.3Hz to 40Hz. These potential changes are detected by Ag / AgCl electrodes and amplified by instrumentation amplifiers (Figure 2). To increase Fig. 4. EOG Based Interactive Communication System IJAMEC, 2017, Special Issue , 1–4 | 2 Fig. 7. Eye Movements This journal is © Advanced Technology & Science IJAMEC, 2017, Special Issue , 1–4 | 3 3. Results Accessing and Controlling Modules with EOG Modules Arrival Time (s) Departure Time (s) Conversation 6.16 8.18 Communication 0.84 3.03 Swift messages 2.87 3.14 Media 1.16 1.88 Social media 5.18 6.11 The Analog Discovery 2 data logger, manufactured by Digilent, was connected to both outputs of the EOG circuit to test that the hardware was tracking the movements of the eyeball. It is desirable for the person to make eye movements in accordance with the commands specified at a distance of about 80 cm. Figure 7 shows the response of the eye sphere to the sagittal and transverse axis commands. During the eye blinking movements, there are 180° phase different signals between the signals from both channels. The obtained data was analyzed by signal processing techniques and converted to a coordinate data to be detected by the monitor and transferred to the PC. IJAMEC, 2017, Special Issue , 1–4 | 3 This journal is © Advanced Technology & Science 4. Discussion and Conclusions Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease which is caused by loss of function of the spinal cord and brain stem nerve cells. Loss of function in the nerve cells leads to weakness and eruption (atrophy) in the muscles. This situation eliminates the movement functions so patient needs to the assistance of a companion. Although there are limitations of the movement, studies on the methods that increase the daily life quality of ALS patients who are not different from healthy individuals as consciousness continue. In this study, the interactive PC software was controlled by the EOG signals. EOG- based interactive software which was developed using the C # programming language has provided to patients' daily requirements, social media accounts etc. The system has been tested on healthy subjects and it has been seen that people can control the software by eye movements. As a result, system will be useful not only for ALS patients, but also for permanent or partially bedridden patient (MS, Hemiplegia, etc.) groups. And also, while the quality of life in the patients' own habitats is being raised, they will also be able to benefit from health services within the scope of home health services. [12] M. Yılmaz, F. Sametoğlu, G. Akmeşe, A. Tak, B. Yağbasan, S. Gökçay, S. Erdem, “In-Home Health Services as an Alternative Form of Presentation of Patient Care,” Istanbul Med Journal, 2010. [13] F. Koç and E. Tan, “Amyotrophic Lateral Sclerosis,” TND Neuromuscular Diseases Scientific Working Group. Acknowledgements This study is supported by TÜBİTAK BİDEB Authority under the project numbered 2209/A program 1919B011601485. We would like to thank TUBITAK Scientific Human Resource Support Program for their support in conducting this research. IJAMEC, 2017, Special Issue , 1–4 | 4 This journal is © Advanced Technology & Science [12] M. Yılmaz, F. Sametoğlu, G. Akmeşe, A. Tak, B. Yağbasan, S. Gökçay, S. Erdem, “In-Home Health Services as an Alternative Form of Presentation of Patient Care,” Istanbul Med Journal, 2010. [13] F. Koç and E. Tan, “Amyotrophic Lateral Sclerosis,” TND Neuromuscular Diseases Scientific Working Group. References [1] R. Barea, L. Boquete, M. Mazo, E. López, “System for assisted mobility using eye movements based on electrooculography,” IEEE Transactions on Neural Systems and Rehabilitation Engineering, 2002, 10(4), 209-218. [2] A. R. Kherlopian, J. P. Gerrein, M. Yue, K. E. Kim, J.W. Kim, M. Sukumaran, P. Sajda, “Electrooculogram based system for computer control using a multiple feature classification model,” In Engineering in Medicine and Biology Society, 2006, pp. 1295-1298. gy y pp [3] H. S. Dhillon, R. Singla, N. S. Rekhi, R. Jha, “EOG and EMG based virtual keyboard: A brain-computer interface,” In Computer Science and Information Technology, 2009, pp. 259-262. [4] A. Bulling, D. Roggen, G. Tröster, “Wearable EOG goggles: Seamless sensing and context-awareness in everyday environments,” Journal of Ambient Intelligence and Smart Environments, 2009, 1(2), 157-171. [5] M. Yan, H. Tamura, K. Tanno, “Development of mouse cursor control system using electrooculogram signals and its applications in revised hasegawa dementia scale task,” In World Automation Congress (WAC), 2012, pp. 1-6. [6] A. Ayvaz, G. Ertaş, “Design of an EOG Based Electronic Circuit to Control Actuators,” National Congress of Medical Technology’14, Turkey, 2014, 22-25. [7] S. Gürkan, P. A. Öner, A. İstanbullu, S. Doğan, “Designing a Portable Data Acquisition System for Human-Computer Interface Applications,” National Congress of Medical Technology’15, Turkey, 2015, 165-168. [8] S. Gürkan, G. Gürkan, A. Kaya, A. B. Uşaklı, “A Novel Eye-Blınk Based Easy Communıcatıon Devıce for Amyotrophıc Lateral Sclerosıs Patıents: Eyepad®,” 2015. [9] C. Mondal, M. K. Azam, M. Ahmad, S.K. Hasan, M.R. Islam, “Design and implementation of a prototype electrooculography based data acquisition system,” In Electrical Engineering and Information Communication Technology (ICEEICT), 2015 International Conference on, pp. 1-6. , pp [10] H. Tamura, M. Yan, K. Sakurai, K. Tanno, “EOG-sEMG Human Interface for Communication,” Computational Intelligence and Neuroscience, 2016. [11] Ministry of Health and its Affiliates Implementing Regulation On The Presentation of Home Health Care Services, 27.02.2015. This journal is © Advanced Technology & Science
https://openalex.org/W2606506288
https://www.nature.com/articles/srep46601.pdf
English
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The pretreatment lymphocyte to monocyte ratio predicts clinical outcome for patients with hepatocellular carcinoma: A meta-analysis
Scientific reports
2,017
cc-by
6,035
The pretreatment lymphocyte to monocyte ratio predicts clinical outcome for patients with hepatocellular carcinoma: A meta- analysis received: 23 September 2016 accepted: 21 March 2017 Published: 18 April 2017 Wei Song1,*, Chuan Tian2,*, Kai Wang1, Run-jin Zhang1 & Shu-bing Zou1 The lymphocyte-to-monocyte ratio (LMR) has been reported to predict clinical outcomes in multiple malignancies. The aim of this study was to assess the prognostic role of pretreatment LMR in hepatocellular carcinoma (HCC). A total of seven studies comprising 2,738 patients were included in the meta-analysis. Pooled results showed that elevated LMR was significantly associated with increased overall survival (OS) (HR: 0.31, 95% CI: 0.20–0.47, p < 0.001), disease-free survival (DFS)/recurrence- free survival (RFS) (HR: 0.57, 95% CI: 0.49–0.67, p < 0.001). The favorable prognostic impact of high LMR on OS was observed in all subgroup with different sample size, type of publication, NOS score, and the cut-off value of LMR. In addition, low LMR was significantly correlated with TNM stage and BCLC stage. We therefore conclude that elevated pretreatment LMR could be a favorable prognostic factor for clinical outcomes in patients with HCC. According to the American Cancer Society, hepatocellular carcinoma (HCC) is the fifth most common malig- nancy and the second cause of cancer-related mortality in the United States in 20151,2. Around the world, there were an estimated 500,000 to 1 million people died of HCC per year3. Liver resection is the most effective therapy for the majority of patients with resectable HCC, but unfortunately most of patients are not eligible for curative resection at the time of diagnosis4. In addition, postoperative recurrence rate for patients with early stage HCC can be high at 50% to 70% after liver resection5. Therefore, the overall prognosis of HCC patients remains poor. It is vital to develop novel potential biomarker to select appropriate treatment strategies and predict prognosis.ll Systemic inflammatory responses can largely influence the formation and recurrence of HCC6–8. Several inflammatory biomarkers, such as neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR) are identified as prognostic indicators in a wide variety of solid tumors9–12. These tests are simple and inexpensive to perform, and they are readily available in daily oncologic practice. LMR is a readily available, routinely measured, and inexpensive inflammatory biomarker, which can be easily applied into in daily oncologic practice. Accumulating evidence shows that lymphocytes are known to play a crucial role in suppressing HCC progression through immunoselection in an immunosuppressive network, which dictates immune responses to tumors13. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Search strategies.  We se Search strategies. We searched MEDLINE, EMBASE, PubMed, and Cochrane databases from inception up to February 2017. Search terms included “HCC” or “hepatocellular carcinoma” or “liver cancer” or “liver tumor” or “liver neoplasms” or “liver cell carcinoma”, “LMR” or “lymphocyte monocyte ratio” or “lymphocyte to monocyte ratio” or “lymphocyte-to-monocyte ratio” or “lymphocyte-monocyte ratio”, “survival” or “prognostic” or “prognosis” or “recurrence” or “clinical outcome”. The references of eligible studies, relevant systematic reviews and meta-analyses in this field were manually retrieved. Inclusion criteria. The criteria for inclusion were listed as follows: (1) the diagnosis of HCC were confirmed by pathology; (2) studies assessing the association of pretreatment LMR with OS, DFS/RFS, or CSS; (3) the cut-off value of LMR was reported; and (4) studies supplied sufficient information for calculating hazard ratio (HR) and 95% confidence interval (CI). Exclusion criteria. The exclusion criteria were as follows: (1) reviews, letters, case reports, and comments; (2) reporting insufficient data for calculating an HR and 95% CI; and (3) overlapping or duplicate data. Data extraction and Assessment of risk of bias. The two reviewers independently reviewed all eligible studies and extracted data. Any disagreement was resolved by a third reviewer. The following information was captured using data abstraction forms:i Study characteristics included first author’s name, year of publication, study design (prospective or retrospec- tive), type of publication, country, ethnicity, survival analysis methods (multivariate, univariate), and time of follow-up. Patient characteristics included patient ages and genders, number of patients, disease stage, treatment, and cut-off value. Outcome measures included HRs for OS, DFS, RFS, or CSS as well as their 95% CIs. HRs were extracted from multivariate or univariate analyses or estimated from Kaplan-Meier survival curves20.hh The study quality was evaluated in accordance with the Newcastle-Ottawa Scale (NOS)21. This included an assessment of subject selection, comparability of groups, and clinical outcome. A total of nine items were extracted and each item scored 1. The maximum score is 9 and those studies with a NOS score ≥​7 were consid- ered as high-quality studies. Statistical analysis. The meta-analysis was conducted using RevMan 5.3 software (Cochrane Collaboration, Copenhagen, Denmark). Heterogeneity between studies was estimated using Cochrane’s Q statistic and I2 statistic22. A p-value <​ 0.1 for the Q-test or I2 >​ 50% indicated significant heterogeneity. When there was no statistically significant heterogeneity, we used the fixed-effects model for pooling the results; otherwise, the random-effects model was applied. The pretreatment lymphocyte to monocyte ratio predicts clinical outcome for patients with hepatocellular carcinoma: A meta- analysis A low lymphocyte count is associated with the systemic inflammatory responses and is known to promote cancer progression through effects upon cell-mediated immunity14. Conversely, acti- vated circulating monocytes can secrete multiple proinflammatory cytokines, which are involved in tumor devel- opment and progression15,16. As a consequence high monocyte counts are associated with microvascular invasion and poor prognosis in patients with HCC17. Moreover, a high LMR, which is defined as absolute lymphocyte counts divided by monocyte counts, is reported to correlate with favorable prognosis in patients with HCC18,19. 1Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Nanchang University, No.1 Minde Road, Nanchang, China. 2Department of Nuclear Medicine, Guizhou Provincial People’s Hospital, Guiyang, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.-B.Z. (email: zousb999@163.com) 1 Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 www.nature.com/scientificreports/ Nevertheless, there has been no meta-analysis to assess the correlation between pretreatment LMR and the survival of HCC patients. Therefore, we conducted a meta-analysis to assess the effects of pretreatment LMR on OS and DFS/ RFS in HCC. In addition, the correlations between LMR and the clinicopathological features was also examined. Search strategies.  We se HRs and their 95% CIs were searched in the original articles or extrapolated using meth- ods described by Tierney and Parmar20,23. The log HR and standard error (SE) were used for aggregation of the survival results23. The associations between LMR and clinicopathologic features were expressed as odds ratios (ORs) and its 95% CIs. P <​ 0.05 was defined as statistically significant. Subgroup analyses were conducted for: the sample size, type of publication, NOS score, and the cut-off value of LMR. Sensitivity analyses were carried out to evaluate result stability excluding each study. Results Search results. Our search strategy yielded 63 potentially relevant records. After duplicates removal (21 records), 42 articles were screened for eligibility. Of these, 32 were excluded through titles and abstracts, leaving 10 articles for further evaluation. Subsequently, 3 articles did not meet the inclusion criteria and were therefore excluded. Eventually, seven studies, comprising a total of 2,507 patients, were considered eligible for the present meta-analysis18,24–29. The PRISMA flow diagram of the study selection process was shown in Fig. 1. Characteristics of the included studies. According to the publication type, 6 and 1 studies were pub- lished in full-text and abstract forms, respectively. All included studies were from China and were published between 2014 and 2017. The sample sizes ranged from 208 to 1,020. There were six studies for OS, and all for DFS/ RFS. The cut-off values for LMR ranged from 0.83 to 3.77. All included studies consisted of two groups: high and low LMR. HR and 95% CI was extracted directly from the seven studies. In methodological quality of studies, the NOS scores of the included studies ranged from 5 to 8. Detailed patient characteristics and methodological quality are shown in Table 1. Meta-analysis. LMR and OS in HCC. Six of the included studies reported the data of LMR and OS in HCC. Overall, elevated LMR had a significant association with increased OS (HR: 0.31, 95% CI: 0.20–0.47, p <​ 0.001) with significant heterogeneity (p =​ 0.002, I2 =​ 74%; Fig. 2). Meta-analysis. LMR and OS in HCC. Six of the included studies reported the data of LMR and OS in HCC Overall, elevated LMR had a significant association with increased OS (HR: 0.31, 95% CI: 0.20–0.47, p <​ 0.001) with significant heterogeneity (p =​ 0.002, I2 =​ 74%; Fig. 2).i i To detect the potential heterogeneity, subgroup analyses stratified by sample size, type of publication, NOS score, and the cut-off value of LMR (Table 2). Subgroup analysis revealed that high LMR predicted increased OS in patient with HCC, regardless of the sample size (≥​300 and <​300), type of publication (full-text and abstract), NOS score (≥​7 and <​7), and the cut-off value of LMR (≥​3 and <​3). Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ LMR and DFS/RFS in HCC. All included studies reported the data of LMR and DFS/RFS in HCC. Results A combined analysis demonstrated that elevated LMR was significantly correlated with increased DFS/RFS (HR: 0.57, 95% CI: 0.49–0.67, p <​ 0.001), with no significant heterogeneity between studies (p =​ 0.21, I2 =​ 28%; Fig. 3). Figure 1. Flow diagram of the study selection process. Author Year Country Type of publication Follow-up (months) Treatment No. of patients Stage Cut-off value Survival analysis Analysis NOS score Li 2017 China Full-text 33 (6–85) Surgery 253 Mixed 3.0 OS/RFS MV/UV 8 Liao 2016 China Full-text 44 (1.5–84) Surgery 387 Mixed 3.3 OS/RFS MV 8 Lin (1) 2015 China Full-text 34.8 (1.7–106.6) Surgery 210 Mixed 3.23 OS/RFS MV/UV 7 Lin (2) 2014 China Abstract 34 (2–106) Surgery 210 — 3.2 OS/RFS MV/UV — Tang 2016 China Full-text 38 (1.5–82.2) Surgery 208 Mixed 0.83 OS/RFS MV/UV 5 Wu 2016 China Full-text 45.5 (2–93) Surgery 450 Mixed 3.77 OS/RFS MV/UV 7 Yang 2017 China Full-text NA Surgery 1020 Mixed 3.23 DFS MV/UV 6 Table 1. Characteristics of the studies included in the meta-analysis. OS: overall survival; DFS: disease-free survival; RFS: recurrence-free survival; MV: multivariate; UV: univariate NA: not available. Figure 1. Flow diagram of the study selection process. Figure 1. Flow diagram of the study selection process. Author Year Country Type of publication Follow-up (months) Treatment No. of patients Stage Cut-off value Survival analysis Analysis NOS score Li 2017 China Full-text 33 (6–85) Surgery 253 Mixed 3.0 OS/RFS MV/UV 8 Liao 2016 China Full-text 44 (1.5–84) Surgery 387 Mixed 3.3 OS/RFS MV 8 Lin (1) 2015 China Full-text 34.8 (1.7–106.6) Surgery 210 Mixed 3.23 OS/RFS MV/UV 7 Lin (2) 2014 China Abstract 34 (2–106) Surgery 210 — 3.2 OS/RFS MV/UV — Tang 2016 China Full-text 38 (1.5–82.2) Surgery 208 Mixed 0.83 OS/RFS MV/UV 5 Wu 2016 China Full-text 45.5 (2–93) Surgery 450 Mixed 3.77 OS/RFS MV/UV 7 Yang 2017 China Full-text NA Surgery 1020 Mixed 3.23 DFS MV/UV 6 Table 1. Characteristics of the studies included in the meta-analysis. OS: overall survival; DFS: disease-free survival; RFS: recurrence-free survival; MV: multivariate; UV: univariate NA: not available. Table 1. Characteristics of the studies included in the meta-analysis. OS: overall survival; DFS: disease-free survival; RFS: recurrence-free survival; MV: multivariate; UV: univariate NA: not available. LMR and DFS/RFS in HCC. All included studies reported the data of LMR and DFS/RFS in HCC. Discussion Hepatocellular carcinoma (HCC) is mainly caused by viral infections such as hepatitis B virals (HBV) and hepati- tis C virals (HCV)3. Mounting studies indicated that infiltration of inflammatory cells in the tumor microenviron- ment significantly affected the biological behavior of HCC25. In the tumor microenvironment, the inflammatory immune cells have been implicated in HCC progression30,31. The pretreatment LMR, as a promising inflammatory biomarker, is reportedly linked to prognosis in patients with HCC18,19,28. Therefore, LMR may serve as a promis- ing prognostic factors for HCC patients. To our knowledge, this is the first meta-analysis to investigate the prog- nostic effect of pretreatment LMR in HCC.i f p Our meta-analysis provides strong evidence that the presence of high LMR significantly increases OS and DFS/RFS in patients with HCC. Stratified analysis demonstrated that high LMR was significantly correlated with increased OS in patient with HCC, regardless of the sample size, type of publication, NOS score, and the cut-off value of LMR. In addition, we further assessed the correlation between pretreatment LMR and clinicopathologi- cal features. The pooled data showed low LMR was linked with TNM stage and BCLC stage. Therefore, pretreat- ment LMR may serve as a promising prognostic biomarker for estimating HCC prognosis. Furthermore, we also performed sensitivity analysis and found that no study had a significant effect on the observed effect size (pooled HR), indicating the robustness of our findings.h gi g The exact molecular mechanisms responsible for the prognostic impact of LMR in HCC are unclear. It has been suggested that cross-talk between the inflammatory response and tumor progression play a critical role in the initiation and progression of HCC14,32,33. In the tumor microenvironment, inflammatory infiltrates have a large influence on the biological behavior of HCC34,35. Tumor-infiltrating lymphocytes (TILs), as representative component of the immune microenvironment, are implicated in several stages of HCC progression, and TIL phenotypes may be a predictor for favorable prognosis36,37. Conversely, low lymphocyte counts might result in an insufficient immunological reaction, which lead to inferior survival in multiple cancers38,39. In addition, infil- trated CD4+​ and CD8+​ T cells interaction among each other are essential to the antitumor immune response by inducing tumor cell apoptosis40,41. Monocytes infiltrating tumor tissue are also involved in HCC development and progression42. Activated monocytes in HCC microenvironments can trigger and polarize T-cell responses and faciliate inflammation-induced tumor development43. Tumor-associated macrophages (TMAs) are derived from circulating monocytes. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Fi 2 F t l t f th i ti b t LMR d OS Figure 2. Forest plots for the association between LMR and OS. Figure 2. Forest plots for the association between LMR and OS. Figure 2. Forest plots for the association between LMR and OS. Subgroup No. of studies No. of patients Effects model HR (95% CI) P value Heterogeneity I2 (%) Ph Overall 6 1718 Random 0.31 (0.20–0.47) <​0.001 74 0.002 Sample size   ≥​300 2 837 Random 0.23 (0.08–0.65) 0.005 84 0.01   <​300 4 881 Random 0.34 (0.19–0.61) <​0.001 77 0.005 Cut-off for LMR   ≥​3 5 1510 Random 0.28 (0.17–0.48) <​0.001 78 0.001   <​3 1 208 — 0.43 (0.26–0.71) 0.001 — — Type of publication   Full-text 5 1508 Random 0.27 (0.17–0.44) <​0.001 74 0.004   Abstract 1 210 — 0.35 (0.24–0.50) 0.006 — — NOS score   ≥​7 4 1300 Random 0.28 (0.17–0.48) <​0.001 78 0.001   <​7 1 208 — 0.43 (0.26–0.71) 0.001 — — Table 2. Pooled hazard ratios (HRs) for OS according to subgroup analyses. Table 2. Pooled hazard ratios (HRs) for OS according to subgroup analyses. Results A combined analysis demonstrated that elevated LMR was significantly correlated with increased DFS/RFS (HR: 0.57, 95% CI: 0.49–0.67, p <​ 0.001), with no significant heterogeneity between studies (p =​ 0.21, I2 =​ 28%; Fig. 3). LMR and DFS/RFS in HCC. All included studies reported the data of LMR and DFS/RFS in HCC. A combined analysis demonstrated that elevated LMR was significantly correlated with increased DFS/RFS (HR: 0.57, 95% CI: 0.49–0.67, p <​ 0.001), with no significant heterogeneity between studies (p =​ 0.21, I2 =​ 28%; Fig. 3). LMR and clinicopathological features. To further explore the impact of LMR on the clinical features in HCC, we identified 9 clinical factors in HCC. The pooled analysis demonstrated that low LMR was significantly correlated with TNM stage (III-IV vs. I-II; HR =​ 1.78, 95% CI: 1.19–2.68, P =​ 0.005) and BCLC stage (B/C vs. A; HR =​ 1.70, 95% CI: 1.21–2.39, P =​ 0.002). Whereas no significant association was found with gender, AFP, liver cirrhosis, tumor differentiation, tumor number, tumor size, and vascular invasion. The correlation between LMR expres- sion and clinicopathological parameters of HCC is shown in Table 3. Sensitivity analysis. In order to assess the influence of single studies on the overall estimate, the sensitivity anal- ysis was performed. Each single study was removed each time to estimate the influence of individual data sets on the combined HR for OS. The results showed that no study had a significant effect on the observed effect size (pooled HR), indicating the robustness of our findings. Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Forest plots for the association between LMR and DFS/RFS. Figure 3. Forest plots for the association between LMR and DFS/RFS. Characteristics No. of studies No. of patients OR (95% CI) p Heterogeneity I2 (%) Ph Gender (male vs. female) 3 913 1.16 (0.64–2.09) 0.62 54 0.11 AFP (>​400 ng/mL vs. <​400 ng/mL) 2 660 1.61 (0.81–3.20) 0.17 73 0.05 Liver cirrhosis (yes vs. no) 3 913 1.66 (0.97–2.83) 0.06 52 0.12 Differentiation (low vs. moderate/high) 3 913 1.04 (0.74–1.46) 0.82 0 0.88 Tumor number (multiple vs. single) 2 660 0.96 (0.62–1.50) 0.87 0 0.61 Tumor size (>​5 cm vs. <​5 cm) 3 913 1.36 (0.79–2.35) 0.27 69 0.04 TNM stage (III-IV vs. I-II) 2 703 1.78 (1.19–2.68) 0.005 33 0.29 BCLC stage (B/C vs. A) 2 703 1.70 (1.21–2.39) 0.002 0 0.38 Vascular invasion (pos vs. neg) 2 660 1.47 (0.48–4.49) 0.50 84 0.01 Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Figure 3. Forest plots for the association between LMR and DFS/RFS. Characteristics No. of studies No. of patients OR (95% CI) p Heterogeneity I2 (%) Ph Gender (male vs. female) 3 913 1.16 (0.64–2.09) 0.62 54 0.11 AFP (>​400 ng/mL vs. <​400 ng/mL) 2 660 1.61 (0.81–3.20) 0.17 73 0.05 Liver cirrhosis (yes vs. no) 3 913 1.66 (0.97–2.83) 0.06 52 0.12 Differentiation (low vs. moderate/high) 3 913 1.04 (0.74–1.46) 0.82 0 0.88 Tumor number (multiple vs. single) 2 660 0.96 (0.62–1.50) 0.87 0 0.61 Tumor size (>​5 cm vs. <​5 cm) 3 913 1.36 (0.79–2.35) 0.27 69 0.04 TNM stage (III-IV vs. I-II) 2 703 1.78 (1.19–2.68) 0.005 33 0.29 BCLC stage (B/C vs. A) 2 703 1.70 (1.21–2.39) 0.002 0 0.38 Vascular invasion (pos vs. neg) 2 660 1.47 (0.48–4.49) 0.50 84 0.01 Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Figure 3. Forest plots for the association between LMR and DFS/RFS. Characteristics No. of studies No. of patients OR (95% CI) p Heterogeneity I2 (%) Ph Gender (male vs. female) 3 913 1.16 (0.64–2.09) 0.62 54 0.11 AFP (>​400 ng/mL vs. <​400 ng/mL) 2 660 1.61 (0.81–3.20) 0.17 73 0.05 Liver cirrhosis (yes vs. no) 3 913 1.66 (0.97–2.83) 0.06 52 0.12 Differentiation (low vs. moderate/high) 3 913 1.04 (0.74–1.46) 0.82 0 0.88 Tumor number (multiple vs. single) 2 660 0.96 (0.62–1.50) 0.87 0 0.61 Tumor size (>​5 cm vs. www.nature.com/scientificreports/ <​5 cm) 3 913 1.36 (0.79–2.35) 0.27 69 0.04 TNM stage (III-IV vs. I-II) 2 703 1.78 (1.19–2.68) 0.005 33 0.29 BCLC stage (B/C vs. A) 2 703 1.70 (1.21–2.39) 0.002 0 0.38 Vascular invasion (pos vs. neg) 2 660 1.47 (0.48–4.49) 0.50 84 0.01 Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. cancers45,46. Given this background, the observed favorable of high pretreatment LMR on OS of cancer patients may reflect the critical function of LMR in an inflammatory tumor microenvironment that inhibits tumor progression. cancers45,46. Given this background, the observed favorable of high pretreatment LMR on OS of cancer patients may reflect the critical function of LMR in an inflammatory tumor microenvironment that inhibits tumor progression. Nevertheless, there were several limitations to the present study. First, excessive heterogeneity existed among studies. Subgroup analyses didn’t find the potential sources of heterogeneity. In addition, we performed sensitivity analysis. The results showed that no study had a significant effect on the observed effect size. Second, the cut-off value for LMR was not unified in each study. Third, all included studies were retrospective analysis. 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Intratumoral regulatory T cells alone or in combination with cytotoxic T cells pre carcinoma after resection. Med. Oncol. 29, 1817–1826, doi: 10.1007/s12032-011-0006-x (2012). t , ( ) 37. Zhou, J. et al. Increased intratumoral regulatory T cells are related to intratumoral macrophages and poor prognosis in hepatocellular carcinoma patients. Int. J. Cancer 125, 1640–1648, doi: 10.1002/ijc.24556 (2009).f 37. Zhou, J. et al. Increased intratumoral regulatory T cells are related to intratumoral carcinoma patients. Int. J. Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 6 www.nature.com/scientificreports/ How to cite this article: Song, W. et al. The pretreatment lymphocyte to monocyte ratio predicts clinical outcome for patients with hepatocellular carcinoma: A meta-analysis. Sci. Rep. 7, 46601; doi: 10.1038/srep46601 (2017). How to cite this article: Song, W. et al. The pretreatment lymphocyte to monocyte ratio predicts clinical outcome for patients with hepatocellular carcinoma: A meta-analysis. Sci. Rep. 7, 46601; doi: 10.1038/srep46601 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. 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Built structures influence patterns of energy demand and CO2 emissions across countries
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Built structures influence patterns of energy demand and CO2 emissions across countries Helmut Haberl 1,5 , Markus Löw1,5, Alejandro Perez-Laborda2,5, Sarah Matej 1, Barbara Plank 1, Dominik Wiedenhofer 1, Felix Creutzig 3,4, Karl-Heinz Erb 1 & Juan Antonio Duro2 Helmut Haberl 1,5 , Markus Löw1,5, Alejandro Perez-Laborda2,5, Sarah Matej 1, Barbara Plank 1, Dominik Wiedenhofer 1, Felix Creutzig 3,4, Karl-Heinz Erb 1 & Juan Antonio Duro2 Built structures, i.e. the patterns of settlements and transport infrastructures, are known to influence per-capita energy demand and CO2 emissions at the urban level. At the national level, the role of built structures is seldom con- sidered due to poor data availability. Instead, other potential determinants of energy demand and CO2 emissions, primarily GDP, are more frequently assessed. We present a set of national-level indicators to characterize patterns of built structures. We quantify these indicators for 113 countries and statis- tically analyze the results along with final energy use and territorial CO2 emissions, as well as factors commonly included in national-level analyses of determinants of energy use and emissions. We find that these indicators are about equally important for predicting energy demand and CO2 emissions as GDP and other conventional factors. The area of built-up land per capita is the most important predictor, second only to the effect of GDP. Increasing global temperature driven by growing greenhouse gas (GHG) emissions is a major global concern1. GHGs mainly result from the energy-related combustion of fossil fuels2. The question emerges, which factors drive energy demand and emissions, and to what extent they are malleable. This question is relevant at many levels, from products to individuals, households, and cities up to continental and even global totals. We here focus on countries, because many decision-making processes occur at the national level. A widespread approach for cross-country analyses classifies the factors influencing resource use and emissions into population, affluence, and technology3–5. These factors are captured in the STIRPAT (Stochastic Impacts by Regression on Population, Affluence and Technology) framework3, derived from the classical IPAT (impact = population × affluence × technology) approach5,6. Increasing global temperature driven by growing greenhouse gas (GHG) emissions is a major global concern1. GHGs mainly result from the energy-related combustion of fossil fuels2. The question emerges, which factors drive energy demand and emissions, and to what extent they are malleable. This question is relevant at many levels, from products to individuals, households, and cities up to continental and even global totals. Article https://doi.org/10.1038/s41467-023-39728-3 1Institute of Social Ecology, University of Natural Resources and Life Sciences, Vienna, Austria. 2Economics Department and Eco-SOS, Universitat Rovira i Virgili, Tarragona, Spain. 3Mercator Research Institute on Global Commons and Climate Change, EUREF 19, 10829 Berlin, Germany. 4Technical University Berlin, Straße des 17 Junis 135, 10623 Berlin, Germany. 5These authors contributed equally: Helmut Haberl, Markus Löw, Alejandro Perez-Laborda. e-mail: helmut.haberl@boku.ac.at Built structures influence patterns of energy demand and CO2 emissions across countries The distance from the vertical axis either to the right (positive corre- lation) or to the left (inverse correlation) depicts the additional explanatory power of the respective indicator over a model con- sidering only GDP and DENS. Several indicators provide additional explanations over GDP and DENS alone. The area of built-up land (both BLcap and BLfract) is positively correlated with TFC and CO2, as are the density of rail and road infrastructures, especially in rural areas. UPOP is inversely correlated with TFC and CO2, as is urban road density (not significant) and BLmono. PGAS, which had not been significant in the bivariate correlations, emerges as an important factor, which is also observed for RDurban and other indicators. UPdens lose importance, most likely due to its high correlation with GDP. Despite these insights from urban studies, material stock pat- terns are seldom considered in debates on national-level analyses of factors determining levels of energy demand and emissions, as well as their possible decoupling from GDP11,15. A systematic review of the empirical literature on these questions15,37 revealed only one study38 considering material stock patterns in analyzing transport-related emissions. Hence little is known about the effects of material stock patterns on energy demand and CO2 emissions beyond the city level. This results from a scale mismatch: maps of material stock patterns provide fine-grained spatial detail30, often focused on specific regions, that cannot be included in national-level analyses of factors driving energy demand and emissions. For cross-country analyses, consistent indicators need to be developed from spatial data and then aggregated at the national level in a manner which preserves key information on patterns and supports comparative analyses across countries and world regions. In this work, we develop national-level indicators of character- istics of built structures that, based on urban studies, can be potential determinants of resource use and emissions. We quantify them for 113 countries comprising 91.2% of the world population and 97.3% of global GDP. We analyze two independent variables: (1) yearly total per- capita final energy consumption (abbreviated as TFC) and (2) yearly per-capita CO2 emissions (abbreviated as CO2). We test material stock pattern indicators against other variables thathave been widely used in national-level analyses of determinants of energy use and emissions, here denoted as conventional factors. Built structures influence patterns of energy demand and CO2 emissions across countries The price of gasoline (abbreviated as PGAS) is an energy price indicator that is strongly related to settlement patterns21. Some studies used urban population as a percent of the total population (abbre- viated as UPOP) as a development indicator22. p In terms of their Pearson coefficients in bivariate correlations, several indicators of material stock patterns are as strongly correlated with TFC and CO2 as the conventional factors (Fig. 2a). GDP is positively correlated with both TFC and CO2. HDD and the fraction of the urban population also show the expected pattern, while PGAS and DENS are largely uncorrelated. Almost all material stock pattern indicators are correlated with both TFC and CO2. The extent of built-up land (BLcap and BLfract) is positively correlated with both TFC and CO2, as are total and rural road density and the dispersion of built-up land and most railway-related indicators. The correlation coefficients of BLcap with TFC and CO2 are both ~0.7; BLcap is the second-best predictor of both TFC and CO2 after GDP. As expected from the urban literature, urban population density (UPdens) is inversely correlated with CO2 and energy, whereas the share of urban population (UPOP) is strongly positively correlated with CO2 and TFC. Inverse relations prevail for BLmono, BLcomp, and the urban-to-rural relations of infrastructure density. At the urban scale, the influence of population density and the spatial layout of urban areas on cities’ resource demand has been widely studied20,23–28. There are several reasons why the extent and spatial layout (density and form) of built structures—henceforth denoted as material stock patterns29, i.e., the spatial patterns of societies’ material stocks in infrastructures and buildings—could affect energy demand and CO2 emissions. The accumulation of material stocks requires massive amounts of resources such as steel-reinforced concrete, mortar, bricks, timber, plastics, glass, gravel, or sand30–33, which are associated with high GHG emissions34. Heating, cooling, and lighting of buildings and production processes in industrial plants require much energy20,23,26,35, as does the mobility of goods and people on roads and railways33,36. Semi-partial correlations of the material stock pattern indicators controlled for GDP and population density (DENS) are shown in Fig. 2b. The part of each material stock pattern indicator correlated with GDP and DENS is removed, revealing the strength of the linear correlation between TFC or CO2 and the remaining part of the respective variable. Built structures influence patterns of energy demand and CO2 emissions across countries We here focus on countries, because many decision-making processes occur at the national level. A widespread approach for cross-country analyses classifies the factors influencing resource use and emissions into population, affluence, and technology3–5. These factors are captured in the STIRPAT (Stochastic Impacts by Regression on Population, Affluence and Technology) framework3, derived from the classical IPAT (impact = population × affluence × technology) approach5,6. be reduced while GDP is growing. This may be possible e.g., through more efficient technologies, but the debate is so far inconclusive10,11. While recent studies revealed examples of growing national econo- mies where policies implemented in the last decade achieved reduc- tions in energy demand and CO2 emissions12–14, neither those studies nor a meta-analysis15 yielded evidence for reductions in energy use and GHG emissions consistent with ambitious climate targets. Other potential determinants of energy demand and emissions receive less attention than GDP, even though additional entry points to accelerate decarbonization are urgently required. Population density has been studied with varied outcomes. An econometric study of OECD countries 1980–2011 suggested an inverse relation between population density and CO2 emissions16; a regression analysis of materials used in >100 countries in 2000 found a similar effect17. A global regression analysis18 found no effect of population density on energy demand, whereas a panel analysis of 11 Asian countries from 1960–200419 to conditions that influence energy demand for heating or cooling of buildings20, fuel prices affecting demand for transport energy21, and the urbanization rate (urban population as percent of the Economic activity (called affluence in IPAT-style analyses, usually measured as Gross Domestic Product or GDP) is acknowledged as a major determinant of energy use and greenhouse gas (GHG) emissions7–9. The discussion mainly focuses on whether GDP can be decoupled from emissions, i.e., whether energy use and emissions can 1Institute of Social Ecology, University of Natural Resources and Life Sciences, Vienna, Austria. 2Economics Department and Eco-SOS, Universitat Rovira i Virgili, Tarragona, Spain. 3Mercator Research Institute on Global Commons and Climate Change, EUREF 19, 10829 Berlin, Germany. 4Technical University Berlin, Straße des 17 Junis 135, 10623 Berlin, Germany. 5These authors contributed equally: Helmut Haberl, Markus Löw, Alejandro Perez-Laborda. e-mail: helmut.haberl@boku.ac.at Nature Communications| (2023) 14:3898 1 https://doi.org/10.1038/s41467-023-39728-3 Article total population). A suitable proxy for the climate dependency of heating energy demand are heating-degree days (abbreviated as HDD)20. Built structures influence patterns of energy demand and CO2 emissions across countries As conventional factors, we use GDP/cap/yr (abbreviated as GDP), population density (DENS), UPOP as a development indicator, HDD as a proxy of climate dependency of energy demand, and PGAS as an energy price indicator. Extensive variables are expressed as per-capita values to facilitate country comparisons and remove countries’ population numbers from the analysis. Multivariate lasso analysis The capability of the material stock pattern indicators to add insights beyond conventional factors is further analyzed in Table 2. We use the least absolute shrinkage and selection operator (lasso) approach to select variables for multivariate statistical models capable of predict- ing cross-country patterns of TFC and CO2. Lasso is a widely- used procedure for automatically performing variable selection in linear regression models39,40. It overcomes the drawbacks of overfitting and multicollinearity associated with ordinary least square (OLS) methods41. The lasso method penalizes complexity to derive the best parsimonious model for any predefined value of λ (the factor deter- mining how strongly model complexity is penalized; for detail, see Methods section), thereby allowing to identify the factors that most strongly affect TFC and CO2. The standard procedure to select λ is cross-validation, where randomly chosen samples of countries are used to develop a model used for the prediction of patterns in out-of- sample countries. The procedure gradually reduces λ, which generally results in more indicators being selected41. Alternative Lasso schemes and other variable selection techniques, such as stepwise regression, yield very similar results (SI). Nature Communications| (2023) 14:3898 tional-level indicators of material stock patterns National-level indicators of material stock patterns p We test three hypotheses based on aggregated indicators of material stock patterns (Fig. 1). Material stock patterns are represented by three types of indicators: (1) The area of built-up land is represented by two indicators, one as a fraction of a nation’s inhabited land, the other per inhabitant. Other indicators describe patterns of built-up areas, including their spatial clustering, form, and distribution, which reflect geomorphological factors as well as historical contingencies. (2) Road indicators that describe the density (length per unit area) of roads in urban and rural regions and the relations between urban and rural road lengths and densities. (3) Railway indicators are defined in the same manner as those for roads (Table 1). The leftmost three columns of Table 2 show the knots in the lasso paths, i.e., the λ values at which indicators are added (or removed due to collinearity). Knots are arranged in decreasing order of λ, with indicators being ranked in order of selection. In the first column, the λ value marked with an asterisk (*) indicates the optimal model identified by the cross-validation. GDP is always selected as the first indicator, but various material stock pattern indicators appear very early on (i.e., at high λ values), and remain active in the optimal model. BLcap is the second-chosen indicator for both CO2 and TFC, and several other Nature Communications| (2023) 14:3898 2 Article https://doi.org/10.1038/s41467-023-39728-3 g. 1 | Workflow of this study. National-level indicators of the extent and spatial tterns of settlements and infrastructures (material stock patterns) were derived om global maps, here illustrated using Paraguay, the UK, Kenia, and Bangladesh as amples. Results were statistically analyzed along with the conventional factors assumed to co-determine energy use and CO2 emissions. The main aim was the hypotheses formulated in the lower-right box. Copyright for administr boundaries: © Eurogeographics. Fig. 1 | Workflow of this study. National-level indicators of the extent and spatial patterns of settlements and infrastructures (material stock patterns) were derived from global maps, here illustrated using Paraguay, the UK, Kenia, and Bangladesh as examples. Results were statistically analyzed along with the conventional factors assumed to co-determine energy use and CO2 emissions. The main aim was to test the hypotheses formulated in the lower-right box. Copyright for administrative boundaries: © Eurogeographics. tional-level indicators of material stock patterns Table 1 | Indicators of the extent and patterns of built-up land and transport infrastructures material stock pattern indicators are selected much earlier than widely-used conventional factors such as population density (DENS). Heating-degree days (HDD) and the price of gas (PGAS) are important for both CO2 and TFC, whereas UPOP is important for predicting CO2 but not selected by lasso for TFC. type approaches, and will enable researchers to broaden their ana- lysis and scenario modeling capabilities by including material stock patterns as crucial factors for analyzing the possible decoupling of energy use and emissions from GDP. Population density plays a smaller role than widely assumed, while many material stock patterns strongly influence the cross-country differences in energy demand and CO2 emissions. The material stock pattern indicator with the most consistent predictive power across all analyses is the area of built-up land per capita (BLcap), which emerges as the second-most important variable (after GDP) in most of our statistical analyses, even in analyses considering the GDP effect. This is plausible because infrastructures and buildings require energy for being built and used, which results in CO2 emissions in current fossil fuel-dominated energy systems34,43. Higher BLcap also means larger floor size and longer distances between destinations, which all raises energy demand in buildings and transport. These findings corroborate and expand on previous analyses (that used entirely different models, did not capture spatial patterns, and mostly referred to temporal trends), suggesting that challenges for climate-change mitigation strongly depend on the past and future accumulation of material stocks in buildings and infrastructures43–46. This is worrying because material stocks are growing globally largely in unison with GDP32,46. The rightmost three columns of Table 2 report the estimated coefficients of models selected by cross-validation as well as measures of in-sample and out-of-sample fit (r2). Models A comprises only vari- ables selected by lasso (path shown in the left part of the table) among all indicators. For the benchmark Models B, only conventional factors are selected. Including material stock pattern indicators in Model A yields better prediction than Models B for both CO2 and TFC in terms of in-sample and out-of-sample goodness of fit, and improves the Bayesian Information Criterion (BIC) of model selection. Other criteria to develop optimal models, and alternative variable selection proce- dures yielded similar results (section 5 in the SI). tional-level indicators of material stock patterns Nature Communications| (2023) 14:3898 3 3 https://doi.org/10.1038/s41467-023-39728-3 Article Table 1 | Indicators of the extent and patterns of built-up land and transport infrastructures Name Abbreviation Description and interpretation Unit (1) Indicators for the extent and pattern of a nation’s built-up land Fraction of built-up land BLfract Built-up land (buildings & infrastructures) as % of the inhabited land area. m²/m² Built-up land per capita BLcap Built-up land per capita. m²/cap Dispersion of built-up land BLdisp Index based on the average distance of each patch of built-up land to the nearest adjacent patch. High values indicate strong dispersion. – Monocentricity of built-up land BLmono Area of the largest contiguous built-up patch as % of the sum of the areas of the ten largest patches. High values indicate dominance of one large center. m2/m2 Compactness of built-up land BLcomp Index describing how round or compact the shapes of a nation’s built-up land patches are on average. – Urban population density UPdens Urban population per unit of urban built-up land cap/m2 (2) Indicators of road density and distribution Road density RDtotal Length of roads per unit area of inhabited land. m/m² Urban road density RDurban Length of roads in urban areas per unit area of urban areas. m/m² Rural road density RDrural Length of roads in rural areas per unit area of rural areas; proxy of rural accessibility and connectivity between urban centers. m/m² Ratio of urban-to-rural road lengths RLurb-rur Ratio of urban to rural road lengths, indicating the extent to which roads are concentrated in cities. – Ratio of urban-to-rural road density RDurb-rur Ratio of RDurban and RDrural, indicating the difference between urban and rural road densities. – (3) Indicators of railway density and distribution Railway density RWDtotal Length of railways per unit area of inhabited land. m/m² Urban railway density RWDurban Length of railways in urban areas per unit area of urban areas. m/m² Rural railway density RWDrural As RDrural for railways. m/m² Ratio of urban-to-rural railway lengths RWLurb-rur As RLurb-rur for railways. – Ratio of urban-to-rural railway density RWDurb-rur As RDurb-rur for railways. – These indicators condense spatially explicit information in maps to national-level indicator values assumed to co-determine a nation’s per-capita level of energy demand and CO2 emissions. Global maps showing the indicator values are in Supplementary Figs. 5–7 (SI). tional-level indicators of material stock patterns Even models that include only material stock pattern indicators and exclude all con- ventional factors (including GDP) achieve good predictions (out-of- sample r2 of 0.65 for TFC and 0.62 for CO2; see results for Model C shown in section 5.2 of the SI). Nature Communications| (2023) 14:3898 Discussion Our indicators and results create options for analyzing which of the characteristics of material stock patterns are most important in determining and predicting energy demand and emissions in cross- country analyses. Analyses presented in Fig. 2, Table 2 as well as the SI clearly show that many specific aspects of material stock patterns play a role in determining cross-country differences in energy demand and emissions. The multivariate analysis also shows that these patterns interact in many ways that are difficult to disentangle due to the col- linearities of the material stock pattern indicators. Future research could employ refined study designs addressing how changes over time and space affect these relationships and elucidate the different causal pathways involved. The analysis shows that extent and spatial patterns of built struc- tures, here denoted as material stock patterns, play an important role in co-determining and predicting the level of resource use, here TFC and CO2, in a cross-country analysis. This implies that insights from urban studies23–26,42 generally hold at the national scale. Despite the unavoidable loss of information resulting from the aggregation of maps to the national scale, the indicators in Table 1 maintain key information representing important characteristics of material stock patterns that strongly affect cross-country patterns of TFC and CO2. The indicators have substantial additional explanatory and pre- dictive power over conventional factors. Discussion Correlations were not significant for between mate for GDP and D ficient of the s correlation co indicate insign UPOP HDD PGAS BLfract BLcap UPdens BLdisp BLmono BLcomp RDtotal RDurban RDrural RLurb-rur RDurb-rur RWDtotal RWDurban RWDrural RWLurb-rur RWLurb-rur -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 Zero correlation Semi-partial correlation Semi-partial correlations for TFC UPOP HDD PGAS BLfract BLcap UPdens BLdisp BLmono BLcomp RDtotal RDurban RDrural RLurb-rur RDurb-rur RWDtotal RWDurban RWDrural RWLurb-rur RWDurb-rur -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 Zero correlation Semi-partial correlation Semi-partial correlations for CO2 Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and per-capita CO2 emissions (CO2) with conventional factors and material stock pattern indicators. a Pearson’s zero correlation coefficients of correlations between TFC (left) and CO2 (right) and material stock pattern indicators as well as conventional factors. Natural logarithms of the variables were analyzed. Squaring the correlation coefficients gives the percentage of the cross-country variation of CO2 or TFC explained by the respective indicator alone. Correlations were not significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations between material stock pattern indicators and TFC (left) and CO2 (right) controlling for GDP and DENS. Distance from the vertical axis indicates the correlation coef- ficient of the semi-partial correlation, and distance from the horizontal axis is the correlation coefficient of the bivariate (uncontrolled) correlation. Red contours indicate insignificant results (significance level p < 0.1). Semi-partial correlations for CO2 Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and per-capita CO2 emissions (CO2) with conventional factors and material stock pattern indicators. a Pearson’s zero correlation coefficients of correlations between TFC (left) and CO2 (right) and material stock pattern indicators as well as conventional factors. Natural logarithms of the variables were analyzed. Squaring the correlation coefficients gives the percentage of the cross-country variation of CO2 or TFC explained by the respective indicator alone. Correlations were not Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and per-capita CO2 emissions (CO2) with conventional factors and material stock significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations between material stock pattern indicators and TFC (left) and CO2 (right) controlling for GDP and DENS. Discussion They can help develop much stronger models of national-level TFC and CO2 than usual IPAT- Nature Communications| (2023) 14:3898 4 https://doi.org/10.1038/s41467-023-39728-3 Article (a) (b) 0,88 0,72 0,67 0,67 0,65 0,59 0,53 0,47 0,46 0,44 0,37 0,27 0,12 0,08 -0,10 -0,11 -0,12 -0,31 -0,44 -0,45 -0,52 -1,00 -0,50 0,00 0,50 1,00 GDP BLcap UPOP RWDtotal RWDrural RWDurban HDD RDrural RDtotal BLfract RWLurb-rur BLdisp PGAS* RLurb-rur* DENS* RDurban* RWDurb-rur BLmono BLcomp UPdens RDurb-rur Bivariate correlation with TFC 0,82 0,72 0,69 0,68 0,67 0,56 0,55 0,50 0,49 0,47 0,38 0,30 0,19 0,01 -0,05 -0,08 -0,19 -0,32 -0,36 -0,43 -0,50 -1,00 -0,50 0,00 0,50 1,00 GDP UPOP BLcap RWDtotal RWDrural RWDurban HDD BLfract RDtotal RDrural RWLurb-rural BLdisp RLurb-rur PGAS* RDurban* DENS* RWDurb-rur BLmono BLcomp UPdens RDurb-rur Bivariate correlation with CO2 UPOP HDD PGAS BLfract BLcap UPdens BLdisp BLmono BLcomp RDtotal RDurban RDrural RLurb-rur RDurb-rur RWDtotal RWDurban RWDrural RWLurb-rur RWLurb-rur -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 Zero correlation Semi-partial correlation Semi-partial correlations for TFC UPOP HDD PGAS BLfract BLcap UPdens BLdisp BLmono BLcomp RDtotal RDurban RDrural RLurb-rur RDurb-rur RWDtotal RWDurban RWDrural RWLurb-rur RWDurb-rur -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 Zero correlation Semi-partial correlation Semi-partial correlations for CO2 Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and per-capita CO2 emissions (CO2) with conventional factors and material stock pattern indicators. a Pearson’s zero correlation coefficients of correlations between TFC (left) and CO2 (right) and material stock pattern indicators as well as conventional factors. Natural logarithms of the variables were analyzed. Squaring the correlation coefficients gives the percentage of the cross-country variation of CO2 or TFC explained by the respective indicator alone. Correlations were not significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations between material stock pattern indicators and TFC (left) and CO2 (right) controlling for GDP and DENS. Distance from the vertical axis indicates the correlation coef- ficient of the semi-partial correlation, and distance from the horizontal axis is the correlation coefficient of the bivariate (uncontrolled) correlation. Red contours indicate insignificant results (significance level p < 0.1). Discussion Distance from the vertical axis indicates the correlation coef- ficient of the semi-partial correlation, and distance from the horizontal axis is the correlation coefficient of the bivariate (uncontrolled) correlation. Red contours indicate insignificant results (significance level p < 0.1). significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations between material stock pattern indicators and TFC (left) and CO2 (right) controlling for GDP and DENS. Distance from the vertical axis indicates the correlation coef- ficient of the semi-partial correlation, and distance from the horizontal axis is the correlation coefficient of the bivariate (uncontrolled) correlation. Red contours indicate insignificant results (significance level p < 0.1). pattern indicators. a Pearson’s zero correlation coefficients of correlations between TFC (left) and CO2 (right) and material stock pattern indicators as well as conventional factors. Natural logarithms of the variables were analyzed. Squaring the correlation coefficients gives the percentage of the cross-country variation of CO2 or TFC explained by the respective indicator alone. Correlations were not Our results have implications for countries pursuing ambitious climate targets2,43. They demonstrate that insights from urban-level studies translate to the national scale, which could so far not be investigated at that level due to lacking data. This suggests that the indicators presented above offer opportunities to investigate the importance of built structures also for other aspects of national economies’ resource use than those analyzed here. Empirical urban studies have consistently shown that urban form and infrastructure affect travel demand and, therefore, GHG emissions47. The relationship between built structures and GHG emissions has also been predicted by theoretical analyses in urban economics21,48. This suggests that our national-level observations—e.g., that large, sprawling material stocks in built structures represent an important determinant of a nation’s per-capita level of energy demand and emissions—are very likely underpinned by similar causal relationships. Of course, more research, in particular on how these relationships change over time, would be desirable. Because high-resolution maps of decadal trends in built structures are gradually becoming available49,50, such analyses may between built structures and GHG emissions has also been predicted by theoretical analyses in urban economics21,48. This suggests that our national-level observations—e.g., that large, sprawling material stocks in built structures represent an important determinant of a nation’s per-capita level of energy demand and emissions—are very likely underpinned by similar causal relationships. Of course, more research, in particular on how these relationships change over time, would be desirable. Discussion (a) (a) (b) 0,88 0,72 0,67 0,67 0,65 0,59 0,53 0,47 0,46 0,44 0,37 0,27 0,12 0,08 -0,10 -0,11 -0,12 -0,31 -0,44 -0,45 -0,52 -1,00 -0,50 0,00 0,50 1,00 GDP BLcap UPOP RWDtotal RWDrural RWDurban HDD RDrural RDtotal BLfract RWLurb-rur BLdisp PGAS* RLurb-rur* DENS* RDurban* RWDurb-rur BLmono BLcomp UPdens RDurb-rur Bivariate correlation with TFC 0,82 0,72 0,69 0,68 0,67 0,56 0,55 0,50 0,49 0,47 0,38 0,30 0,19 0,01 -0,05 -0,08 -0,19 -0,32 -0,36 -0,43 -0,50 -1,00 -0,50 0,00 0,50 1,00 GDP UPOP BLcap RWDtotal RWDrural RWDurban HDD BLfract RDtotal RDrural RWLurb-rural BLdisp RLurb-rur PGAS* RDurban* DENS* RWDurb-rur BLmono BLcomp UPdens RDurb-rur Bivariate correlation with CO2 Bivariate correlation with CO2 (b) UPOP HDD PGAS BLfract BLcap UPdens BLdisp BLmono BLcomp RDtotal RDurban RDrural RLurb-rur RDurb-rur RWDtotal RWDurban RWDrural RWLurb-rur RWLurb-rur -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 Zero correlation Semi partial correlation Semi-partial correlations for TFC UPOP HDD PGAS BLfract BLcap UPdens BLdisp BLmono BLcomp RDtotal RDurban RDrural RLurb-rur RDurb-rur RWDtotal RWDurban RWDrural RWLurb-rur RWDurb-rur -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 Zero correlation Semi partial correlation Semi-partial correlations for CO2 UPOP HDD PGAS BLfract BLcap UPdens BLdisp BLmono BLcomp RDtotal RDurban RDrural RLurb-rur RDurb-rur RWDtotal RWDurban RWDrural RWLurb-rur RWLurb-rur -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 Zero correlation Semi-partial correlation Semi-partial correlations for TFC P -0,4 -0 Zero correlation Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and per-capita CO2 emissions (CO2) with conventional factors and material stock pattern indicators. a Pearson’s zero correlation coefficients of correlations between TFC (left) and CO2 (right) and material stock pattern indicators as well as conventional factors. Natural logarithms of the variables were analyzed. Squaring the correlation coefficients gives the percentage of the cross-country variation of CO2 or TFC explained by the respective indicator alone. Discussion In the first column, the λ value marked with an asterisk denotes the optimal model (Model A) emerging from the cross-validation. CV MSPE is the cross-validated mean-square prediction error evaluated with tenfolds (for detail, see Methods section). For Model B, only conventional factors are selected. The same folds are used for the assessment of all models. BIC is the Bayesian Information Criterion for model selection. r2 is the goodness of fit within the sample of countries, and oSr2 refers to the (cross-validated) out-of-sample goodness of fit. resolution land-cover data53. The indicators refer to the years 2015 (built-up land) and 2020 (roads and railroads). To reduce random fluctuations for the energy and emission data as well as the conven- tional factors, we calculated averages for as many years in the period 2015-2020 as were available in the statistical sources. Extensive vari- ables were represented by per-capita values to facilitate comparisons between different-sized countries, following the convention to regard population as a scaling factor with an elasticity of one in STIRPAT analyses5. soon be feasible. We conclude that the area and patterns of built-up land emerge as an important entry point for efforts at reducing energy demand and CO2 emissions at national levels51, suggesting that limiting built-up area per capita could be a worthwhile policy goal, not only from a land-use perspective, but also to limit future GHG emissions. Discussion Because high-resolution maps of decadal trends in built structures are gradually becoming available49,50, such analyses may Nature Communications| (2023) 14:3898 5 https://doi.org/10.1038/s41467-023-39728-3 Article Table 2 | Multivariate analysis using cross-validation lasso | y g Models predicting TFC Lasso path for model A Estimated coefficients λ (A)dded, (R)emoved CV MPSE Model A Model B 0.711 GDP(A) 0.703 GDP 0.494 0.572 0.371 BLcap(A) 0.319 DENS −0.017 −0.039 0.161 HDD(A), RWDtot(A) 0.179 UPOP 0.121 RDurb-rur(A) 0.166 HDD 0.021 0.051 0.111 PGAS(A) 0.161 PGAS −0.352 −0.288 0.101 RDurb(A) 0.155 BLcap 0.184 0.076 RWDurb(A) 0.135 BLmono −0.017 0.063 RWDtot(R) 0.125 RDurb −0.392 0.048 DENS(A) 0.115 RLurb-rur −0.024 0.030 BLmono(R) 0.108 RDurb-rur −0.074 0.027 RLurb-rur(A) 0.107 RWDurb 0.045 0.017* (Unchanged) 0.105 Intercept 3.457 2.542 0.016 RWDurb-rur(A) 0.105 0.013 BLcomp(A) 0.105 Measures of in-and-out-of-sample fit 0.011 (Unchanged) 0.106 BIC 84.17 100.32 r2 0.900 0.851 oSr2 0.865 0.833 Models predicting CO2 λ (A)dded, (R)emoved CV MPSE variable Model A Model B 0.867 GDP(A) 1.278 GDP 0.449 0.582 0.544 BLcap(A) 0.801 DENS −0.061 0.023 0.496 UPOP(A) 0.738 UPOP 0.466 0.558 0.452 RWDtot(A) 0.683 HDD 0.055 0.109 0.312 HDD(A) 0.530 PGAS −0.680 −0.688 0.215 PGAS(A) 0.446 BLfract 0.201 0.135 RDurb-rur(A) 0.338 BLcap 0.176 0.123 BLfract(A) 0.323 BLcomp 0.489 0.085 RWLurb-rur(A) 0.289 RDurb −0.201 0.070 BLfract(R) 0.280 RWDurb 0.130 0.048 BLcomp(A) 0.269 RWDrur 0.017 0.044 RDurb(A) 0.266 Intercept −2.723 −4.294 0.037 RWDurb(A) 0.262 0.025 BLfract (A) 0.256 Measures of in-and-out-of-sample fit 0.023 DENS(A) 0.255 BIC 178.44 190.82 0.021 RWLurb-rur(R) 0.254 r2 0.873 0.812 0.016 RWDrur(A) 0.251 oSr2 0.817 0.785 0.016 RDurb-rur(R) 0.251 0.014 RWDtot(R) 0.250 0.012* (Unchanged) 0.249 0.006 (Unchanged) 0.250 The leftmost three columns show the lasso path for predicting cross-country patterns of TFC (above) and CO2 (below) using all variables. In the first column, the λ value marked with an asterisk denotes the optimal model (Model A) emerging from the cross-validation. CV MSPE is the cross-validated mean-square prediction error evaluated with tenfolds (for detail, see Methods section). For Model B, only conventional factors are selected. The same folds are used for the assessment of all models. BIC is the Bayesian Information Criterion for model selection. r2 is the goodness of fit within the sample of countries, and oSr2 refers to the (cross-validated) out-of-sample goodness of fit. The leftmost three columns show the lasso path for predicting cross-country patterns of TFC (above) and CO2 (below) using all variables. Deriving the built-up land layer To calculate IH, we first cut out high-altitude regions from the country’s total territory. We calculated the elevational distribution of BL features and excluded all areas above the area-weighted 99th per- centile of BL-elevations. In a second step, we excluded the LC100 land cover types bare/sparse vegetation (deserts and rocks), moss & lichen, snow & ice, and permanent water bodies. Thirdly, we spatially inter- sected the map resulting from the previous two steps with a synthetic layer that represents the gapless inhabited land area. To derive this layer, we applied an area-dependent buffer for all BL features. See Eq. (1) for the dynamic BL buffer width (wBL); ABL feature denotes the area of an exemplary built-up land feature. g p y The built-up land vector (BL) for every country is one primary data product derived by the vectorized LC100 grid. This standard BL layer comprised all national built-up features and was used to derive the BL- related indicators shown in part (1) of Table 1. To map each country’s urban agglomerations, and thereby distinguish urban from rural land respectively infrastructures (see Supplementary Figs. 2–4, SI), we clustered features of the BL vector layer using an empirical growing- neighborhood approach: We started with the country’s largest BL feature and created convex hulls, which were buffered with the fifth of the area-equal radius. We then identified intersecting BL features within this buffered hull and created a new hull and buffer. These two steps were repeated as long as the BL area of all intersecting features reached 33.3% of the area of the buffered hull (used to check the intersections), and the BL area increased at least 0.5%, compared to the BL area of the previous iteration. If these criteria were not given, the growing procedure terminated and the collected BL features dissolved to one BL agglomeration. The algorithm subsequently went on with the next-biggest BL feature, which had not been assigned to an already- created agglomeration, again starting the growing-neighborhood procedure. The whole process terminated when the next BL feature (to restart the growing-neighborhood procedure again) represented less than 0.1% of the total BL area of the respective country. The maps ð1Þ maxfwBL : 100kmg Finally, we added cropland areas (from LC100) and the original BL areas to re-include those BL areas excluded by the elevation threshold. The infrastructure layers The preprocessed OSM database comprises globally consistent road (R) and railway vector data (RW), but the availability of OSM data varies strongly between countries. While the OSM data in countries of the global North (industrial or even postindustrial countries) also include minor road and train track categories (e.g., cycleways, steps, or private gauges), OSM data in countries of the global South only comprise the main road and railway network. To reduce this inconsistency, we excluded minor OSM classes in order to derive a more comparable global database (Supplementary Table 3). The maps showing the road network indicators are in Supplementary Fig. 6 (SI). Fractions of the area refer to an inhabited land. The railway indicators were derived from OSM in the same manner as those for roads and are shown in Supple- mentary Fig. 7 (SI). The railway types considered are defined in Sup- plementary Table 4. Fractions refer to an inhabited land. The planar extent of road and railway networks was calculated using width data reported in Supplementary Table 5. The distinction between urban and rural infrastructures was based on a spatial intersection of OSM road and railway data with the BL features, which resulted in layers of urban and rural networks (example shown in Supplementary Fig. 4, SI). Reference layer for inhabited land Reference layer for inhabited land The definition of some material stock pattern indicators requires a reference area (AREF). The area of the total national territory (ANT) may not be suitable, given that in some countries (almost), the entire area is inhabitable, whereas other countries contain large tracts of land unsuitable for human habitation and hence largely uninhabited. We, therefore, developed the proxy layer inhabited land (IH) as a more suitable area reference (AIH). In contrast to existing similar datasets69, this IH mainly uses land cover information of the LC100 grid. This guarantees thematic consistency in spatial intersections with BL data that were derived using the same dataset. The IH includes not only area that is covered with settlements or infrastructures, but also cropland areas and areas with ambiguous land cover that fall within a zone of influence around existing built-up areas, which is approximated by a buffer that depends on the area of a built-up land feature (Eq. 1). The IH is based on the current settlement and cropland extent, as well as the spatial distribution, density, and elevation occurrence of built-up areas according to the LC100 grid. Data used to derive spatial indicators The spatial indicators presented in this study rely on three spatially explicit datasets; (1) built-up features and urban agglomerations, (2) main infrastructure features (road and railway), and (3) an inhabited land area layer used for reference. All variables that express quan- tities as fractions of a country’s area, relate to inhabited land. We used the LC100 grid of the Copernicus Global Land Cover Service53 to derive relevant land cover information. The choice of input data and data quality checks are discussed in the SI; see also Supplementary Fig. 1 (SI). In contrast to other built-up land datasets54–56, the LC100 not only provides globally consistent information on built-up areas, it also includes all other complementary land cover types at a global scale. For data preparation, we vectorized the LC100 grid to allow further spatial intersection procedures (e.g., to clip to national bor- ders). The NOAA-DEM grid was used as a global digital elevation model. We utilized the Geofabrik-download hub57 to obtain the entire global OSM data. The GISCO archive from EUROSTAT58 provides the applied country borders. Details on the data sources are given in Supplementary Table 1 (SI); datasets59 and software code60 are available online. Methods The material stock pattern indicators were derived from crowd- sourced data on infrastructures (roads and railways)52 as well as high- Nature Communications| (2023) 14:3898 Nature Communications| (2023) 14:3898 6 Article https://doi.org/10.1038/s41467-023-39728-3 in Supplementary Fig. 5 (SI) show the built-up (BL) land indicators. Fractions of land areas refer to inhabited land. in Supplementary Fig. 5 (SI) show the built-up (BL) land indicators. Fractions of land areas refer to inhabited land. Sources of energy and emission data as well as conventional factors Gross domestic product (GDP) in constant 2015 US$ was sourced from UN Statistics Division National Accounts61. Population data used to calculate population density (DENS) and urban population rates (UPOP) were taken from official census data 2017 of the World Bank62,63. The World Bank database was also used to source data on pump prices for gasoline (PGAS)64. Heating-degree days (HDD) were calculated as a population-weighted average of °C days above 18 °C and sourced from the International Energy Agency (IEA) Weather for Energy Tracker65. Territorial CO2 emissions from fossil fuel combus- tion and cement production (CO2) were sourced from the Global Carbon Project database66,67 and total final energy consumption (TFC) from the IEA energy balances, which could only be accessed for the latest year 201768. The record date for the download of these indicators was 25 March 2021. We always used the latest available year for our cross-sectional analysis, but also conducted robustness checks using arithmetic averages over the latest five years for all indicators to exclude potential bias from outstanding annual values. Details on the data are available in Supplementary Table 2 in the SI. Data availability Datasets on spatial data on patterns of global infrastructure and set- tlements, the inhabited land layer, as well as the indicator values of dependent and independent variables used in the statistical analyses is freely available here: https://doi.org/10.5281/zenodo.5876941. An interim result that was too large to be uploaded as zenodo archive is available from the authors for non-commercial research purposes upon reasonable request (for detail, see ref. 59). The semi-partial correlation is calculated by first fitting a linear regression of Y on X and computing the coefficient as: rY Xi∣Xi ð Þ = sign tð Þ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi t2 1  R2   n  k v u u t ð2Þ ð2Þ References 1. IPCC. Climate Change 2021, The Physical Science Basis. (Working Group I contribution to the Sixth Assessment Report of the Inter- governmental Panel on Climate Change, 2021). 1. IPCC. Climate Change 2021, The Physical Science Basis. (Working Group I contribution to the Sixth Assessment Report of the Inter- governmental Panel on Climate Change, 2021). 2. IPCC. Global Warming of 1.5 °C. An IPCC special report on the impacts of global warming of 1.5 °C above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate poverty. (WMO, UNEP, 2018). Pearson correlations The Pearson correlation is a measure of linear association between two variables. The coefficient can be obtained from bivariate data X1,Y 1   ,:::, Xn,Y n     as rXY = SXY=SXSY , where SXY and Si denote the sample covariance and standard deviation. The correlation coefficient is between −1 and 1. Correlations equal to 1 (or −1) indicate a perfect linear association, with data points lying exactly on a positive (nega- tive) line. A value equal to zero indicates the absence of any linear association. The squared correlation coefficient r2 xy is the coefficient of determination (R-squared) of the linear regression of variable x on variable y; it measures the fraction of the variance explained by the regression line. The first part of the term (3) is the in-sample squared error mini- mized by a classical least-squares approach. Lasso also includes the absolute sum of coefficients in the objective function, which penalizes complexity driving some of the estimated coefficients to zero. The value of λ is typically chosen so that the estimated model satisfies a predetermined condition. Several criteria can be employed, the most common of which is cross-validation. Cross-validation selects the λ to minimize the out-of-sample prediction error. First, sample observations are split into K random folds (validation sets). For each validation set, the model is fitted using data from the other folds, and the out-of-sample deviance for the observations in the validation set is computed (i.e., using data not employed for estimation). Finally, the overall out-of-sample performance of the model in all the validation sets is assessed by the mean-square prediction error (MSPE), a statis- tical parameter in squared (log) units required for model selection. Cross-validation selects the λ over a grid of possible values such that the corresponding model has the minimum MSPE71. In Table 2, MSPE is transformed into r2, i.e., the goodness of fit within the sample of countries, and oSr2, i.e., the cross-validated out-of-sample goodness of fit for the optimal models. If no variable is added or removed at λ*, this is reported in the left columns of Table 2 as Unchanged. Note that beyond λ*, more variables could be added but would not further improve the out-of-sample prediction. Definitions of spatial pattern indicators The spatial pattern indicators were derived from the built-up, road, and railway layers. Details on the definitions of the indicators (Table 1) are given in the SI in section 3, Supplementary Tables 6–8. 1 2N Y  XB ð Þ0 Y  XB ð Þ + λ XP j = 1∣Bj∣ ð3Þ ð3Þ Code availability h l l In (2) t is the t-statistic of variable X i in the previous regression, R2 is the R-squared, k is the number of independent variables plus the constant, and n is the sample size. Finally, the significance level is given by 2=Pr tnk > ∣t∣   , where Pr is the probability, t is as described above and tnk follows a Student’s t distribution with n  k degrees of free- dom. Further details on the correlation techniques used are available e.g. here70. The code used for calculations of maps is freely available here: https:// doi.org/10.5281/zenodo.5883652. The code used for calculations of maps is freely available here: https:// doi.org/10.5281/zenodo.5883652. Deriving the built-up land layer Please note that for spatial indicators that depend on AREF, the specific spatial pattern (shape) of IH is not relevant: we just use the national total area of IH instead of the area of the national territory as reference value. The potential usefulness of this IH layer for other research Nature Communications| (2023) 14:3898 7 Article https://doi.org/10.1038/s41467-023-39728-3 questions, in particular where its spatial accuracy is of high impor- tance, needs to be tested and is not in focus in this study. model with the maximum complexity. In general, the larger the λ, the smaller the number of non-zero coefficients. Consider a linear specification Y = B0 + B1X1 + . . . + BPXP + ϵ, where variables have been previously standardized to account for differences in scales. Lasso finds estimates for model coefficient B keeping the model sparse by minimizing the following term: Semi-partial correlations Suppose that Y is determined by X = X1,::::,Xk   . 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Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs
BMC Medical research methodology
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University of Wollongong University of Wollongong Research Online Research Online University of Wollongong University of Wollongong Research Online Research Online Faculty of Informatics - Papers (Archive) Faculty of Engineering and Information Sciences 2010 Investigation of relative risk estimates from studies of the same population Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs with contrasting response rates and designs Nicole M. Mealing University of Western Sydney Emily Banks Australian National University Louisa R. Jorm University of Western Sydney David G. Steel University of Wollongong, dsteel@uow.edu.au Mark S. Clements University of Western Sydney See next page for additional authors Follow this and additional works at: https://ro.uow.edu.au/infopapers Part of the Physical Sciences and Mathematics Commons Recommended Citation Recommended Citation Mealing, Nicole M.; Banks, Emily; Jorm, Louisa R.; Steel, David G.; Clements, Mark S.; and Rogers, Kris D.: Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs 2010, 26-38. https://ro.uow.edu.au/infopapers/1470 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Faculty of Engineering and Information Sciences Faculty of Informatics - Papers (Archive) y p ( ) 2010 Investigation of relative risk estim Investigation of relative risk estim with contrasting response rates with contrasting response rates Nicole M. Mealing University of Western Sydney Emily Banks Australian National University Louisa R. Jorm University of Western Sydney David G. Steel University of Wollongong, dsteel@uow.edu.au Mark S. Clements University of Western Sydney See next page for additional authors Follow this and additional works at: https://ro Part of the Physical Sciences and Mathem Recommended Citation Recommended Citation Mealing, Nicole M.; Banks, Emily; Jorm, Louis Investigation of relative risk estimates from s rates and designs 2010, 26-38. https://ro.uow.edu.au/infopapers/1470 Research Online is the open access institutional re contact the UOW Library: research-pubs@uow.edu Investigation of relative risk estimates from studies of the same population Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs with contrasting response rates and designs Follow this and additional works at: https://ro.uow.edu.au/infopapers Follow this and additional works at: https://ro.uow.edu.au/infopapers Part of the Physical Sciences and Mathematics Commons Disciplines Disciplines Physical Sciences and Mathematics Physical Sciences and Mathematics Recommended Citation Recommended Citation Mealing, Nicole M.; Banks, Emily; Jorm, Louisa R.; Steel, David G.; Clements, Mark S.; and Rogers, Kris D.: Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs 2010, 26-38. Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Abstract Abstract Background Background: There is little empirical evidence regarding the generalisability of relative risk estimates from studies which have relatively low response rates or are of limited representativeness. The aim of this study was to investigate variation in exposure-outcome relationships in studies of the same population with different response rates and designs by comparing estimates from the 45 and Up Study, a population-based cohort study (self-administered postal questionnaire, response rate 18%), and the New South Wales Population Health Survey (PHS) (computer-assisted telephone interview, response rate ~60%). Methods Methods: Logistic regression analysis of questionnaire data from 45 and Up Study participants (n = 101,812) and 2006/ 2007 PHS participants (n = 14,796) was used to calculate prevalence estimates and odds ratios (ORs) for comparable variables, adjusting for age, sex and remoteness. ORs were compared using Wald tests modelling each study separately, with and without sampling weights. Results Results: Prevalence of some outcomes (smoking, private health insurance, diabetes, hypertension, asthma) varied between the two studies. For highly comparable questionnaire items, exposure-outcome relationship patterns were almost identical between the studies and ORs for eight of the ten relationships examined did not differ significantly. For questionnaire items that were only moderately comparable, the nature of the observed relationships did not differ materially between the two studies, although many ORs differed significantly. Conclusions Conclusions: These findings show that for a broad range of risk factors, two studies of the same population with varying response rate, sampling frame and mode of questionnaire administration yielded consistent estimates of exposure-outcome relationships. However, ORs varied between the studies where they did not use identical questionnaire items. Publication Details Publication Details Mealing, N., Banks, E., Jorm, L., Steel, D., Clements, M. & Rogers, K. (2010). Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs. BMC Medical Research Methodology, 10 26-38. Keywords Keywords Investigation, relative, risk, estimates, from, studies, same, population, contrasting, response, rates, designs Investigation, relative, risk, estimates, from, studies, same, population, contrasting, response, rates, designs Disciplines Disciplines Physical Sciences and Mathematics BioMed Central © 2010 Mealing et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: There is little empirical evidence regarding the generalisability of relative risk estimates from studies which have relatively low response rates or are of limited representativeness. The aim of this study was to investigate variation in exposure-outcome relationships in studies of the same population with different response rates and designs by comparing estimates from the 45 and Up Study, a population-based cohort study (self-administered postal questionnaire, response rate 18%), and the New South Wales Population Health Survey (PHS) (computer-assisted telephone interview, response rate ~60%). Methods: Logistic regression analysis of questionnaire data from 45 and Up Study participants (n = 101,812) and 2006/ 2007 PHS participants (n = 14,796) was used to calculate prevalence estimates and odds ratios (ORs) for comparable variables, adjusting for age, sex and remoteness. ORs were compared using Wald tests modelling each study separately, with and without sampling weights. Results: Prevalence of some outcomes (smoking, private health insurance, diabetes, hypertension, asthma) varied between the two studies. For highly comparable questionnaire items, exposure-outcome relationship patterns were almost identical between the studies and ORs for eight of the ten relationships examined did not differ significantly. For questionnaire items that were only moderately comparable, the nature of the observed relationships did not differ materially between the two studies, although many ORs differed significantly. Conclusions: These findings show that for a broad range of risk factors, two studies of the same population with varying response rate, sampling frame and mode of questionnaire administration yielded consistent estimates of exposure-outcome relationships. However, ORs varied between the studies where they did not use identical questionnaire items. Authors Authors Nicole M. Mealing, Emily Banks, Louisa R. Jorm, David G. Steel, Mark S. Clements, and Kris D. Rogers Nicole M. Mealing, Emily Banks, Louisa R. Jorm, David G. Steel, Mark S. Clements, and Kris D. Rogers This journal article is available at Research Online: https://ro.uow.edu.au/infopapers/1470 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Open Access * Correspondence: Nicole.Mealing@saxinstitute.org.au 1 The Sax Institute, P.O. Box 123, Broadway NSW 2007, Australia Full list of author information is available at the end of the article Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 (male or female), remoteness (major city, inner regional, outer regional or remote) and age (five year age groups from 45-85 years or ≥85 years). have relatively high response rates within these groups, recent response rates to population-based cohort studies are usually below 50% [8-12]. Furthermore, cohort study participants are generally healthier and more health con- scious than non-participants [3,13-16]. Concern is often expressed at the low response rates for cohort studies, or the selectiveness of the group under study, and the gener- alisability of their results [17]. The 45 and Up Study The 45 and Up Study is a population-based cohort study of more than 260,000 men and women aged 45 years and over in New South Wales (NSW), Australia [10]. Partici- pants were randomly selected from the database that is used to administer the national universal health insur- ance scheme (Medicare Australia), which has almost complete coverage of the population. Equal numbers of males and females were selected for participants less than 80 years old. Individuals aged 80 years or over and resi- dents in rural areas were oversampled by a factor of two, males aged 80 years or over were oversampled compared to females and all residents in remote areas were com- pletely enumerated. Participants entered the study by completing a baseline postal questionnaire and providing written consent to have their health followed over time. The study questionnaire is available at http:// www.45andUp.org.au. The survey was available only in English. The current overall response rate to the baseline questionnaire is estimated to be 17.9% [10]. The final ana- lytic sample consisted of 44,851 men and 52,961 women joining the study up to July 2008 after excluding 125 respondents who had a missing Accessibility Remoteness Index of Australia (ARIA+) [19] score. The NSW Population Health Survey The NSW Population Health Survey (PHS) is an ongoing survey on the health of people in NSW using computer assisted telephone interviewing [21]. Independent sam- ples of NSW households with private telephones are drawn each year using random digit dialling, and one per- son is randomly selected to participate in the survey. Informed consent was obtained from participants by their willingness to complete the telephone interview. The survey questionnaire is available at http:// www.health.nsw.gov.au/publichealth/surveys/phs.asp. The survey is administered in 6 languages. In 2006 partic- ipants were asked all survey questions and in 2007 they were asked a random subset of the survey questions. We report analyses of data for 5,766 men and 9,030 women aged 45 years or over who responded to the 2006 (n = 5,480) or 2007 (n = 9,316) PHS, with response rates of 59.3% [22] and 63.6% [23] respectively. Direct empirical data to support the assumption that internal comparisons remain reliable, despite low response rates or highly selected study groups, is lacking. Furthermore, concerns are also expressed that elements of study design, such as sampling methods and use of postal questionnaires versus interviews, may influence the observed relationships [18]. This paper investigates whether or not cross-sectional estimates of exposure- outcome relationships are affected by survey aspects (response rate, sampling frame and mode of question- naire administration) or the wording of questionnaire items, by comparing estimates computed from two inde- pendent studies of the same target population with diver- gent response rates and different designs. Weights were assigned to each year of data to adjust for the differences in the probability of selection within the household, number of residential telephone connections to the house and the varying sampling fraction between each of the 8 NSW area health services to provide esti- mates that were representative of the NSW population [21]. These area health services can include several remoteness categories. Post-stratification weights were also assigned according to sex (male or female) and age (five year age groups from 45-85 years or 85-110 years) using 2005 and 2007 mid-year population statistics released by the ABS for each area health service [22,23]. After weighting, Indigenous people are slightly under- represented in the PHS sample, and Australian-born peo- ple slightly over-represented, compared to the overall NSW population [22,23]. Background sarily required for reliable estimates of relative risk based on internal comparisons within study populations [3-5]. Indeed, having a greater proportion of respondents in extreme categories compared to the population of inter- est may often be necessary, in order to yield sufficient information about specific exposure-outcome relation- ships [5]. A key issue is whether there is any nonresponse bias after conditioning on the covariates included in the analysis. The aim of most epidemiological studies is to obtain esti- mates that can be generalised to a population of interest. For surveys concerned with disease prevalence, the main means to achieve this is to draw a sample that is suffi- ciently representative of the target population. However, few surveys have perfect response rates and any level of nonresponse can potentially lead to biased estimates of prevalence [1,2]. Cohort studies generally require more extensive data collection than one-off surveys, as well as the provision of identifying details and a long-term commitment to fol- low-up. While cohort studies often focus on selected population groups (e.g. occupational groups) [6,7] and In contrast, much epidemiological practice is based around the principle that representativeness is not neces- * Correspondence: Nicole.Mealing@saxinstitute.org.au 1 The Sax Institute, P.O. Box 123, Broadway NSW 2007, Australia Full list of author information is available at the end of the article Page 2 of 12 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Analysis Before analyses commenced, twenty exposure-outcome pairs were selected for inclusion in our analyses. These were selected on the basis of demonstrating relationships across a wide range of domains of research interest. This consisted of i) ten pairs where both the exposure and the outcome variables were highly comparable across the two studies; and ii) ten pairs where the exposure and/or out- come variables were only moderately comparable across the two studies. Participants were classified as having hypertension, diabetes and/or asthma if they reported that these condi- tions had ever been diagnosed by a doctor (both studies) or at a hospital (PHS only). Only participants who answered version two of the 45 and Up baseline question- naire were asked if they had ever been diagnosed with asthma (n = 65,522). Unweighted and survey weighted prevalence estimates with 95% confidence intervals (CI) were calculated for each study for all highly and moderately comparable vari- ables used in the exposure-outcome relationship analy- ses. Odds ratios (ORs) were used to approximate relative risks and logistic regression analyses were used to calcu- late the 20 pre-determined exposure-outcome relation- ships for each study; separated into highly and moderately comparable ORs. In each case two sets of ORs were calculated; namely the crude OR and the OR adjusting for age, sex and remoteness since these were the sampling variables common to both surveys. Unweighted and weighted comparisons of these two types of ORs by study are presented in Additional files 2 and 3 respec- tively (45 and Up Study) and Additional files 4 and 5 respectively (PHS). Indicator variables were constructed for being born in Australia, missing all natural teeth, speaking a language other than English at home, having fallen in the past 12 months, having private health insurance (excluding Medicare) and having a hysterectomy. Daily fruit consumption was grouped into participants who don't eat fruit, participants who eat fruit but less than two serves per day, and participants who eat two or more serves per day. An indicator for females who were breast screened in the past two years was ascertained from responses to ever having a mammogram and the year of (45 and Up Study) or time interval (PHS) since their last mammogram. Psychological distress was evaluated using the Kessler (K10) measure [26] ascertained as the sum of responses for 10 questions. Moderately comparable questionnaire items The wording of questions across the two studies differed for household income before tax (45 and Up Study included benefits, pensions and superannuation), bowel screening (screening tests varied by study) and current smoking status (45 and Up Study participants recorded whether they were 'regular smokers' currently without a definition for regular, whereas PHS participants recorded if they 'smoke daily' where smoking was defined to include cigarettes, cigars and pipes). Questions on mammography screening and hysterec- tomy were only asked in the 2006 PHS and hypertension and bowel screening in the 2007 PHS. Highly comparable questionnaire items The response categories varied across the two studies for highest level of educational attainment (for these analyses similar categories were constructed) and self- rated health status (the PHS had an additional response category - for these analyses the categories "poor" and "very poor" on the PHS were combined). Remoteness was determined using the mean ARIA+ score for the postcode of the participant's residential address and categorised as major city, inner regional, outer regional or remote, according to the Australian Institute of Health and Welfare [24]. Self reported height and weight were used to calculate participants' body mass index (BMI) as weight in kilo- grams divided by the square of height in meters. BMI was categorised as underweight (BMI < 18.5 kg/m2), normal range (BMI 18.5-24.9 kg/m2), overweight (BMI 25.0-29.9 kg/m2) or obese (BMI ≥ 30 kg/m2) according to the World Health Organisation [25]. Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Page 3 of 12 those with a score less than 22 as having low/moderate levels of psychological distress [27]. of participants a question of interest then the same restriction was applied to the other study. Data are reported on falls in the past 12 months for participants aged 60 years and over, hysterectomy operation in females less than 70 years, mammography screening in the past two years for females less than 80 years and bowel screening in the past 5 years for all persons aged 50 years and over. Questionnaire items We obtained the original questionnaires from the 45 and Up Study and the 2006 and 2007 PHS and compared the wording of questions and response categories. We classi- fied questionnaire items as highly comparable, moder- ately comparable or not comparable, based on whether the item was expected to yield identical, similar or non- comparable responses, respectively, for a given individ- ual. Analyses focused on items considered highly or mod- erately comparable; items used in these analyses are compared in Additional file 1. All variables used in these analyses were derived from self-reported data except postcode (45 and Up Study only). Post-stratification estimation weights were assigned to the 45 and Up baseline survey to adjust the sample to account for the differences in selection probabilities and response rates and give consistency with 2006 population estimates produced by the Australian Bureau of Statistics (ABS) [20]. The post-strata were formed according to sex All analyses included all participants in both studies, unless otherwise stated. If one study only asked a sub-set Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Analysis If a respondent answered nine of the 10 items then the missing item was imputed as the average of the other nine responses. If a respondent answered less than nine items their K10 score was set to missing. Partic- ipants with a K10 score of 22 or greater were assigned as having high/very high levels of psychological distress and In the figures, the squares and lines represent each OR estimate and CI, with the area of each square being pro- portional to the sample size used for each estimate. Wald chi-square statistics were computed to test for differences in the log odds ratios between the two surveys for each of the 20 exposure-outcome pairs and compared to a chi-square distribution with degrees of freedom equal to the number of categories minus one in the expo- Page 4 of 12 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 sure variable. Each study was modelled separately and then the Wald statistics were calculated by combining the two sets of estimated parameters, variances and covari- ances. Analyses were conducted with and without using sampling weights. With survey data the Wald test can be unreliable if the degrees of freedom on the estimated covariance matrix are small [28]. In this case the samples were large and the designs relatively simple. distress was observed (Wald test P = 0.02). Similar obser- vations were seen when the ORs from these ten relation- ships were calculated using sampling weights (Additional file 6). The ten exposure-outcome relationships where the exposure and/or outcome variables were only moderately comparable across the two studies are presented in Figure 2, with ORs adjusted for age, sex and remoteness. Each exposure-outcome pair had a similar relationship pattern for both studies and all OR estimates were in the same direction and of similar magnitude, except when self- rated health status was the exposure variable. The rela- tionships did not differ significantly for 4 out of 10 of the exposure-outcome associations. Significant but relatively minor differences in ORs were observed for smoking and educational attainment and pre-tax income in relation to psychological distress, private health insurance and remoteness of residence. Results The distributions of social and demographic characteris- tics and of health risk factors and conditions in the two studies are shown in Tables 1 and 2, respectively. Younger persons and/or those living in major cities were under- represented in both surveys as were males in the PHS (the 45 and Up Study sample was stratified by sex). The prevalence confidence intervals were narrower on the 45 and Up Study compared to the PHS, because of the larger sample size. The weighted estimates of prevalence were similar across the two studies for variables such as age, sex, and remoteness (the variables used for weighting), country of birth, educational attainment, fruit consumption, body- mass-index and falls. However, the prevalence of speak- ing a language other than English at home and of holding private health insurance was higher in the 45 and Up Study compared to the PHS, while the prevalence of smoking, high/very high psychological distress, ever diagnosed with hypertension, ever diagnosed with diabe- tes and ever diagnosed with asthma was lower (Table 1; Table 2). The PHS tended to have less missing data than the 45 and Up Study, particularly for variables relating to mammography screening, K10 score and household income before tax. Prevalence estimates for self-rated health status varied across the two studies with the pro- portion who reported the lowest category of self-rated health status on the 45 and Up baseline questionnaire (i.e. "poor") being similar to the proportion who reported the lowest category on the PHS (i.e. "very poor")(Table 2). Following adjustment for age, sex and remoteness, additional weighting of the OR for age, sex and remote- ness did not change any of the ORs from the 45 and Up Study materially (i.e. no changes were >10%) (Additional files 2, 3). This is because the weighting is determined by the variables used in the logistic regression. Weighting the PHS resulted in some changes to the ORs because not all variables used to determine the weighting (i.e. house- hold size to account for the selection of a person from each selected household and the 8 area health services) were used in the logistic regression (Additional files 4, 5). Weighting did not change the general nature of the observed relationships. Analysis In spite of the similarity in the shape of the relationship, substantial heterogeneity and large differences in ORs were observed for relationships with self-rated health (where the PHS had an additional response category, "very poor"). Similar observations were seen when the ORs from these ten relationships were calculated using sampling weights (Additional file 7). Analyses were carried out using SAS, version 9 [29]. This study has the approval of the University of New South Wales Ethics Committee and the NSW Population and Health Services Research Ethics Committee. Discussion Discussions around epidemiological methods often con- clude that representativeness is not necessarily required for reliable estimates of relative risk based on internal comparisons within study populations [4]. By their nature, cohort studies tend not to be directly representa- tive of the general population, however over time, their results have usually been shown to be both reproducible and generalisable to the larger population [6,7]. Miettinen explains that "an empirical relation is not distorted by any manipulation of the distribution of the study base accord- ing to the elements in the occurrence relation - the deter- minant, the modifiers and/or confounders. For example, the empirical relation of body weight to gender does not The ten exposure-outcome relationships where both the exposure and the outcome variables were highly com- parable across the two studies are presented in Figure 1, with ORs adjusted for age, sex and remoteness. The observed relationships were virtually identical between the two studies. For 8 out of the 10 relationships there was no significant difference between the results from the different studies. There was borderline evidence of a dif- ference in the risk of falling according to BMI across the two studies (Wald test P = 0.04) and minor heterogeneity in the relationship of age to high/very high psychological Mealing et al. depend on the gender distribution in the study base." [[5], p. 56] depend on the gender distribution in the study base." [[5], p. 56] pleted the survey in languages other than English. It was not possible to definitively separate the individual effects of sampling frame, response rate and mode of adminis- tration, since response rates and aspects of study design are closely linked [33]. It is generally accepted that in order to produce results that are generalisable, studies should exhibit sufficient variability in the determinant and modifiers to be studied and a limited range for confounders [4,5]. Nevertheless the possibility of bias cannot be excluded, and empirical data on how exposure-outcome relationships might vary according to the degree of nonresponse are lacking. We were unable to locate other empirical comparisons of relative risk estimates in independent studies with divergent response rates and different study designs that were drawn from the same target population. Indirect evidence supporting our findings comes from studies that have observed consistent ORs in study respondents and non-respondents using linked data [34] and in initial cohort study participants and participants responding to a subsequent questionnaire [35]. Two studies found only small biases in relative risk estimates due to nonresponse, in cross-sectional ORs from a cohort study relating to cardiovascular disease [31], and in cohort analyses relat- ing to reproductive outcomes [12]. One study found con- sistent ORs related to smoking in respondents recruited by postal survey and those recruited through postal and telephone surveys and home visits [18]. Nonresponse is a form of self-selection. Selection solely by the exposure or outcome variable does not bias the estimates of ORs in logistic regression [14,30,31] and selection solely on the basis of covariates in the logistic regression also leads to unbiased ORs. Although evidence from simulation supports the principle of generalisability [12], specific scenarios may result in significant bias if selection criteria and dependent variables are closely related [32]. In particular, biases can occur if selection depends on both the exposure and outcome [14,17]. We found that although some prevalence estimates var- ied between the two studies of the same population inves- tigated here, exposure-outcome relationships did not differ materially, where the variables used were highly comparable. Discussion BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Page 5 of 12 Table 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS populations 45 and Up Study PHS Social and Demographics Characteristics a N Crude % (95% CI) Weighted b % (95% CI) N Crude % (95% CI) Weighted c % (95% CI) Sex Male 48851 48.0 (47.7, 48.3) 48.0 (47.6, 48.4) 5766 39.0 (38.2, 39.8) 48.4 (47.4, 49.5) Female 52961 52.0 (51.7, 52.3) 52.0 (51.6, 52.4) 9030 61.0 (60.2, 61.8) 51.6 (50.5, 52.6) Age 45-49 years 12399 12.2 (12.0, 12.4) 18.9 (18.6, 19.2) 1803 12.2 (11.7, 12.7) 19.3 (18.3, 20.3) 50-54 years 16120 15.8 (15.6, 16.1) 17.1 (16.8, 17.4) 2043 13.8 (13.3, 14.4) 17.5 (16.6, 18.4) 55-59 years 17350 17.0 (16.8, 17.3) 15.9 (15.7, 16.2) 2188 14.8 (14.2, 15.4) 16.2 (15.4, 17.0) 60-64 years 15342 15.1 (14.8, 15.3) 12.6 (12.4, 12.8) 2252 15.2 (14.6, 15.8) 12.5 (11.9, 13.1) 65-69 years 12800 12.6 (12.4, 12.8) 10.1 (9.9, 10.3) 1939 13.1 (12.6, 13.6) 10.2 (9.6, 10.8) 70-74 years 9564 9.4 (9.2, 9.6) 8.3 (8.1, 8.5) 1710 11.6 (11.0, 12.1) 8.5 (8.0, 8.9) 75-79 years 7235 7.1 (6.9, 7.3) 7.3 (7.2, 7.5) 1490 10.1 (9.6, 10.6) 7.3 (6.9, 7.8) 80-84 years 7545 7.4 (7.2, 7.6) 5.4 (5.3, 5.6) 922 6.2 (5.8, 6.6) 5.9 (5.4, 6.3) ≥ 85 years 3457 3.4 (3.3, 3.5) 4.3 (4.1, 4.4) 449 3.0 (2.8, 3.3) 2.6 (2.3, 2.9) Remoteness (ARIA+) Major City 44146 43.4 (43.1, 43.7) 69.1 (68.8, 69.4) 6718 45.4 (44.6, 46.2) 60.6 (59.6, 61.6) Inner Regional 36640 36.0 (35.7, 36.3) 22.7 (22.5, 23.0) 4351 29.4 (28.7, 30.1) 24.4 (23.5, 25.2) Outer Regional 18926 18.6 (18.4, 18.8) 7.6 (7.5, 7.7) 3086 20.9 (20.2, 21.5) 12.2 (11.6, 12.7) Remote 2100 2.1 (2.0, 2.1) 0.6 (0.5, 0.6) 456 3.1 (2.8, 3.4) 1.3 (1.2, 1.5) Missing 0 - - 185 1.3 (1.1, 1.4) 1.5 (1.2, 1.8) Language other than English spoken at home No 92230 90.6 (90.4, 90.8) 87.4 (87.1, 87.7) 13379 93.0 (92.6, 93.4) 89.5 (88.7, 90.3) Yes 9580 9.4 (9.2, 9.6) 12.6 (12.3, 12.9) 986 6.9 (6.4, 7.3) 10.4 (9.6, 11.2) Missing 2 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 17 0.1 (0.0, 0.2) 0.1 (0.0, 0.2) Country of Birth Australia 75821 74.5 (74.2, 74.7) 70.8 (70.5, 71.1) 11332 76.6 (75.9, 77.3) 71.1 (70.1, 72.2) Not Australia 24964 24.5 (24.3, 24.8) 28.2 (27.9, 28.6) 3433 23.2 (22.5, 23.9) 28.7 (27.7, 29.7) Missing 1027 1.0 (0.9, 1.1) 1.0 (0.9, 1.0) 24 0.2 (0.1, 0.2) 0.2 (0.1, 0.2) Private Health Insurance No 38300 37.6 (37.3, 37.9) 33.9 (33.6, 34.3) 6766 45.7 (44.9, 46.5) 41.5 (40.4, 42.5) Yes 63508 62.4 (62.1, 62.7) 66.1 (65.7, 66.4) 7973 53.9 (53.1, 54.7) 58.2 (57.1, 59.3) Missing 4 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 57 0.4 (0.3, 0.5) 0.3 (0.2, 0.4) ble 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS populations Mealing et al. Discussion BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Page 6 of 12 Educational Attainment No School Certificate 12385 12.2 (12.0, 12.4) 10.9 (10.7, 11.1) 2017 13.7 (13.1, 14.2) 11.7 (11.1, 12.3) School Certificate 22608 22.2 (22.0, 22.5) 20.3 (20.0, 20.6) 3732 25.3 (24.6, 26.0) 20.6 (19.8, 21.4) Trade/Certificate/Diploma 32289 31.7 (31.4, 32.0) 31.0 (30.6, 31.3) 1631 26.5 (25.8, 27.2) 29.5 (28.5, 30.5) Higher School Certificate 9787 9.6 (9.4, 9.8) 10.1 (9.9, 10.3) 3902 11.1 (10.6, 11.6) 10.9 (10.2, 11.6) Tertiary Qualification 22802 22.4 (22.1, 22.7) 25.9 (25.6, 26.3) 3096 21.0 (20.3, 21.7) 25.0 (24.0, 26.0) Missing 1941 1.9 (1.8, 2.0) 1.8 (1.8, 1.9) 360 2.4 (2.2, 2.7) 2.3 (2.0, 2.6) Household Income <$20,000 p.a 20633 20.3 (20.0, 20.5) 17.9 (17.6, 18.1) 4505 30.4 (29.7, 31.2) 22.2 (21.4, 22.9) $20,000-$39,999 p.a 18386 18.1 (17.8, 18.3) 15.3 (15.1, 15.6) 3145 21.3 (20.6, 21.9) 20.3 (19.5, 21.1) ≥$40,000 p.a 39792 39.1 (38.8, 39.4) 44.6 (44.2, 44.9) 4768 32.2 (31.5, 33.0) 41.4 (40.3, 42.5) Missing 23001 22.6 (22.3, 22.8) 22.2 (21.9, 22.5) 2378 16.1 (15.5, 16.7) 16.2 (15.4, 17.0) ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; p.a, per annum a Characteristics above the line-break are highly comparable across the two surveys. Characteristics below the line-break are moderately comparable across the two surveys. Weighted prevalence estimates for age, sex and remoteness differ across the two studies since the weighting schemes vary. b Weighted by age, sex and remoteness c Weighted by area health service, probability of selection in the household and the number of residential connections to the house Table 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS populations (Continued) nd demographic characteristics of the 45 and Up Study and the NSW PHS populations (Continued) Table 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS popula depend on the gender distribution in the study base." [[5], p. 56] This was despite major differences between the studies, including varying response rates, sampling frames and modes of administration; the PHS had a smaller proportion of missing and invalid responses due to the nature of the computer assisted telephone inter- viewing system and it included respondents who com- Having established the lack of any major differences attributable to response rate and study design (including sampling frame and mode of questionnaire administra- tion), the comparison of exposure-outcome relationships containing moderately comparable variables across the two studies can be seen as illustrating the additional effect of the specific questionnaire items used. Our find- ings demonstrate that an apparently minor difference in Mealing et al. depend on the gender distribution in the study base." [[5], p. 56] BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Page 8 of 12 Last Mammography Screening Not within past 2 years 12622 26.2 (25.9, 26.6) 28.6 (28.1, 29.1) 1018 34.4 (32.7, 36.1) 36.7 (34.5, 39.0) Within past 2 years 30056 62.5 (62.1, 62.9) 60.1 (59.6, 60.6) 1925 65.1 (63.4, 66.8) 62.8 (60.6, 65.1) Missing 5413 11.3 (11.0, 11.5) 11.3 (11.0, 11.7) 15 0.5 (0.3, 0.8) 0.4 (0.2, 0.7) Hysterectomy No 29487 73.9 (73.4, 74.3) 76.6 (76.1, 77.0) 1583 71.1 (69.2, 73.0) 74.2 (72.0, 76.4) Yes 10431 26.1 (25.7, 26.6) 23.4 (23.0, 23.9) 630 28.3 (26.4, 30.2) 25.0 (22.8, 27.2) Missing 0 - - 13 0.6 (0.3, 0.9) 0.8 (0.3, 1.3) Psychological Distress Low\Moderate 81542 80.1 (79.8, 80.3) 80.7 (80.4, 81.0) 9503 88.8 (88.2, 89.4) 88.9 (88.1, 89.7) High\Very high 6663 6.5 (6.4, 6.7) 6.8 (6.6, 7.0) 1101 10.3 (9.7, 10.9) 10.2 (9.4, 11.0) Missing 13607 13.4 (13.2, 13.6) 12.5 (12.2, 12.7) 100 0.9 (0.8, 1.1) 0.9 (0.7, 1.2) Current Smoker No 93685 92.0 (91.9, 92.2) 91.9 (91.7, 92.1) 9468 87.9 (87.3, 88.6) 88.1 (87.3, 89.0) Yes 7575 7.4 (7.3, 7.6) 7.6 (7.4, 7.8) 1292 12.0 (11.4, 12.6) 11.8 (11.0, 12.7) Missing 552 0.5 (0.5, 0.6) 0.5 (0.5, 0.6) 7 0.1 (0.0, 0.1) 0.1 (0.0, 0.1) Last Bowel Screening Not within past 5 years 49036 54.8 (54.5, 55.2) 55.9 (55.6, 56.3) 2497 53.5 (52.1, 54.9) 54.6 (52.7, 56.4) Within past 5 years 35990 40.3 (39.9, 40.6) 39.2 (38.8, 39.6) 2098 45.0 (43.5, 46.4) 43.3 (41.5, 56.4) Missing 4387 4.9 (4.8, 5.0) 4.9 (4.7, 5.0) 72 1.5 (1.2, 1.9) 2.2 (1.4, 2.9) Self-reported Health Status Excellent 14920 14.7 (14.4, 14.9) 15.5 (15.2, 15.8) 2433 17.8 (17.2, 18.5) 18.1 (17.2, 18.9) Very Good 36046 35.4 (35.1, 35.7) 35.4 (35.0, 35.7) 3815 28.0 (27.2, 28.7) 28.5 (27.5, 29.5) Good 33178 32.6 (32.3, 32.9) 32.1 (31.7, 32.4) 3990 29.2 (28.5, 30.0) 29.8 (28.7, 30.8) Fair 11878 11.7 (11.5, 11.9) 11.4 (11.2, 11.6) 2265 16.6 (16.0, 17.2) 16.0 (15.2, 16.8) Poor 2090 2.1 (2.0, 2.1) 2.0 (1.9, 2.1) 863 6.3 (5.9, 6.7) 5.8 (5.3, 6.3) Very Poor d N/A 239 1.8 (1.5, 2.0) 1.6 (1.3, 1.9) Missing 3700 3.6 (3.5, 3.7) 3.7 (3.5, 3.8) 44 0.3 (0.2, 0.4) 0.3 (0.2, 0.4) CI, Confidence Interval; NSW, New South Wales; a Variables above the line-break are highly comparable across the two surveys. depend on the gender distribution in the study base." [[5], p. 56] BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Page 7 of 12 Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS populations 45 and Up Study PHS Variable a N Crude % (95% CI) Weighted b % (95% CI) N Crude % (95% CI) Weighted c % (95% CI) Fruit Consumption Don't eat fruit 6620 6.5 (6.4, 6.7) 6.5 (6.4, 6.7) 321 6.1 (5.5, 6.8) 6.1 (5.2, 6.9) < 2 serves per day 31707 31.1 (30.9, 31.4) 31.0 (30.7, 31.4) 1719 32.8 (31.6, 34.1) 33.4 (31.8, 35.1) ≥ 2 serves per day 57620 56.6 (56.3, 56.9) 56.7 (56.3, 57.0) 3135 59.9 (58.5, 61.2) 59.4 (57.7, 61.2) Missing 5865 5.8 (5.6, 5.9) 5.7 (5.6, 5.9) 61 1.2 (0.9, 1.5) 1.0 (0.7, 1.3) Teeth Some/all natural teeth 88501 86.9 (86.7, 87.1) 88.0 (87.8, 88.2) 9321 86.7 (86.0, 87.3) 90.3 (89.7, 90.9) No natural teeth 9953 9.8 (9.6, 10.0) 8.7 (8.5, 8.9) 1427 13.3 (12.6, 13.9) 9.7 (9.1, 10.3) Missing 3358 3.3 (3.2, 3.4) 3.3 (3.2, 3.5) 6 0.1 (0.0, 0.1) 0.0 (0.0, 0.1) Body Mass Index Underweight 1461 1.4 (1.4, 1.5) 1.5 (1.4, 1.6) 230 2.1 (1.9, 2.4) 1.9 (1.6, 2.3) Normal Range 35219 34.6 (34.3, 34.9) 35.6 (35.2, 35.9) 4214 39.0 (38.1, 40.0) 38.4 (37.2, 39.6) Overweight 37373 36.7 (36.4, 37.0) 36.6 (36.2, 36.9) 3774 35.0 (34.1, 35.9) 36.0 (34.8, 37.2) Obese 20271 19.9 (19.7, 20.2) 19.2 (18.9, 19.5) 2205 20.4 (19.7, 21.2) 20.5 (19.4, 21.5) Missing 7488 7.4 (7.2, 7.5) 7.1 (6.9, 7.3) 373 3.5 (3.1, 3.8) 3.2 (2.8, 3.7) Ever diagnosed with hypertension No 72023 70.7 (70.5, 71.0) 72.1 (71.7, 72.4) 1189 52.1 (50.1, 54.2) 55.2 (52.6, 57.8) Yes 25144 24.7 (24.4, 25.0) 22.9 (22.6, 23.2) 1086 47.6 (45.6, 49.7) 44.6 (42.0, 47.2) Missing 4645 4.6 (4.4, 4.7) 5.0 (4.9, 5.2) 6 0.3 (0.1, 0.5) 0.2 (0.0, 0.4) Ever diagnosed with diabetes No 88056 86.5 (86.3, 86.7) 86.6 (86.3, 86.8) 9450 88.4 (87.8, 89.0) 89.2 (88.4, 90.0) Yes 9111 8.9 (8.8, 9.1) 8.4 (8.2, 8.6) 1202 11.2 (10.6, 11.8) 10.5 (9.7, 11.2) Missing 4645 4.6 (4.4, 4.7) 5.0 (4.9, 5.2) 36 0.3 (0.2, 0.4) 0.3 (0.2, 0.4) Ever diagnosed with asthma No 55034 84.0 (83.7, 84.3) 83.8 (83.5, 84.1) 8753 82.0 (81.3, 82.8) 83.2 (82.2, 84.1) Yes 7653 11.7 (11.4, 11.9) 11.4 (11.1, 11.7) 1894 17.8 (17.0, 18.5) 16.7 (15.8, 17.6) Missing 2835 4.3 (4.2, 4.5) 4.8 (4.6, 5.0) 22 0.2 (0.1, 0.3) 0.1 (0.1, 0.2) Fallen in the past 12 months No 42038 75.1 (74.8, 75.5) 74.1 (73.7, 74.6) 2957 77.3 (76.0, 78.7) 77.5 (75.9, 79.1) Yes 10757 19.2 (18.9, 19.6) 20.0 (19.6, 20.4) 856 22.4 (21.1, 23.7) 22.1 (20.6, 23.7) Missing 49017 5.6 (5.4, 5.8) 5.8 (5.6, 6.1) 11 0.3 (0.1, 0.5) 0.4 (0.1, 0.6) Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS populations Mealing et al. b Weighted by age, sex and remoteness c Weighted by area health service, probability of selection in the household and the number of residential connections to the house d The category 'Very Poor' was only available on the NSW Population Health Survey bove the line-break are highly comparable across the two surveys. Variables below the line-break are moderately compara ss the two surveys b d depend on the gender distribution in the study base." [[5], p. 56] Variables below the line-break are moderately comparable but similar across the two surveys b Weighted by age, sex and remoteness c W i ht d b h lth i b bilit f l ti i th h h ld d th b f id ti l ti t th h Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS populations (Continued) lth risk factors and conditions of the 45 and Up Study and the NSW PHS populations (Continued) Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS popu Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Page 9 of 12 Figure 1 Odds ratios a by study where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS b. ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; PHS, Population Health Survey. a Ad- justing for age, sex and remoteness.b Black squares represent ORs with area inversely proportional to the sample size contributing to the OR and the corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for a difference between the two studies for the specific exposure-outcome pair and outcome variables were highly comparable across the 45 and Up Study and the Australia; CI, Confidence Interval; NSW, New South Wales; PHS, Population Health Survey. a Ad- esent ORs with area inversely proportional to the sample size contributing to the OR and the rval. c P-value from Wald chi-square test: testing for a difference between the two studies for Figure 1 Odds ratios a by study where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS b. ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; PHS, Population Health Survey. a Ad- justing for age, sex and remoteness.b Black squares represent ORs with area inversely proportional to the sample size contributing to the OR and the corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for a difference between the two studies for the specific exposure-outcome pair the wording of questions can significantly influence mea- sures of prevalence and estimates of risks. depend on the gender distribution in the study base." [[5], p. 56] This empha- sizes the critical importance of maintaining the consistency of survey questions if valid comparisons are to be made and is consistent with previous studies [36- 38]. Although most differences attributable to question wording resulted in minor heterogeneity, highly signifi- cant heterogeneity was evident for the question on self- rated health status, where the response categories varied across the two studies. However, despite differences between questionnaire items, the observed ORs would lead to similar conclusions regarding the nature of the exposure-outcome relationships. bias. Under ideal circumstances we could use census data; however the Australian Census includes only very limited health data. Additionally, these findings relate to two large studies with considerable variability in the fac- tors included in our analyses. This ensured that there were substantial numbers of participants from each study in each exposure-outcome category, and allowed for adjustment for multiple factors. Although these findings support the principle of generalisability of findings from a relatively select group of participants, it remains possible that they are less applicable to smaller, less heterogeneous studies. These findings relate to cross-sectional analyses; prospective, longitudinal analyses are less prone to the potential biases investigated here, since baseline selection cannot be influenced by outcome status. One shortcoming of our study is the lack of strict gold- standard measures for the study variables. The PHS has a 40% nonresponse rate and may be subject to nonresponse Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 Page 10 of 12 Figure 2 Odds Ratios a by study where either the exposure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS b. ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; p.a, per an- num; PHS, Population Health Survey. a Adjusting for age, sex and remoteness. b Black squares represent ORs with area inversely proportional to the sample size contributing to the OR and the corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for a difference between the two studies for the specific exposure-outcome pair Figure 2 Odds Ratios a by study where either the exposure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS b. depend on the gender distribution in the study base." [[5], p. 56] ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; p.a, per an- num; PHS, Population Health Survey. a Adjusting for age, sex and remoteness. b Black squares represent ORs with area inversely proportional to the sample size contributing to the OR and the corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for a difference between the two studies for the specific exposure-outcome pair Applying weights to survey data to calculate prevalence estimates that account for the differences in probability of selection is standard practice. However, use of sampling weights is less common when calculating relative risks from cohort study data; instead adjustments are usually made to account for potential confounders. The relative risk estimates adjusting for age, sex and remoteness from the 45 and Up Study were not altered materially by fur- ther weighting. Hence weighting did not appear to be necessary when the variables used in calculating the weights were used as covariates in the analysis. Weighting is potentially important in the PHS because of the role of household size and area health service in the weighting. Conclusions These findings show that broad ranges of exposure-out- come relationships estimated from two studies of the same population remained consistent regardless of the underlying response rate or mode of questionnaire administration. They provide empirical support for the basic epidemiological principle that results based on internal comparisons remain generalisable even when study subjects are drawn from a relatively select group. They emphasize the crucial importance of maintaining the consistency of question wording in order to permit comparisons between studies. Page 11 of 12 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 The authors declare that they have no competing interests. , ( ) 12. Nohr EA, Frydenberg M, Henriksen TB, Olsen J: Does low participation in cohort studies induce bias? Epidemiology 2006, 17(4):413-8. cohort studies induce bias? Epidemiology 2006, 17(4):413-8 Authors' contributions 13. Eastwood BJ, Gregor RD, MacLean DR, Wolf HK: Effects of recruitment strategy on response rates and risk factor profile in two cardiovascular surveys. Int J Epidemiol 1996, 25(4):763-9. NM, EB, LJ and KR participated in the study concept and design and were involved in the acquisition of data. NM and EB carried out the literature review and drafted the manuscript. All authors were involved in the analysis and inter- pretation of data. NM performed the statistical analysis and DS, MC and KR pro- vided statistical advice. All authors critically revised the manuscript for important intellectual content. EB and LJ obtained funding for this study and provided study supervision. All authors read and approved the final manu- script. 14. Criqui MH: Response bias and risk ratios in epidemiologic studies. Am J Epidemiol 1979, 109(4):394-9. 15. Melton LJ, Dyck PJ, Karnes JL, O'Brien PC, Service FJ: Nonresponse bias in studies of diabetic complications - the Rochester Diabetic Neuropathy Study. Journal of Clinical Epidemiology 1993, 46(4):341-8. 16. Heilbrun LK, Nomura A, Stemmermann GN: The effects of nonresponse in a prospective-study of cancer - 15-year follow-up. Int J Epidemiol 1991, 20(2):328-38. References Additional file 5 Ten crude and adjusted ORs from the NSW PHS (unweighted and weighted), where either the exposure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS. Additional file 5 Ten crude and adjusted ORs from the NSW PHS (unweighted and weighted), where either the exposure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS. 8. Lee C, Dobson AJ, Brown WJ, Bryson L, Byles J, Warner-Smith P, Young AF: 8. Lee C, Dobson AJ, Brown WJ, Bryson L, Byles J, Warner-Smith P, Young AF: Cohort profile: the Australian Longitudinal Study on Women's Health. Int J Epidemiol 2005, 34(5):987-91. Additional file 6 Weighted odds ratios by study where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS. Additional file 6 Weighted odds ratios by study where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS. Additional file 6 Weighted odds ratios by study where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS. 9. Day N, Oakes S, Luben R, Khaw KT, Bingham S, Welch A, Wareham N: EPIC- Norfolk: study design and characteristics of the cohort. European prospective investigation of cancer. Br J Cancer 1999, 80(Suppl 1):95-103. Additional file 7 Weighted odds Ratios by study where either the expo- sure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS. Additional file 7 Weighted odds Ratios by study where either the expo- sure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS. Additional file 7 Weighted odds Ratios by study where either the expo- sure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS. 10. 45 and Up Study Collaborators: Cohort profile: the 45 and up study. Int J Epidemiol 2008, 37(5):941-7. 10. 45 and Up Study Collaborators: Cohort profile: the 45 and up study. Int J Epidemiol 2008, 37(5):941-7. 11. Watts G: UK Biobank gets 10% response rate as it starts recruiting volunteers. BMJ 2007, 334(7595):659. 11. Watts G: UK Biobank gets 10% respons volunteers. BMJ 2007, 334(7595):659. Acknowledgements Canberra 2004. Acknowledgements The authors are grateful to Ms Margo Barr and Mrs Sandra Carlson for their expert assistance and advice on the NSW Population Health Survey and use of the NSW Population Health Survey data. We thank the men and women partic- ipating in the 45 and Up Study, and the NSW Department of Health for permit- ting use of the 2006 and 2007 NSW Population Health Survey Datasets. The 45 and Up Study is run by The Sax Institute in collaboration with the Can- cer Council of New South Wales; the New South Wales Division of the National Heart Foundation of Australia; the New South Wales Department of Health; beyondblue: the national depression initiative and the New South Wales Depart- ment of Ageing, Disability and Home Care. This work was also supported by the National Health and Medical Research Council [402810]. NM was employed by the NSW Department of Health on the NSW Biostatistical Officer Training Program at the time this work was conducted. 17. Greenland S: Response and Follow-up Bias in Cohort Studies. Am J Epidemiol 1977, 106(3):184-7. 18. Siemiatycki J, Campbell S: Nonresponse bias and early versus all responders in mail and telephone surveys. Am J Epidemiol 1984, 120(2):291-301. 19. Department of Health and Aged Care (GISCA): Measuring remoteness: accessibility/remoteness index of Australia (ARIA). Canberra 2001. 20. Population Health Division: The health of the people of New South Wales - Report of the Chief Health Officer [http://www.health.nsw.gov.au/ publichealth/chorep/]. Sydney: NSW Department of Health Accessed 4 March 2009 21. Barr M, Baker D, Gorringe M, Fritsche L: NSW Population Health Survey: Description of Methods Sydney: NSW Department of Health; 2008. The concept of this study was developed as part of the Study of Economic and Environmental Factors in health (SEEF) project. The SEEF investigators are: Adrian Bauman, Sally Redman, Emily Banks, Mark Harris, Deborah Schofield, Tony McMichael, Hilary Bambrick, John Beard, Dorothy Broom, Julie Byles, Judith Clark, Bin Jalaludin, Louisa Jorm, Don Nutbeam, Bryan Rodgers, Heather Bowles, Jack Chen, PH Phongsavan, Lynne Parkinson. 22. Centre for Epidemiology and Research: 2006 Report on Adult Health from the New South Wales Population Health Survey Sydney: NSW Department of Health; 2007. 23. Centre for Epidemiology and Research: 2007 Report on Adult Health from the New South Wales Population Health Survey Sydney: NSW Department of Health; 2008. 24. Australian Institute of Health and Welfare: Rural, Regional and Remote Health: A guide to remoteness classifications. References 1. Burkell J: The dilemma of survey nonresponse. Libr Inform Sci Res 2003, 25(3):239-63. Additional file 1 Questionnaire items rated as highly or moderately com- parable on the 45 and Up Study and the NSW PHS and used in the analyses. Additional file 1 Questionnaire items rated as highly or moderately com- parable on the 45 and Up Study and the NSW PHS and used in the analyses. Additional file 1 Questionnaire items rated as highly or moderately com- parable on the 45 and Up Study and the NSW PHS and used in the analyses. Additional file 2 Ten crude and adjusted ORs from the 45 and Up Study (unweighted and weighted), where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS. 2. Van Goor H, Verhage AL: Nonresponse and recall errors in a study of absence because of illness: An analysis of their effects on distributions and relationships. Quality & Quantity 1999, 33(4):411-28. Additional file 2 Ten crude and adjusted ORs from the 45 and Up Study (unweighted and weighted), where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS. 3. Breslow NE, Day NE: Statistical methods in cancer research. Volume II-- The design and analysis of cohort studies. IARC Sci Publ 1987, 82:1-406. 4. Ponsonby AL, Dwyer T, Couper D: Is this finding relevant? Generalisation and epidemiology. Aust N Z J Public Health 1996, 20(1):54-6. Additional file 3 Ten crude and adjusted ORs from the 45 and Up Study (unweighted and weighted), where either the exposure or outcome or both variables were only moderately comparable across the 45 and Up Study and the NSW PHS. 5. Miettinen OS: Theoretical epidemiology: principles of occurrence research in medicine New York: Wiley; 1985. 6. Doll R, Peto R, Boreham J, Sutherland I: Mortality in relation to smoking: 50 years' observations on male British doctors. BMJ 2004, 328(7455):1519. Additional file 4 Ten crude and adjusted ORs from the NSW PHS (unweighted and weighted), where the exposure and outcome variables were highly comparable across the 45 and Up Study and the NSW PHS. 7. Colditz GA, Manson JE, Hankinson SE: The Nurses' Health Study: 20-year contribution to the understanding of health among women. J Womens Health 1997, 6(1):49-62. Received: 14 December 2009 Accepted: 1 April 2010 Published: 1 April 2010 This article is available from: http://www.biomedcentral.com/1471-2288/10/26 © 2010 Mealing 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 re BMC Medical Research Methodology 2010, 10:26 Author Details 25. WHO/IASO/IOTF: The Asia-Pacific perspective: redefining obesity and its treatment Melbourne: Health Communications Australia; 2000. 1The Sax Institute, P.O. Box 123, Broadway NSW 2007, Australia, 2School of Medicine, University of Western Sydney, Campbelltown Campus, Locked Bag 1797, Penrith South DC NSW 1797, Australia, 3National Centre for Epidemiology and Population Health, Australian National University, ACT 0200, Australia and 4Centre for Statistical and Survey Methodology, University of Wollongong, Northfields Avenue, North Wollongong NSW 2522, Australia 26. Kessler RC, Andrews G, Colpe LJ, Hiripe E, Mroczek DK, Normand SL, Walters EE, Zaslavsky AM: Short screening scales to monitor population prevalences and trends in non-specific psychological distress. Psychol Med 2002, 32(6):959-76. 27. Andrews G, Slade T: Interpreting scores on the Kessler Psychological Distress Scale (K10). Aust N Z J Public Health 2001, 25(6):494-7. 27. Andrews G, Slade T: Interpreting scores on the Kessler Psychological Distress Scale (K10). Aust N Z J Public Health 2001, 25(6):494-7. Received: 14 December 2009 Accepted: 1 April 2010 Published: 1 April 2010 This article is available from: http://www.biomedcentral.com/1471-2288/10/26 © 2010 Mealing 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 rep BMC Medical Research Methodology 2010, 10:26 Received: 14 December 2009 Accepted: 1 April 2010 Published: 1 April 2010 This article is available from: http://www.biomedcentral.com/1471-2288/10/26 © 2010 Mealing 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 rep BMC Medical Research Methodology 2010, 10:26 Page 12 of 12 Mealing et al. BMC Medical Research Methodology 2010, 10:26 http://www.biomedcentral.com/1471-2288/10/26 28. Rao JNK, Thomas DR: Analysis of categorical response data from complex surveys: An appraisal and update. In Analysis of survey data Edited by: Chambers RL, Skinner CJ. New York: Wiley; 2003:92. 29. SAS Institute Inc: SAS version 9 NC, USA: Cary. 30. Scott AJ, Wild CJ: Fitting logistic-models under case-control or choice based sampling. Journal of the Royal Statistical Society Series B- Methodological 1986, 48(2):170-82. 31. Austin MA, Criqui MH, Barrett-Connor E, Holdbrook MJ: The effect of response bias on the odds ratio. Am J Epidemiol 1981, 114(1):137-43. 31. Austin MA, Criqui MH, Barrett-Connor E, Holdbrook MJ: The effect of response bias on the odds ratio Am J Epidemiol 1981 114(1) 137 43 32. Brehm J: The phantom respondent: Opinion surveys and political representation Michigan: The University of Michigan Press; 1993. 33. Perkins JJ, Sanson-Fisher RW: An examination of self- and telephone- administered modes of administration for the Australian SF-36. J Clin Epidemiol 1998, 51(11):969-73. 34. Martikainen P, Laaksonen M, Piha K, Lallukka T: Does survey non- response bias the association between occupational social class and health? Scand J Public Health 2007, 35(2):212-5. 35. Van Loon AJM, Tijhuis M, Picavet HSJ, Surtees PG, Ormel J: Survey non- response in the Netherlands: Effects on prevalence estimates and associations. Ann Epidemiol 2003, 13(2):105-10. 36. Goodman R, Iervolino AC, Collishaw S, Pickles A, Maughan B: Seemingly minor changes to a questionnaire can make a big difference to mean scores: a cautionary tale. Soc Psychiatry Psychiatr Epidemiol 2007, 42:322-27. 37. Kalton G, Schuman H: The effect of the question on survey responses: A review. Journal of the Royal Statistical Society Series A (General) 1982, 145(1):42-73. 37. Kalton G, Schuman H: The effect of the question on survey responses: A review. Journal of the Royal Statistical Society Series A (General) 1982, 145(1):42-73. 38. Picavet HSJ, Bos GAM Van den: Comparing survey data on functional disability: The impact of some methodological differences. J Epidemiol Community Health 1996, 50(1):86-93. 38. Picavet HSJ, Bos GAM Van den: Comparing survey data on functional disability: The impact of some methodological differences. J Epidemiol Community Health 1996, 50(1):86-93. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2288/10/26/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2288/10/26/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2288/10/26/prepub doi: 10.1186/1471-2288-10-26 Cite this article as: Mealing et al., Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs BMC Medical Research Methodology 2010, 10:26
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Variáveis associadas à ansiedade gestacional em primigestas e multigestas
Temas em Psicologia
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Abstract This study aimed to describe and compare anxiety on primigravidae and multiparous in the third quarter, as well as to identify, among sociodemographic variables and of pregnancy, which ones were associated with high anxiety. There were 479 pregnant women participants who responded to an anxiety instru- ment (STAI) and a questionnaire on sociodemographic variables and about pregnancy. The groups were statistically compared e a logistic regression model was composed to evaluate associations. The results indicated that 36% of pregnant women presented symptoms of high anxiety on third gestational trimes- ter, with a predominance of the multiparous in relation to the primigravidae. For the multiparous, the highest chance for high anxiety was associated with low income and for the primigravidae to the threat of miscarriage in early pregnancy. For both primigravidae and multiparous, wanting the baby appeared as a protective factor for high anxiety. The expressive percentage of pregnant women with anxiety symptoms justifi es the offering of prevention services and promotion of mental health for pregnant women, with a differentiated attention to primigravidae and multiparous. Keywords: Anxiety, pregnant, primigravidae, multiparous. * Mailing address: Rua Quintino Bocaiúva, s/n, Aparecida de São Manuel, São Manuel, SP, Brazil 18650-000 Phone (fax) 2(14) 3841-3766. E-mail: professora.schiavo@gmail.com, olgarolim@fc.unesp.br and gimol@ fmb.unesp.br Agradecemos a Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo auxílio fi nanceiro para execução desse estudo. Article Article Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas Rafaela de Almeida Schiavo*, 1, 2 Orcid.org/0000-0001-7290-3610 Olga Maria Piazentin Rolim Rodrigues3 Orcid.org/0000-0002-5332-256X Gimol Benzaquen Perosa4 Orcid.org/0000-0002-7825-2537 Rafaela de Almeida Schiavo*, 1, 2 Orcid.org/0000-0001-7290-3610 Olga Maria Piazentin Rolim Rodrigues3 Orcid.org/0000-0002-5332-256X Gimol Benzaquen Perosa4 Orcid.org/0000-0002-7825-2537 1Universidade Paulista, Bauru, SP, Brasil 2Instituto Municipal de Ensino Superior de São Manuel, São Manuel, SP, Brasil 3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brasil 4Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Brasil 1Universidade Paulista, Bauru, SP, Brasil 2Instituto Municipal de Ensino Superior de São Manuel, São Manuel, SP, Brasil 3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brasil 4Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Brasil Trends in Psychology/Temas em Psicologia ISSN: 2358-1883 Sociedade Brasileira de Psicologia Trends in Psychology/Temas em Psicologia ISSN: 2358-1883 Sociedade Brasileira de Psicologia Trends in Psychology/Temas em Psicologia ISSN: 2358-1883 Schiavo, Rafaela de Almeida; Rodrigues, Olga Maria Piazentin Rolim; Perosa, Gimol Benzaquen Variáveis Associadas à Ansiedade Gestacional em Primigestas e Multigestas Trends in Psychology/Temas em Psicologia, vol. 26, núm. 4, Outubro-Dezembro, 2018, pp. 2091-2104 Sociedade Brasileira de Psicologia DOI: 10.9788/TP2018.4-14Pt Disponível em: http://www.redalyc.org/articulo.oa?id=539658227014 Disponível em: http://www.redalyc.org/articulo.oa?id=539658227014 Como citar este artigo Número completo Mais artigos Home da revista no Redalyc Sistema de Informação Científica Redalyc Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto Sistema de Informação Científica Redalyc Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto Trends in Psychology / Temas em Psicologia ISSN 2358-1883 (online edition) DOI: 10.9788/TP2018.4-14En ISSN 2358-1883 (online edition) 1Universidade Paulista, Bauru, SP, Brasil 2Instituto Municipal de Ensino Superior de São Manuel, São Manuel, SP, Brasil 3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brasil 4Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Brasil Resumo Este estudo teve por objetivo descrever e comparar a ansiedade de primigestas e multigestas, no terceiro trimestre gestacional, bem como identifi car, dentre as variáveis sociodemográfi cas e de gestação, as que p gradecemos a Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Coordenação d perfeiçoamento de Pessoal de Nível Superior (CAPES) pelo auxílio fi nanceiro para execução desse estudo Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Resumen Este estudio tuvo como objetivo describir y comparar la ansiedad en el primigestas y multigestas en tercer trimestre, y para identifi car, entre las variables sociodemográfi cas y el embarazo, que se asocian a mayor ansiedad. Participaron en 479 mujeres embarazadas que respondieron a un instrumento de an- siedad (STAI) y un cuestionario sobre variables sociodemográfi cas y el embarazo. Los investigadores compararon los grupos estadísticamente y montaron un modelo de regresión logística para evaluar las asociaciones. Los resultados indicaron que el 36% de las mujeres embarazadas tenían síntomas altos de ansiedad en tercer trimestre, con un predominio de multigestas en relación con el primigestas. Para multigestas la mayor oportunidad para alta ansiedad se asoció con bajas ingresos y para el primigestas con un aborto involuntario en el embarazo temprano. Ambos primigestas y multigestas quieren el bebé apareció como un factor protector para alta ansiedad. Un porcentaje signifi cativo de mujeres embaraza- das con síntomas de ansiedad justifi ca los servicios dirigidos a la prevención y promoción de la salud mental de las mujeres embarazadas, con una especial atención a las primigestas y multigestas. Palabras clave: Ansiedad, embarazo, primigestas, multigestas. Anxiety is part of the process of human development, and can be present in all periods of the life cycle. It is a characteristic that occurs in times of fear, danger or tension. However, in some moments of life, anxiety is more intense than in others. Depending on the period, it is considered to be an expected emotional state during a change of employment, faced with the result of a job application or medical examination, marriage, pregnancy and the birth of a child, among others. being higher than in the puerperium (Agrati et al., 2015; Baptista et al., 2006) for both fathers and mothers (Figueiredo & Conde, 2011). Due to the negative regulation of placental 11β-HSD2, which increases fetal exposure to maternal cortisol (O’Donnell et al., 2012), high levels of anxiety in pregnancy can result in complications and intercurrences such as premature birth and low birth weight (Bener, 2013; Schetter & Tanner, 2012). Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2106 se associaram para alta ansiedade. Participaram 479 gestantes que responderam a um instrumento de ansiedade (IDATE) e um questionário sobre variáveis sociodemográfi cas e da gestação. Compararam- se os grupos estatisticamente e montou-se um modelo de regressão logística para avaliar o peso das associações (p<0,05). Os resultados indicaram que 36% das gestantes apresentaram sintomas de alta ansiedade no terceiro trimestre gestacional, com um predomínio das multigestas em relação às primi- gestas. Para as multigestas, a maior chance para alta ansiedade esteve associada à baixa renda e, para as primigestas, à ameaça de aborto no início da gestação. Tanto para primigestas como para multigestas, desejar o bebê apareceu como fator de proteção para alta ansiedade. A expressiva porcentagem de ges- tantes com sintomas de ansiedade justifi ca o oferecimento de serviços voltados à prevenção e promoção de saúde mental das gestantes, com atenção diferenciada às primigestas e multigestas. Palavras-chave: Ansiedade, gravidez, primigesta, multigesta. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2107 Melchiori, Salgado, & Tavano, 2015). It may also affect growth and cause delays in childhood development (Beltrami, Moraes, & Souza, 2013; O’Donnell, Glover, Barker, & O’Connor, 2014; Schetter & Tanner, 2012). Melchiori, Salgado, & Tavano, 2015). It may also affect growth and cause delays in childhood development (Beltrami, Moraes, & Souza, 2013; O’Donnell, Glover, Barker, & O’Connor, 2014; Schetter & Tanner, 2012). of being a mother, which can increase anxiety. Due to cultural issues, this new responsibility is often accompanied by guilt and anxiety, with the woman being insecure about whether she will fully correspond to the ideal of mothering spread in Western society, expecting perfect mothers, housewives and wives (Pitilin, Haracemiw, Marcon, & Pelloso, 2013). Regarding the maternal mental health, gestational anxiety has been associated with gestational depression (Schetter & Tanner, 2012) and is indicative of puerperal anxiety and depression (Agrati et al., 2015; Coelho, Murray, Royal-Lawson, & Cooper, 2011; Faisal-Cury et al., 2010) coinciding with the studies of Airosa and Silva (2013) and Nardi et al. (2015) who observed that gestational anxiety left women vulnerable to the emergence of emotional disturbances. However, in the study by Saviani-Zeoti and Petean (2015), elevated levels of gestational anxiety did not present any impairment in the attachment relationship with the fetus. For Simas et al. (2013), primigravidas and multigravidas are equally anxious, however, they present anxiety for different reasons: the primigravidas are more distressed by their inexperience in the task of being a mother while the multigravidas are more ambivalent with the unplanned pregnancy. On the other hand, for Lopes et al. (2012), it is a myth to believe that the experience of motherhood with the fi rst-born would facilitate the relationship with the second child. According to the authors, multigravidas, especially those who are economically vulnerable and have to assume countless costs, experience a silenced social violence, where there is no room for error, anger and sadness, which may justify the high anxiety rates. According to the literature, some of the risk factors for high gestational anxiety are low maternal age, low educational level (Araújo, Pacheco, Pimenta, & Kac, 2008), low income (Simas, Souza, & Scorsolini-Comin, 2013) and threat of abortion (Alves, Alves, Ibiapina, Busgaib, & Costa, 2010). Fonseca-Machado, Alves, Freitas, Monteiro, and Gomes-Sponholz (2014), in an integrative literature review, identifi ed that domestic violence during the pregnancy had negative repercussions on the mental health of pregnant women. Resumen Gestational anxiety is also associated with shorter exclusive breastfeeding (Adedinsewo, Fleming, Steiner, Meaney, & Girard, 2014; Ystrom, 2012), which may have negative consequences for the mother/baby relationship (Airosa & Silva, 2013; Nardi, Rodrigues, Anxiety in pregnancy is one of the most common psychological changes (Baptista, Baptista, & Torre, 2006; Faisal-Cury, Araya, Zugaib, & Menezes, 2010), varying in intensity in different societies and cultures, with its indices Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Participants Between 2010 and 2015, 485 pregnant women in the third trimester that were waiting for prenatal care at the Primary Health Units (PHUs) were invited to participate in the study. Of these, 479 agreed to participate in the study and, while waiting, completed the instruments individually, in an appropriate room. There were 222 primigravidas and 257 multigravidas. Method This was a cross-sectional study that evaluated the sociodemographic characteristics, the perception of motherhood and the anxiety of pregnant women. To compose the sample, locations were selected that concentrated a large number of consultations for the pregnant woman through the Brazilian Nation Health System (Sistema Único de Saúde - SUS), in three medium-sized cities in the state of São Paulo. Data Collection Procedure The recruitment of the participants took place in the PHUs of the three cities of São Pau- lo state. The researcher, the fi rst author of this study, approached all the pregnant women who were in the third trimester and who waited in the waiting room to be attended by the obstetrician and invited them to participate in the study, stat- Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2108 women breastfed in the fi rst hour after birth than primiparous women. cerned the medical history (health and mental health prior to gestation, health during gestation) and two were related to the pregnancy (threat of abortion of the current pregnancy and previous abortion). The pregnant women were also asked if they and their partners had wanted the baby (or not). As can be seen, studies comparing anxiety levels between primigravidas and multigravidas, as well as its associated factors, are still scarce with differing results, possibly due to the samples, evaluation instruments and diverse contexts of investigation. These fi ndings suggest the need for further investigation in order to assist in offering differentiated support services to primiparous and multiparous pregnant women, focused on the control of maternal anxiety in this period of the life cycle. Based on the above considerations, this study had the objective of describing and comparing the anxiety of primigravidas and multigravidas in the third gestational trimester, as well as identifying the sociodemographic and gestational variables associated with high anxiety. For the assessment of anxiety, the State- Trait Anxiety Inventory (STAI), developed by Spielberger, Gorsuch, and Lushene (1970) and translated and adapted into Portuguese by Bi- aggio and Natalício (1979/2003), was used. It is an self-application instrument, for youths and adults, composed of two scales that evalu- ate State anxiety (S-anxiety) and Trait anxiety (T-anxiety). From 20 items, individuals have to evaluate themselves, according to a four-point Likert-type scale. Regarding its psychometric qualities, its reliability in the test-retest was 0.74 for State anxiety and 0.83 for Trait anxiety. The internal consistency, assessed through Cronbach’s Alpha, was 0.88 in relation to female State anxiety and 0.87 for female Trait anxiety (Biaggio & Natalício, 1979/2003). For the present study, internal consistency was calculated through Cronbach’s Alpha for state and trait anxiety in the third trimester. Alphas of 0.67 for State anxiety and 0.63 for Trait anxiety were obtained, indicating that the consistency indices for the Portuguese version of the STAI applied with pregnant women were moderate to good. As the indication of symptoms of high anxiety, the cut-off point used was a score equal to or above the 75th percentile, as previously used in other studies (Perosa, Canavez, Silveira, Padovani, & Peraçou, 2009; Perosa, Silveira, & Canavez, 2008). Instruments The authors used a single form with 11 questions, fi ve of which were related to sociode- mographic data (age, marital status, schooling, family income, number of children), three con- Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. In the review carried out for the present article, no references were found for studies that emphasized the protective factors for high anxiety. In the puerperium, the results are also controversial. In Brazil, Faisal-Cury and Menezes (2006) identifi ed that multiparous women presented higher indices of anxiety than primiparous women. In the study by Verissimo (2010) in Portugal, primiparas were found to have greater anxiety. In addition to the incipient literature and the inconclusive data regarding the possible mental health problems in primigravidas and multigravidas, some research has been interested in the repercussion that the number of children can have on the quality of life of the couple as well as their relationship with the children (Camarneiro & Justo, 2012; Pereira, Fonseca, Oliveira, Souza, & Mello, 2013; Saviani-Zeoti & Petean, 2015). There are data indicating that primigravidas have higher marital satisfaction, greater preoccupation with the fetus and better perception of their quality of life than multigravidas (Camarneiro & Justo, 2012). On the other hand, in the study by Pereira et al. (2013), a higher percentage of multiparous Few studies have addressed gestational anxiety in multigravidas and primigravidas and the results are still inconclusive, sometimes associating high anxiety with primigravidas (Alves et al., 2007), or with multigravidas (Lopes, Vivian, Oliveira, Pereira, & Piccinini, 2012), or presenting no differences between the two groups (Simas et al., 2013). According to Alves et al. (2007), primiparity, even though it is a period of adaptive psychic normality, it is also a critical moment marked by anguish due to the prominence of having to assume a new role, that Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Data Analysis Procedure The sociodemographic, clinical and gesta- tional, numerical and categorical variables were binarized and organized in the Statistical Pack- age for the Social Sciences (SPSS, version 21.0, Chicago INc II, USA) database for further data analysis. The criterion to binarize the age was the one proposed by the World Health Organiza- tion (WHO, 1986) that defi nes the ages between 10 and 19 years as adolescence. The STAI was corrected according to the specifi c norms of the manual and the binarized results, considering the 75th percentile as a marker of high anxiety. The numerical variables (maternal age and number of children) and categorical variables (all dummy type) were analyzed using descriptive statistics (frequency and percentage). Before proceeding to the inferential statistical analysis, the Shap- iro-Wilk test was applied to test the normality of the variables. The chi-square test was used to compare the primigravidas and multigravidas re- garding the sociodemographic variables related to the clinical and gestational history and high anxiety. To identify possible variables that were predictors of risk or protection for high anxiety, a logistic regression model with all dichotomous variables in a single block was applied for the total sample and, separately, for the primigravi- das and multigravidas. The signifi cance level ad- opted was p<.05. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2109 Ethical Procedures ing that they had to respond to two instruments, and that one would assess their level of anxiety. The pregnant women who agreed to participate, after signing the consent form, completed the Form and the STAI in a room of the health unit reserved for this purpose. For the non-literate pregnant women and those who, for some rea- son, preferred not to read, the researcher read the inventory questions and fi lled out the answer sheet of the instrument. All the pregnant women were informed about the result of the STAI soon after its application, the ones that scored for high anxiety were advised to seek specialized clini- cal psychological care, the researcher gave these women the telephone number and address of the place where they could fi nd this care. The project was approved by the Research Ethics Committee of the Botucatu Medical School Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP (process No. 4166- 2012) and by the Faculty of Sciences of UNESP Bauru campus (process No. 614/46/01/09). The data collection was authorized by the heads of the three centers that attended the pregnant women in primary care. Data collection only took place after the signing of the consent form that was given to the participants. All 479 participants, after responding to the instruments, awaited correction by the researcher at the application site, which immediately provided the result. Those who scored for high anxiety were referred to specialized professional care locations in their city. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. Results Primarily, sociodemographic and maternity data will be presented. This will be followed by the trait and state anxiety characteristics among the primigravidas and multigravidas in the third gestational trimester, the factors associated with high anxiety in the third trimester and the factors associated with high anxiety in primigravidas and multigravidas. From Table 1 it is possible to verify that the multigravidas were signifi cantly older (p<.001), with a mean of 27 years (SD=5.4), while the primigravidas had a mean age of 20 years (SD=5.5). Regarding the number of children, 36.6% of the multigravidas already had one child, 34.4% had two and 42.2%, three or more children. The multigravidas also presented lower levels of education (p=.009) and a higher percentage of the mothers (p=.004) and fathers (p=.049) had not wanted the baby when compared to the primigravidas. Considering the total sample, trait or state, around 36% of the pregnant women presented high anxiety. In Table 2 it can also be observed that, signifi cantly, a higher percentage of mul- tigravidas presented high anxiety, both trait (61.8%) and state (60.9%) in relation to the pri- migravidas. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Results 2110 Table 1 Comparison between Primigravidas and Multigravidas according to Sociodemographic and Maternity Characteristics (N = 479) Associated Variables Parity p – value* Primigravidas n = 222 Multigravidas n = 257 X2 F % F % Total Age 107.395 < .001 < 19 years old 101 88.6 13 11.4 114 (24%) > 20 years old 121 33.2 244 66.8 365 (76%) Marital Status 2.674 .102 Lives with partner 164 44.3 206 55.7 370 (77%) Don´t live with partner 58 53.2 51 46.8 109 (23%) Education 6.479 .011 < nine years old 84 39.8 127 60.2 211 (44%) > nine years old 138 51.5 130 48.5 268 (56%) Paid Occupation 0.018 .893 Yes 73 45.9 86 54.1 159 (33%) No 149 46.6 171 53.4 320 (67%) Family Income 0.628 .428 < 2 minimum wages 206 45.9 243 54.1 449 (94%) > 2 minimum wages 16 53.3 14 46.7 30 (6.3%) Pre-gestational health 2.737 .098 Normal 206 47.6 227 52.4 433 (90%) A problem 16 34.8 30 65.2 46 (10%) Gestational health 1.482 .223 Normal 189 47.6 208 52.4 397 (83%) A problem 33 40.2 49 59.8 82 (17%) Threat of abortion 0.267 .605 Yes 21 42.9 28 57.1 49 (10%) No 201 46.7 229 53.3 430 (90%) Wanted the baby 8.223 .004 Yes 147 51.8 137 48.2 284 (59%) No 75 38.5 120 61.5 195 (41%) Partner wanted the baby 3.892 .049 Yes 158 49.5 161 50.5 319 (67%) No 64 40.0 96 60.0 160 (33%) Note. F – frequency; % - percentage; X2 - Chi-Square. *p-value for the Chi-Square test. Table 1 Comparison between Primigravidas and Multigravidas according to Sociodemographic and Maternity Characteristics (N = 479) een Primigravidas and Multigravidas according to Sociodemographic and Maternity = 479) son between Primigravidas and Multigravidas according to Sociodemographic and Maternity eristics (N = 479) Marital Status Family Income Note. F – frequency; % - percentage; X2 - Chi-Square. q y p *p-value for the Chi-Square test. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. Results 2111 Table 2 Difference between Primigravidas and Multigravidas in High Trait/State Anxiety (N = 479) Parity p – value* Primigravidas Multigravidas Total X2 F % F % F % Trait anxiety 7.316 .007 No high anxiety 156 51.0 150 49.0 306 63.9 With high anxiety 66 38.2 107 61.8 173 36.1 Total 222 46.3 257 53.7 479 100 State anxiety 5.802 .016 No high anxiety 154 50.5 151 49.5 305 63.7 With high anxiety 68 39.1 106 60.9 174 36.3 Total 222 46.3 257 53.7 479 100 Note. F – frequency; % - percentage; X2 - Chi-Square. *p-value for the Chi-Square teste. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2111 able 2 fference between Primigravidas and Multigravidas in High Trait/State Anxiety (N = 479) *p-value for the Chi-Square teste. Table 3 Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimester of Gestation (N = 479) Anxiety trait Anxiety state p-value* OR IC 95% p-value* OR IC 95% Age .490 1.185 0.732 1.921 .068 1.593 0.966 2.628 Marital Status .777 1.071 0.667 1.719 .766 1.079 0.665 1.742 Education .109 1.395 0.929 2.093 .317 1.234 0.818 1.863 Paid Occupation .220 0.757 0.485 1.181 .629 0.896 0.574 1.398 Family Income .247 1.736 0.682 4.419 .082 2.335 0.898 6.069 Pre-gestational health .266 1.464 0.748 2.865 .135 1.672 0.852 3.280 Gestational health .520 1.187 0.704 2.001 .679 0.893 0.521 1.529 Threat of abortion .920 1.034 0.539 1.983 .021 2.126 1.122 4.027 History of abortion .066 1.589 0.970 2.600 .029 1.734 1.057 2.845 Wanted the baby .031 0.571 0.343 0.949 .003 0.455 0.273 0.758 Partner wanted the baby .037 0.568 0.334 0.967 .122 0.655 0.384 1.120 Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimester of N = 479) Table 3 Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimester of Gestation (N = 479) able 3 ssociated Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimest estation (N = 479) Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. tion with high trait or state anxiety. Table 4 Table 4 Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety on Primigravidas in in the Third Trimester of Gestation (n = 222) Primigravidas Anxiety Trait Anxiety State p-value* OR IC 95% p-value* OR IC 95% Age .933 0.970 0.481 1.958 .514 1.273 0.617 2.626 Marital Status .504 0.779 0.374 1.622 .567 1.235 0.600 2.543 Education .734 1.129 0.569 2.226 .789 1.100 0.549 2.204 Paid Occupation .192 0.602 0.281 1.290 .942 0.973 0.462 2.047 Family Income .355 0.554 0.158 1.938 .757 0.816 0.226 2.947 Pre-gestational health .923 1.061 0.323 3.480 .703 1.253 0.393 3.996 Gestational health .412 0.678 0.268 1.716 .381 0.662 0.263 1.666 Threat of abortion .136 2.164 0.785 5.964 .017 3.464 1.245 9.640 History of abortion .196 1.842 0.730 4.651 .086 2.263 0.891 5.749 Wanted the baby .839 0.910 0.365 2.267 .033 0.390 0.164 0.928 Partner wanted the baby .020 0.322 0.124 0.837 .358 0.653 0.263 1.620 Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. Table 4 Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety on Primigravidas in in the Third Trimester of Gestation (n = 222) ors for Greater and Lesser Chance of High Trait/State Anxiety on Primigravidas in in the r of Gestation (n = 222) Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. er Brazilian studies evaluating pregnant and puerpera women (Faisal-Cury & Menezes, 2006; Morais, Lucci, & Otta, 2013; Beltra- mi et al., 2013), which permits comparisons between the present study and others. As in other studies (Borges, Santos, Nascimento, Chofakian, & Gomes-Sponholz, 2016; Con- ceição & Fernandes, 2015), a large number of the pregnant women reported not having wanted the pregnancy (41%), especially the With respect to multigravidas, low income increased the chance of having high trait and state anxiety while having wanted the baby de- creased the chance, for both types of anxiety (Table 5). Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2112 in multigravidas (Table 5) in the third trimester of gestation are presented below. hibiting high trait anxiety in the third trimester of the gestation. Similarly, the mother having wanted the baby decreased the chance of scoring for high state anxiety, in the third trimester of the gestation. The pregnant women who had a clini- cal history of a threat of abortion in early preg- nancy and previous abortion had a higher chance of presenting high state anxiety (Table 3). In Table 4, it can be seen that for the primigravidas, having wanted the baby appeared as a protective factor, while the threat of abortion increased the chance for high state anxiety by 3.464. Regarding trait anxiety, the partner having wanted the baby reduced the chance of the pregnant woman presenting high anxiety. The factors associated with high trait and state anxiety in the primigravidas (Table 4) and Results However, it was found that the mother and her partner hav- ing wanted the baby decreased the chance of ex- In the prediction analysis for the total of the sample, it can be observed that no sociodemo- graphic variable showed a signifi cant associa- Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Discussion The sociodemographic characteristics of the participants were similar to those of oth- Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2113 Table 5 Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety on Multigravidas in in the Third Trimester of Gestation (n = 257) Multigravidas Anxiety Trait Anxiety State p-value* OR IC 95% p-value* OR IC 95% Age .802 0.856 0.253 2.892 .137 2.641 0.735 9.489 Marital Status .328 1.405 0.711 2.773 .758 0.898 0.455 1.775 Education .119 1.553 0.894 2.698 .276 1.355 0.785 2.340 Paid Occupation .761 0.914 0.512 1.632 .655 0.878 0.495 1.557 Family Income .043 5.094 1.050 24.704 .021 6.622 1.337 32.793 Pre-gestational health .236 1.702 0.706 4.104 .167 1.836 0.775 4.352 Gestational health .129 1.727 0.853 3.496 .793 1.098 0.545 2.215 Threat of abortion .146 0.503 0.199 1.270 .360 1.493 0.633 3.525 History of abortion .368 1.327 0.717 2.453 .162 1.538 0.841 2.813 Wanted the baby .007 0.397 0.203 0.776 .015 0.439 0.226 0.850 Partner wanted the baby .641 0.848 0.425 1.693 .303 0.698 0.352 1.384 Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. *p-value for the Logistic Regression. er number of multigravidas presented high anxiety, perhaps associated with their low income and number of children, as will be discussed later. multigravidas. This suggests that, despite the law guaranteeing family planning as a State responsibility, the aim of providing conditions for men and women to access in- formation about contraception (Prates, Abib & Oliveira, 2008) may not be occurring ef- fectively. For some authors, this is due to the limitation and defi ciency in the care infra- structure, the lack of information regarding contraception among women or the resis- tance of the spouse to seek family planning services (Caetano, 2014; Penaforte et al., 2010; Pitilin et al., 2013). This study allowed the identifi cation of approximately 36% of the participants, in the last trimester of gestation, with high trait and state anxiety. This proportion is smaller than that of other national studies, probably due to the cut-off point adopted for high anx- iety in this study being higher than in other studies (Baptista et al., 2006; Faisal-Cury & Menezes, 2006). The multigravidas were older than the primigravidas. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2114 In both the primigravidas and multigrav- idas, the history of abortion signifi cantly in- creased the chance of high anxiety (state), possibly because this experience causes pregnant women to experience the new preg- nancy with ambivalent feelings of attach- ment to the baby, as well as insecurity and fear (Freire & Chatelard, 2009). In the study by Fernandes et al. (2012), both women who spontaneously aborted and those who had provoked an abortion presented anxiety with a new pregnancy associated with fear of an- other abortion. Menezes, 2006; Morais et al., 2013). Studies have highlighted that multiparity is associ- ated with a lower degree of maternal edu- cation, which is, in turn, refl ected in lower wages, lower family income and greater eco- nomic vulnerability (Moura, Silva, Gomes, Almeida, & Evangelista, 2010; Prates et al., 2008; Severinski, Mamula, Severinski, & Mamula, 2009). Confi rming the positions of Simas et al. (2013), the percentage of pregnant women with high anxiety, as well as sociodemo- graphic, clinical and gestational risk factors, differed in both groups: for the multigravi- das low-income appeared to be a risk factor for high anxiety (trait and state), while for the primigravidas it was the threat of abor- tion in early pregnancy (state). While the possibility of losing the child appeared as a risk factor, having wanted the child was associated with less chance of primigravidas and multigravidas present- ing symptoms of high anxiety (trace and state). For Pitilin et al. (2013), most of the time, even if the pregnancy was unplanned and women believe they have become preg- nant at the wrong time, the initial anguish and anxiety become feelings of resigna- tion, adaptation and, during the pregnancy, they start to want the baby. In some stud- ies, women who did not plan the pregnancy due to fi nancial and marital diffi culties had a belief that the baby came to restore the fam- ily relationship, including the conjugal one, and the desire for the child became a satis- factory, even saving experience, increasing their self-esteem and protecting them from emotional problems (Moura et al., 2010; Pri- etsch, González-Chica, Cesar, & Mendoza- Sassi, 2011; Simas et al., 2013). Discussion It was hoped that because the multigravidas were older, indicating greater experience, knowledge and the ability to an- ticipate occurrences acquired in raising other children, they would present lower anxiety compared to the primigravidas. However, in this study, as in Lopes et al. (2012) a high- The multigravidas also had lower educa- tion levels than the primigravidas, possibly due to the multigravidas not being favored by the new educational policies of continu- ous progression, which started in 1996, re- sponsible for higher levels of education of pregnant women and puerperas in recent studies (Beltrami et al., 2013; Faisal-Cury & Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. As 42.2% of the multigravidas had more than three children, the arrival of one more child, especially for the low-income mul- tigravidas, may have led to a new recon- fi guration of the family system, with more responsibilities and possible losses in their perspective for the future, fi nancial diffi cul- ties and reduced quality of life, generating uncertainties and anxiety (Borges, Ferreira, Mariutti, & Almeida, 2011; Prates et al., 2008; Simas et al., 2013). In the study by Pitilin et al. (2013), with multiparas of four or more children, SUS users, it was observed that economic vulnerability was not limited to income deprivation, but also interfered with the possibility of obtaining quality work with adequate remuneration, with ac- cess to public services and with the dynam- ics of family life. The partner wanting the baby decreased the odds for high trait anxiety in the primi- gravidas, showing that the desire for a child and possible paternal involvement during the pregnancy may have attenuated the un- certainties regarding the care, especially for the primiparas. According to Simas et al. (2013), when the partner accepts the preg- nancy and wants to be a father, he becomes more involved in the process, takes respon- sibility for the preparations for the arrival of the baby with the pregnant woman, of- fers emotional support, tries to make contact Thus, fi nancial diffi culties may have increased the tension in the multigravidas faced with the birth of a new child, while the possibility of fetal loss increased the chance of high state anxiety in the primigravidas. Often, the threat of abortion is a condition with unknown pathophysiology that requires several types of intervention that, on one hand, seek to prevent fetal loss and, on the other, increase the sensation of unpredict- ability and anxiety (Alves et al., 2010). Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2115 limits the generalization of the results. It is rec- ommended that, in new investigations, in addi- tion to not binarizing the variables, the subjects should be randomized, increasing the generality of the results. with the baby and listens to the concerns and desires of the women, constituting an impor- tant social support. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. The present study contributed with ad- vances in this area of research, since it com- pared the anxiety of primigravidas and mul- tigravidas in the third gestational trimester, and identifi ed, among the sociodemographic and gestational variables, those that were associated with high anxiety in each group. This can allow socio-educative measures to be developed from the data identifi ed with greater attention to the emotional health of pregnant women, mainly low-income multi- gravidas and primigravidas with the threat of abortion in early pregnancy. Despite the limitations, the present study identifi ed high anxiety in 36% of the pregnant women using the SUS, in the third trimester of pregnancy, mainly among the multigravidas. The already proven association of gestational anxiety with obstetric complications and puerperal de- pression, as well as with damage that can affect childhood growth and development, indicate the need for professionals that work in prenatal care to pay special attention to the emotional health of the pregnant women. The study also reinforces that the risk fac- tors for high anxiety are different in primigravi- das and multigravidas, justifying the provision of differentiated care for women pregnant for the fi rst time and for those that have been through previous pregnancies. In the case of the primi- gravidas, experiencing a threat of abortion in early pregnancy appeared as the main variable predicting high anxiety in the third trimester, which indicates the importance of providing maximum information, along with psychologi- cal support, at that moment. Often, there are no reasons to justify the threat of abortion (Alves et al., 2010), which leads many women to perceive it as an individual fault, associated with feelings of guilt and fantasies of “a deserved punish- ment”, relevant aspects to be considered in the actions of psychological health care. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2116 materno. Cogitare Enfermagem, 12(4), 416-427. Retrieved from revistas.ufpr.br/cogitare/article/ download/10063/6918 structural variable in which health profession- als cannot act, with public policies in the care of these women, therefore, being relevant, in order to favor social-assistance, education and voca- tional resources. In addition, it is possible to act preventively by investing in programs that pro- mote more effi cient and effective family plan- ning for families using the public health system. Advances in contraception have enabled greater decision-making power for couples and the woman herself, with the possibility of choosing when to have a child becoming a decisive factor for a healthy pregnancy and maternity/paternity. Alves, J. A. G., Alves, N. G., Ibiapina, F. L. P., Bus- gaib, R. P. S., & Costa, F. S. (2010). Ameaça de aborto: Conduta baseada em evidências. Femi- na, 38(2), 111-115. Retrieved from http://fi les. bvs.br/upload/S/0100-7254/2010/v38n2/a010. pdf Araújo, D. M. R., Pacheco, A. H. R. N., Pimenta, A. M., & Kac, G. (2008). Prevalência e fatores as- sociados a sintomas de ansiedade em uma coorte de gestantes atendidas em um centro de saúde do município do Rio de Janeiro. Revista Brasileira de Saúde Materno Infantil, 8(3), 333-340. doi: 10.1590/S1519-38292008000300013 It is also hoped that these results can help in the development of new studies and provide sup- port for policies aimed at pregnant users of pub- lic services. The intention is to provide health- care that goes beyond the biomedical scope and takes into account emotional and social vulner- abilities from the development of approaches that ultimately improve the quality of life of this population. Baptista, M. N., Baptista, A. S. D., & Torres, E. C. R. (2006). Associação entre suporte social, de- pressão e ansiedade em gestantes. PSIC. Revista de Psicologia da Vetor Editora, 7(1), 39-48. Re- trieved from http://pepsic.bvsalud.org/pdf/psic/ v7n1/v7n1a06.pdf Beltrami, L., Moraes, A. G., & Souza, A. P. R. A. (2013). Ansiedade materna puerperal e risco para o desenvolvimento infantil. Distúrbios da Comunicação, 25(2), 229-239. Retrieved from http://revistas.pucsp.br/index.php/dic/article/ view/16476/12373 Final Considerations The results obtained bring advances to the knowledge in the area, however, it is important to consider some limitations of this study that could help in the planning of new investigations. The fi rst limitation refers to the instrument used to measure anxiety, the STAI, which was also used in other studies to identify levels of anxiety at the end of gestation (Agrati et al., 2015; Araú- jo et al., 2008; Baptista et al., 2006; O’Donnell et al., 2012; Roos, Faure, Louchner, Vythilingum, & Stein, 2013). Although the reliability calcu- lated for this study through Cronbach’s alpha showed moderate to good indices, the STAI is not a specifi c instrument for pregnant women, since some symptoms evaluated as anxiety could be characteristics expected during pregnancy. It is recommended that further investigations be conducted to identify the best instrument to mea- sure anxiety in pregnant women, or to develop an instrument specifi c for this population. The feelings and fantasies of primigravidas need to be addressed from the moment the threat of abortion occurred to prevent the chronifi ca- tion of the pregnant woman’s emotional state, which may ultimately negatively infl uence both the maternal mental health and the birth, as well as the physical and emotional health of the baby. However, studies are still needed to verify what type of care, individual or group, can more ef- fectively prevent the aggravation of anxiety at the end of gestation. Another possible limitation of the study may have been the fact that all the variables, in- cluding the continuous ones, such as age and in- come, were binarized and, if considered in their continuous form, they would possibly allow a more accurate analysis of anxiety in the different age and socioeconomic groups, in the logistic re- gression. Also, the fact that the sample was not probabilistic, but constituted by convenience, With regard to the multigravidas, a high percentage had not wanted the baby (60%) and high anxiety was associated with low income, a Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2117 Conceição, S. P., & Fernandes, R. A. Q. (2015). In- fl uência da gravidez não planejada no tempo de aleitamento materno. Escola Anna Nery, 19(4), 600-605. doi: 10.5935/1414-8145.20150080 Conceição, S. P., & Fernandes, R. A. Q. (2015). In- fl uência da gravidez não planejada no tempo de aleitamento materno. Escola Anna Nery, 19(4), 600-605. doi: 10.5935/1414-8145.20150080 Freire, T. C. G., & Chatelard, D. S. (2009). O aborto é uma dor narcísica irreparável? Revista Mal Es- tar e Subjetividade, 9(3), 1007-1022. Retrieved from http://pepsic.bvsalud.org/pdf/malestar/ v9n3/12.pdf Camarneiro, A. P. F., & Justo, J. M. R. M. (2012). Efeito do número de fi lhos na satisfação conju- gal e na vinculação pré-natal materna e paterna. International Journal of Developmental and Educational Psychology, 1(1), 19-28. Retrieved from http://infad.eu/RevistaINFAD/wp-content/ uploads/2013/02/INFAD_010124_19-28.pdf Lopes, R. C. S., Vivian, A. G., Oliveira, D. S., Pereira, C. R. R., & Piccinini, C. A. (2012). De- safi os para a maternidade decorrentes da gesta- ção e do nascimento do segundo fi lho. In C. A. Piccinini & P. 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Psychoneuroen- docrinology, 37(6), 818-826. doi: 10.1016/j. psyneuen.2011.09.014 Figueiredo, B., & Conde, A. (2011). Anxiety and de- pression in women and men from early pregnan- cy to 3-months postpartum. Archives of Wom- en’s Mental Health, 14(3), 247-55. doi: 10.1007/ s00737-011-0217-3 Fonseca-Machado, M. O., Alves, L. C., Freitas, P. S., Monteiro, J. C. S., & Gomes-Sponholz, F. (2014). Mental health of women who suffer in- timate partner violence during pregnancy. Inves- tigación y Educación em Enfermaría, 32(2), 291- 305. doi: 10.1590/S0120-53072014000200012 Penaforte, M. C. L. F., Silva, L. R., Esteves, A. P. V. S., Silva, R. F., Santos, I. M. M., & Silva, M. D. B. (2010). Conhecimento, uso e escolha dos mé- todos contraceptivos por um grupo de mulheres de uma unidade básica de saúde em Teresópo- lis, RJ. Cogitare Enfermagem, 15(1), 124-130. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018
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β-ketoenole dyes: Synthesis and study as fluorescent sensors for protein amyloid aggregates
Dyes and pigments
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Svitlana Chernii1, Vladyslava Kovalska1, Mykhaylo Losytskyy1, Yan Dovbii2, Iryna Tretyakova2, Rafal Czerwieniec3, Victor Chernii2, Sergiy Yarmoluk1, Sergiy Volkov2 Svitlana Chernii1, Vladyslava Kovalska1, Mykhaylo Losytskyy1, Yan Dovbii2, Iryna Tretyakova2, Rafal Czerwieniec3 Victor Chernii2, Sergiy Yarmoluk1, Sergiy Volkov2 1Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143 Kyiv, Ukraine 2V.I. Vernadskii Institute of General and Inorganic Chemistry NASU, 32/34 Palladin Av., 03080 Kyiv, Ukraine 3Institut für Physikalische und Theoretische Chemie, Universitätsstr. 31, 93053, Regensburg, Germany Dyes and Pigments 132 (2016) 274-281 DOI: 10.1016/j.dyepig.2016.04.053 aggregates Svitlana Chernii1, Vladyslava Kovalska1, Mykhaylo Losytskyy1, Yan Dovbii2, Iryna Tretyakova2, Rafal Czerwieniec3, Victor Chernii2, Sergiy Yarmoluk1, Sergiy Volkov2 1Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143 Kyiv, Ukraine 2V.I. Vernadskii Institute of General and Inorganic Chemistry NASU, 32/34 Palladin Av., 03080 Kyiv, Ukraine 3Institut für Physikalische und Theoretische Chemie, Universitätsstr. 31, 93053, Regensburg, Germany Regensburg, Germany This work was supported in the framework of Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) program Project Number: 645628 Project Acronym: METCOPH Project title: Metallocomplexes of macrocyclic compounds for photonic devices Project title: Metallocomplexes of macrocyclic compounds for photonic devices ABSTRACT The series of the new β-ketoenole dyes ((2E,5Z,7E,9E)-6-hydroxy-2- (alkylamino)-10-phenyldeca-2,5,7,9-tetraen-4-ones) with variation of alkylamino tail groups was synthesized and studied as potential probes for the sensing of protein aggregates amyloid fibrils. The dyes are low fluorescent when free but able to increase their emission intensity in dozens of times in the presence of fibrillar insulin. The fluorescent response of the dye on fibrillar insulin strongly depends on the nature of the alkylamino tail group. For compounds with propylamino (dye 13) and 2-hydroxyethylamino (dye 14) fragments the fluorescence intensity in the presence of fibrillar insulin exceeds that for the native one in 22 and 66 times correspondingly. However dyes demonstrate from low or moderate exceed of the fluorescence intensity in the presence of aggregated lysozyme compared to native one (up to 8.7 times for the dye 53 bearing methyl ester tail group), due to their pronounced sensitivity to native lysozyme. The dyes in complexes with insulin have rather height quantum yield up to 0.15, the large Stokes shifts values (about 100 nm and more), their binding constant values are about 105 M-1. The dye 14 allows fluorescent detection of the insulin amyloid fibrils in the concentration range 1-50 μg/ml. This causes an interest in the future study of the β-ketoenole as prospective fluorescent amyloid-sensitive molecules. KEYWORDS: β-ketoenole dyes, fluorescent sensing, probes, amyloid fibrils, insulin, lysozime, proteins 1. INTRODUCTION One of the most convenient methods for the analysis of biomolecules is the use of extrinsic fluorescent probes that noncovalently bind to them by electrostatic, van der Waals and hydrophobic interactions. The wide range of fluorescent molecules is developed for high efficient sensing, quantification and visualization of proteins and nucleic acids in in vitro and in vivo assays [1-4]. The spontaneous aggregation of proteins leading to formation of insoluble beta- pleated aggregates (amyloid fibrils) is among the actual targets in the biomedical researches, since these aggregates are connected with the range of harmful human diseases including neurodegenerative ones. This causes an interest in the development of new appropriate analytical tools to be used in the study of this process. The extrinsic fluorescent probes are used for the detection and quantification of amyloid fibrils, monitoring of the kinetics of their formation and study of the factors and agents affecting these processes. For these purposes, amyloid sensitive fluorescent probes Thioflavin T and it`s derivatives are commonly applied. The histological dyes Congo Red and Chrysamine G are used for the staining and study of amyloid formations on tissues [5-7]. Recently we discovered and developed mono and polymethine cyanines dyes as efficient fluorescent probes for the detection of protein β-pleated aggregates [8, 9]. Cyanines with high sensitivity to the amyloid fibrils and wide detection range (1.5-120 µg/ml for trimethine cyanine 7519 [10]) surpassing that of Thioflavine T were proposed. On the base of these cyanine dyes the inhibitory assay for the search of the compounds with anti-fibrillogenic activity was developed and applied. At the same time further search for the amyloid- sensitive probes with high fluorescent response to the fibrillar protein presence is still urgent. One of the necessary requirements for the molecule to be applicable as the fluorescent probe for the amyloid formations detection is high affinity of the complex formation between this molecule and the amyloid fibril. As the most probable mode of such complex formation, the insertion of the dye molecule into the groove of the amyloid fibril is suggested [11]. As a result of such binding, the fluorescent response is observed due to the quite rigid fixation of the dye molecule, and the polarization of the absorbed light is caused by the same orientation the bound dye molecules. 1. INTRODUCTION Thus, in order to obtain an efficient response to the presence of amyloid fibril, the molecule should have shape complimentary to that of the fibril groove and the size fitting the fibril groove (about 6,5-7 Å) [12]. (Fig 1) Figure 1. Scheme of the layout of the dye molecule in the fibrilar groove (left) [11] and AFM image of the insulin amyloid fibrils (right) [13]. Figure 1. Scheme of the layout of the dye molecule in the fibrilar groove (left) [11] and AFM image of the insulin amyloid fibrils (right) [13]. In the present work we firstly studied the β-ketoenole dyes as potential fluorescent probes for the sensing of amyloid aggregates of proteins (Table 1). The β-ketoenoles are the molecules of the elongated shape that is suggested as preferable for fitting to the groove of the amyloid fibril; besides they have rather flexible aliphatic chromophore chain providing the low intrinsic fluorescence intensity to the unbound dye. Unlikely to the majority of the amyloid-sensitive dyes bearing either positive (Thioflavin T, cyanine dyes) or negative (Congo Red) charge, the molecules of β-ketoenoles are uncharged. With this aim the series of ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10- phenyldeca-2,5,7,9-tetraen-4-ones) dyes with variation of alkylamyno substituents was synthesized and the fluorescent properties were characterized for the free dyes as well as in the presence of amyloidogenic proteins lysozyme and insulin in the native and aggregated form. The range of detection of the amyloid aggregates with the most efficient dye was determined. Besides, the fluorescent sensitivity of β- ketoenole dye to the serum albumin able to bind the variety of the small molecules was studied for the comparison. Table 1. Dye structures. 13 14 47 50 14 13 13 14 50 47 47 50 47 51 52 53 54 88 51 52 52 51 54 53 53 54 88 88 2.1. Synthesis and characterization of the dyes 2.1. Synthesis and characterization of the dyes The general procedure of ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10- phenyldeca-2,5,7,9-tetraen-4-ones) synthesis is as follows. The dyes were prepared by mixing the 5mM 4-hydroxy-6-methyl-3-((2E,4E)-5- phenylpenta-2,4-dienoyl)-2H-pyran-2-one in 10 ml of DMF (described in [14]) with an excess of 10% amine. The mixture was heated at 100 ºC for 30 minutes; during the reaction path it changed the color to red. The heating was stopped after the finishing of the carbon dioxide elimination. The cooled reaction mixture was precipitated by water. The formed product was washed twice with water and crystallized from DMF-ethanol mixture, filtered off, washed twice with ethanol on the filter and dried. All obtained compounds are yellow-orange fine-crystalline substances. Structures of investigated compounds were confirmed with 1H NMR and Anal. Calcd. (%). Calcd. (%). (2E,5Z,7E,9E)-6-hydroxy-10-phenyl-2-(propylamino)deca-2,5,7,9-tetraen-4- one. 13. Yield 67%. Mp 255-259°C. 1H NMR (CDCl3, 400 MHz) δ 14.99 (1H, s), 10.43 (1H, s), 7.44 (2H, d, J = 7.4 Hz), 7.34 (2H, dd, J = 13.4, 6.2 Hz), 7.26 (1H, dd, J = 9.8, 4.7 Hz,), 7.17 (1H, dd, J = 15.0, 10.8 Hz), 7.01 – 6.71 (2H, m), 6.01 (1H, d, J = 15.1 Hz), 5.20 (1H, s), 4.78 (1H, s), 3.24 (2H, dd, J = 13.3, 6.7 Hz), 1.98 (3H, s), 1.65 (2H, dd, J = 14.4, 7.2 Hz), 1.09 – 0.96 (3H, m). Found (%): C, 76.79; H, 7.74; N, 4.64; molecular formula C19H23NO2 requires C, 76.73; H, 7.80; N, 4.71. (2E,5Z,7E,9E)-6-hydroxy-2-((2-hydroxyethyl)amino)-10-phenyldeca-2,5,7,9- tetraen-4-one. 14. Yield 69%. Mp 273-275°C. 1H NMR (CDCl3, 400 MHz) δ 14.84 (1H, s), 10.44 (1H, s ), 7.46 (2H, dd, J = 12.9, 7.2 Hz), 7.40 – 7.30 (2H, m ), 7.30 – 7.22 (1H, m ), 7.18 (1H, dd, J = 15.0, 10.8 Hz), 6.91 (1H, ddd, J = 23.8, 20.0, 13.2 Hz), 6.76 (1H, d, J = 15.5 Hz), 6.00 (1H, d, J = 15.0 Hz), 5.21 (1H, s ), 4.83 (1H, s), 3.79 (2H, dt, J = 10.7, 5.2 Hz), 3.45 (2H, dt, J = 11.4, 5.6 Hz), 2.29 (1H, s ), 2.01 (3H, s). Found (%): C, 72.14; H, 7.02; N, 4.63; molecular formula C18H21NO3 requires C, 72.22; H, 7.07; N, 4.68. (2E,5Z,7E,9E)-2-(allylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-one. 47. Yield 44%. M143-144°C. 1H NMR (CDCl3, 400 MHz) δ 15.13 (1H, s ), 10.30 (1H, t, J = 6.0 Hz), 7.52 (2H, d, J = 7.7 Hz), 7.36 (3H, t, J = 7.5 Hz), 7.28 (1H, t, J = 7.3 Hz), 7.06 (2H, dd, J = 9.2, 4.8 Hz), 6.89 (1H, d, J = 14.2 Hz), 6.15 (1H, dd, J = 14.0, 6.4 Hz), 6.02 – 5.86 (1H, m ), 5.34 (2H, s), 4.89 (1H, s ), 3.97 (2H, t, J = 5.6 Hz), 1.98 (3H, s). Found (%): C, 77.11; H, 7.12; N, 4.70; molecular formula C19H21NO2 requires C, 77.26; H, 7.17; N, 4.74. (2E,5Z,7E,9E)-2-((3-(dimethylamino)propyl)amino)-6-hydroxy-10- phenyldeca-2,5,7,9-tetraen-4-one. 50. Yield 40%. Mp 121-123°C. Calcd. (%). 1H NMR (DMSO, 400 MHz) δ 15.19 (1H, s ), 10.32 (1H, s ), 7.52 (2H, d, J = 7.5 Hz), 7.36 (2H, t, J = 7.5 Hz), 7.28 (1H, t, J = 7.3 Hz), 7.11 – 6.98 (2H, m ), 6.88 (1H, dd, J = 15.4, 6.4 Hz), 6.15 (1H, d, J = 14.0 Hz), 5.31 (1H, s ), 4.82 (1H, s ), 3.32 (2H, dd, J = 13.0, 6.3 Hz), 2.25 (2H, t, J = 6.8 Hz), 2.13 (6H, s), 1.99 (3H, s ), 1.75 – 1.57 (2H, m ). Found (%): C, 74.02; H, 8.24; N, 8.19; molecular formula C21H28N2O2 requires C, 74.08; H, 8.29; N, 8.23. (2E,5Z,7E,9E)-2-(sec-butylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4- one. 51. Yield 36%. Mp 159-162°C. 1H NMR (DMSO, 400 MHz) δ 15.05 (1H, s ), 10.30 (1H, d, J = 9.4 Hz), 7.52 (2H, d, J = 7.5 Hz), 7.36 (2H, t, J = 7.5 Hz), 7.28 (1H, t, J = 7.2 Hz), 7.04 (2H, dt, J = 14.3, 7.4 Hz), 6.88 (1H, d, J = 14.7 Hz), 6.23 – 6.08 (1H, m ), 5.31 (1H, s ), 4.81 (1H, s ), 3.68 – 3.53 (1H, m ), 2.01 (3H, s ), 1.50 (2H, dt, J = 14.0, 6.9 Hz), 1.16 (3H, d, J = 6.4 Hz), 0.89 (3H, t, J = 7.3 Hz). Found (%): C, 77.22; H, 7.97; N, 4.46; molecular formula C20H25NO2 requires C, 77.14; H, 8.09; N, 4.50. (2E,5Z,7E,9E)-6-hydroxy-2-(isobutylamino)-10-phenyldeca-2,5,7,9-tetraen-4- one. 52. Yield 45%. Mp 170-173°C. 1H NMR (DMSO, 400 MHz) δ 15.06 (1H, s ), 10.44 (1H, t, J = 5.9 Hz), 7.52 (2H, d, J = 7.4 Hz), 7.37 (2H, dd, J = 14.1, 6.9 Hz), 7.28 (1H, t, J = 7.3 Hz), 7.12 – 6.98 (2H, m ), 6.89 (1H, t, J = 11.3 Hz), 6.27 – 6.03 (1H, m ), 5.32 (1H, s ), 4.85 (1H, s ), 3.15 (2H, t, J = 6.4 Hz), 1.99 (3H, s ), 1.78 (1H, d, J = 6.4 Hz), 0.94 (6H, d, J = 6.7 Hz). Found (%): C, 76.96; H, 8.01; N, 4.39; molecular formula C20H25NO2 requires C, 77.14; H, 8.09; N, 4.50. (2E,5Z,7E,9E)-6-hydroxy-2-((2-methoxyethyl)amino)-10-phenyldeca-2,5,7,9- tetraen-4-one. 53. Yield 65%. Mp 153-155°C. 2.2. Insulin and lysozyme aggregates formation Human insulin (Private Joint Stock Company «On the production of insulin «Indar», Ukraine) was dissolved at 340 µM concentration in 0,1 mM HCl solution (pH 2). Fibrils were formed by incubating the protein solution in a thermomixer incubator at 65 °C for about 5 h. To prepare lysozyme aggregates, lysozyme of hen egg white (Sigma-Aldrich Co. USA) was dissolved at 1 mM concentration in 0,1 mM HCl solution (pH 2) and incubated in a thermomixer incubator at 65 °C for 24 hours. Calcd. (%). 1H NMR (DMSO, 400 MHz) δ 15.15 (1H, s ), 10.30 (1H, s ), 7.52 (2H, d, J = 7.4 Hz), 7.37 (2H, t, J = 7.5 Hz), 7.28 (1H, t, J = 7.3 Hz), 7.14 – 6.96 (2H, m ), 6.98 – 6.80 (1H, m ), 6.16 (1H, dd, J = 13.4, 6.8 Hz), 5.32 (1H, s ), 4.85 (1H, s ), 3.49 (4H, d, J = 6.6 Hz), 3.30 (3H, s ), 2.00 (3H, s ). Found (%): C, 72.89; H, 7.34; N, 4.49; molecular formula C19H23NO3 requires C, 72.82; H, 7.40; N, 4.47. (2E,5Z,7E,9E)-6-hydroxy-2-((3-methoxypropyl)amino)-10-phenyldeca-2,5,7,9- tetraen-4-one. 54. Yield 57%. Mp 106-107°C. 1H NMR (DMSO, 400 MHz) δ 15.18 (1H, s ), 10.30 (1H, t, J = 5.8 Hz), 7.59 – 7.47 (2H, m ), 7.36 (2H, t, J = 7.5 Hz), 7.28 (1H, t, J = 7.3 Hz), 7.15 – 6.97 (2H, m ), 6.89 (1H, dd, J = 14.3, 7.6 Hz), 6.21 – 6.01 (1H, m ), 5.31 (1H, s ), 4.85 (1H, d, J = 9.5 Hz), 3.38 (4H, dd, J = 10.8, 4.7 Hz), 3.25 (3H, d, J = 2.4 Hz), 1.99 (3H, s ), 1.78 (2H, dd, J = 12.7, 6.2 Hz).. Found (%): C, 73.45; H, 7.65; N, 4.25; molecular formula C20H25NO3 requires C, 73.37; H, (%): C, 73.45; H, 7.65; N, 4.25; molecular formula C20H25NO3 requires C, 73.37; H, 7.70; N, 4.28 7.70; N, 4.28 7.70; N, 4.28 (2E,5Z,7E,9E)-2-(heptylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4- one. 88. Yield 62%. Mp 132-134°C. 1H NMR (DMSO, 300 MHz) δ 15.16 (1H, s), 10.35 (1H, s), 7.56 (2H, d, J = 7.3 Hz), 7.40 (3H, t, J = 7.4 Hz), 7.31 (1H, t, J = 7.3 Hz), 7.09 (2H, dt, J = 16.4, 11.3 Hz), 6.91 (1H, d, J = 14.4 Hz), 6.27 – 6.07 (1H, m), 5.34 (1H, s), 4.87 (1H, s), 3.36 – 3.24 (2H, m), 2.02 (2H, s), 1.56 (2H, m), 1.31 (8H, m, J = 4.6 Hz), 0.89 (3H, m, J = 7.1 Hz). Found (%):C, 77.95; H, 8.68; N, 3.92; molecular formula C23H31NO2 requires C, 78.15; H, 8.84; N, 3.96. 2.3. Preparation of dye solutions and protein solutions β-ketoenole stock solutions were prepared by dissolving the weighted amount of the dyes at 2 mM concentration in DMSO or DMF. Working solutions of free dyes were prepared by dilution of the dye stock solutions in 50 mM Tris-HCl buffer (pH 7.9) to the concentration 2 µM. The working solutions of dye-proteins complexes were prepared by adding to the dye 2 µM solution the aliquot of monomer or fibrillar protein stock solution, the protein concentration in the working solution was thus 3,4 µM for insulin and 4 µM for lysozyme. Bovine serum albumin (BSA) was obtained from Sigma-Aldrich Co. USA. To prepare the dye-BSA working solution, to the 0.2mg/ml (3 µM) BSA solution in 50 mM Tris-HCl buffer (pH 7.9) the aliquot of the dye stock solution was added, the dye concentration in the working solution was thus 2 µM. 2.4. Spectral measurements Fluorescence excitation and emission spectra were registered using the fluorescent spectrophotometer Cary Eclipse (Varian, Austria). Fluorescence emission was excited at the maximum wavelength of excitation spectrum of corresponding dye solution. The quantum yield value of the dyes 14 and 50 (2 μM) in the presence of fibrillar insulin (13.6 μM) was determined using Rhodamine 6G solution in ethanol as the reference (quantum yield value 0.95) [15]. All the spectral- luminescent characteristics of unbound dyes in aqueous buffer were studied at room temperature. 2.5. Estimation of equilibrium constants of the dye-to-fibril binding To estimate the equilibrium constant of the dyes 14 and 50 binding to fibrillar insulin, fluorescent titration of the dyes (2 μM) upon addition of 0-45 μM of fibrillar insulin was performed. Taking into account only the points for the concentrations 2 M of protein molecules and higher, we could consider the concentration of the binding sites to be much higher than this of the dyes and thus the concentration of the free protein to be roughly equal to its total concentration. Under this assumption, the equation for the equilibrium constant K of dye-fibril binding could be written as: Cd/Cbd = 1 + (K×CF)–1 (1) (1) where Cd, Cbd and CF are total dye, fibril-bound dye and fibrillar protein concentrations respectively. Further, let us consider the totally unbound and totally bound with fibrils dye solution to have the fluorescence intensity I0 and Imax respectively. In this case the measured dye fluorescence intensity I at the fibrillar protein concentration CF could be written as I = I0×(Cd – Cbd)/Cd + Imax×Cbd/Cd, that can be transformed into: Cd/Cbd = (Imax – I0)/(I – I0) (2) (2) Together with (1), (2) gives Together with (1), (2) gives Together with (1), (2) gives Together with (1), (2) gives I – I0 = A×K×CF/(1+K×CF) (3) (3) I – I0 = A×K×CF/(1+K×CF) (3) A being the denotation for (Imax – I0) difference. Thus the experimental dependence of I – I0 on CF was approximated with the equation (3), A and K being obtained as approximation parameters. Accounting for several assumptions made, the obtained K value could be regarded as a rough estimation of the binding constant value rather than its precise value. Besides it should be reminded that the estimated binding constant is only an apparent value calculated with respect to protein globule concentration and not this of the binding sites that is unknown; actually the estimation of binding constant with respect to protein globule concentration is common for the ligand-fibril binding studies. 2.6. Computer simulations of the dye 14 dimensions 2.6. Computer simulations of the dye 14 dimensions To estimate the dimensions of the dye 14, geometry optimization of the dye structure was first performed using the PM3 method from the HyperChem 6.03 program package. Further the isosurface with the total charge density 0.002 that characterizes the molecular dimensions was built; linear dimensions i.e. length, height and width of the obtained isosurface were then estimated. 3.1. Synthesis of the β-ketoenole dyes In present work we obtained series of new compounds by the pyran ring opening reaction of 4-hydroxy-6-methyl-3 - ((2E, 4E) -5-phenylpenta-2,4-dienoyl) - 2H-pyran-2-one with primary aliphatic amines. The mechanism of this reaction was studied in [14, 16], it occurs through the nucleophilic attack of the 6-carbon atom of the pyran cycle leading to its next opening and decarboxilation. We have found that on the first step of this reaction the interaction of 4-hydroxy-6-methyl-3 - ((2E, 4E) - 5-phenylpenta-2,4-dienoyl) -2H-pyran-2-one with amines (Scheme 1) leads to the formation of the corresponding salt. When heated, it is dissociated and at the same time the amino group attacks a carbon atom of the methyl group in the pyran ring, causing its opening and subsequent decarboxylation [14, 16]. In the case of n-alkyl amines, formation of the corresponding salts, ring opening and decarboxylation occurs quite easily, but iso-amines react considerably worse and require higher reaction temperature. Scheme. 1. Synthesis of the studied β-ketoenole dyes. Scheme. 1. Synthesis of the studied β-ketoenole dyes. In the case of the tert-butylamine, the reaction stopped at the stage of the salt formation, opening of the pyran ring does not occur (Fig. 1), the behavior of - phenyl ethyl amine is similar. This observation indirectly confirms the reaction mechanism proposed in [14, 16]. Such molecules could be divided into two parts: hydrophobic ones containing phenyl moiety and polymethine chain, and hydrophilic molecules containing keto- enol fragment and tail alkylamino group. For related compounds the existence of the set of tautomeric forms of the ketoenol fragment was established by NMR [16]. The compounds described here are also able to the formation of the tautomers. In their NMR spectra in addition to the main groups of signals there are the minor signals with similar morphology corresponding to the presence of 3-6% of admixtures. According to data of LC/MS the molecular ions of these admixtures have the same molecular weight as that of the base compound that also confirms the presence of the tautomeric forms of compounds (data not presented). 3.2. Spectral properties of dyes in buffer and in the presence of native roteins 3.2. Spectral properties of dyes in buffer and in the presence of native proteins proteins The fluorescent characteristics of the 9 β-ketoenole compounds in buffer solution and in the presence of insulin, lysozyme and BSA in their native form are presented in Table 2. The excitation maxima for the studied dyes in the free state are located in the range 414-428 nm; emission maxima are in the range 523–560 nm, except for the dye 88 (emission maximum at 604 nm) and the dye 52 possessing long-wavelength emission maximum (at 606 nm) together with the short-wavelength one (545 nm). The addition of insulin, lysozyme and BSA could cause the shift of the excitation and emission spectrum maxima both to longer or shorter wavelengths for up to 35 nm. Excitation maxima in the presence of proteins are places in the range 408-435 nm respectively, while positions of the emission maxima are at 522-571 nm. Exceptions are the dye 88 with long alkyl tail that has emission bands with maxima in the range 604-611 nm respectively. For the dyes 52 (free) and the dye 47 (in the presence of insulin) emission spectra contain two bands, the long-wavelength one for the both dyes has the maximum at 606-607 nm. The large values of the shift between the excitation and emission maxima for free dyes and the dyes in the presence of proteins (98-190 nm) should be mentioned. Table 2. Spectral-luminescent properties of β-ketoenole dyes in the presence of the native proteins. Name free dye with insulin with lysozyme with BSA λex, nm λem, nm I0, a.u. λex, nm λem, nm IM, a.u. IM/I0 λex, nm λem, nm IM, a.u. IM/I0 λex, nm λem, nm IM, a.u. * – I0 was this at the wavelength 545 nm 3.2. Spectral properties of dyes in buffer and in the presence of native roteins IM/I0 13 427 542 1.7 413 542 3.7 2.2 427 525 17.7 10.4 427 536 1 0.6 14 426 539 1.3 426 539 1.6 1.2 426 540 10 7.7 410 540 7 5.4 47 415 545 2.6 415 433/ 607 4.7/ 7.3 1.8 / 2.8 410 541 1.9 7.3 415 530 5 1.9 50 414 523 0.8 427 526 2 2.5 427 526 7.7 9.6 418 523 0.8 1 51 417 554 5.5 415 539 6.8 1.2 415 543 10 1.8 417 550 3.8 0.7 52 414 545 / 606 7.5 / 15 415 553 6.2 0.8* 415 571 9.3 1.2* 408 555 5 0.7* 53 415 535 0.7 427 522 4.3 6.1 423 525 2.6 3.7 415 535 4 5.7 54 428 560 3.6 420 536 4.9 1.4 427 525 17.7 4.9 428 531 2 0.6 88 414 604 0.7 416 604 0.7 1 410 611 3.2 4.6 435 611 1.5 2.1 λex, λem – maximum wavelength of fluorescence excitation and emission spectra; I0 (IM) - fluorescence emission intensity of the dye in free state (in the presence of native protein); a.u. - arbitrary units. λex, λem – maximum wavelength of fluorescence excitation and emission spectra; λex, λem – maximum wavelength of fluorescence excitation and emission spectra; I0 (IM) - fluorescence emission intensity of the dye in free state (in the presence of native protein); a.u. - arbitrary units. I0 (IM) - fluorescence emission intensity of the dye in free state (in the presence of native protein); * – I0 was this at the wavelength 545 nm * – I0 was this at the wavelength 545 nm Fig.2. The excitation and emission spectra of the dye 50(left) and dye 14(right) in the presence of the monomeric or fibrillar insulin. Fig.2. The excitation and emission spectra of the dye 50(left) and dye 14(right) in the presence of the monomeric or fibrillar insulin. Fig.2. The excitation and emission spectra of the dye 50(left) and dye 14(right) in the presence of the monomeric or fibrillar insulin. As it was suggested, the fluorescence intensity of the free β-ketoenole dyes is low 0.7-7.5 a.u. The enhancement of the dyes fluorescence intensity in the presence of the native proteins does not exceed 10 times, and formed protein-dye complexes are of the quite low intensity. The dyes 51 and 52 with the branched alkyl substituent slightly change their emission upon the addition of proteins. 3.2. Spectral properties of dyes in buffer and in the presence of native roteins Other dyes demonstrate the quite pronounced emission increase (in 3.7-10 times) in the presence of lysozyme. The dye 14 having the tail alkyl hyrdoxy group gives fluorescent response to the presence of both lysozyme the albumin. At the same time, the compound 53 bearing the methyl ester as tail group possesses better sensitivity to insulin and BSA than to lysozyme. 3.3. Spectral properties of dyes in the presence of fibrillar proteins Insertion of the dye molecule into the groove of the amyloid fibril is suggested to be the most possible model of the dye-fibril binding. With the help of the computer simulation, the dimensions of the dye 14 were estimated to be about 23Å×7.8Å×4.8Å, that is characteristic for the dyes of the studied series. Thus the studied dyes fit to the fibrillar groove formed by the β-pleated structure of the fibril, the width of which is believed to be equal to 6.5-7Å [12]. Thus, we can expect the formation of the dye-fibril fluorescent complex due to the fixation of the dye molecules in the fibril groove. The fluorescent characteristics of the 9 β-ketoenole compounds in the presence of fibrillar aggregates of insulin and lysozyme are presented in Table 3. For the majority of the studied β-ketoenole dyes, the addition of the fibrillar insulin results in the long-wavelength shift of the excitation maximum wavelength for up to 22 nm (except of the dye 88 with the 3 nm short-wavelength shift). The short-wavelength shifts of the emission maximum wavelength for up to 26 nm were also observed for the majority of the dyes except 50 (almost no change), 88 (7-nm long-wavelength shift) and 54 (two maxima shifted to the short- and long- wavelength region with respect to the free dye were observed). Table 3. Table 3. Table 3. Spectral-luminescent properties of β-ketoenole dyes in the presence of the aggregated proteins Name with fibrilar insulin with fibrilar lysozyme Spectral-luminescent properties of β-ketoenole dyes in the presence of the aggregated proteins Name with fibrilar insulin with fibrilar lysozyme Spectral-luminescent properties of β-ketoenole dyes in the presence of the λex, nm λem, nm IF, a.u. IF/IM λex, nm λem, nm IF, a.u. IF/IM 13 441 528 80 21.6 441 517 10 0.6 14 430 531 97 60.6 427 540 37 3.7 47 436 528 49.7 6.8* 426 532 5.9 0.3 50 436 524 25 12.5 426 522 27.7 3.6 51 437 528 20.9 3.1 415 543 8.5 0.8 52 435 535 17 2.7 408 555 9.2 1.0 53 434 525 / 591 22 5.1 440 526 22.7 8.7 54 436 545 / 588 33.7 / 39.6 6.9 / 8.1 441 517 10 0.6 88 411 611 1.9 2.7 410 611 3.5 1.1 λex, λem - maxima of fluorescence excitation and emission spectra; λex, λem - maxima of fluorescence excitation and emission spectra; IF - emission intensity of dye in the presence of fibrillar form of the insulin and lysozyme; IF/ IM - emission increasing of the dye in the presence of corresponding proteins; a.u. - arbitrary units; * – IM was this at the wavelength 607 nm The presence of fibrillar insulin leads to the enhancement of the dye fluorescence intensity (IF) as compared to that of both free dye (I0) and the dye in the presence of native insulin (IM); the highest values of the IF/IM ratio were observed for the dyes 13 and 14 containing “short” unbranched propylamino and hydroxyethylamino groups as substituents (22 and 61 times respectively). The rather high fluorescent response with IF/IM between 7 and 13 times was also observed for the dyes 47, 50 and 54 containing allylamino, dimethylaminopropylamino and methoxypropylamino substituents. The other dyes did not demonstrate significant IF/IM values. It should be also mentioned that the fluorescence quantum yield value for the dyes 14 and 50 in the presence if fibrillar insulin is rather high and equals to 0.12 and 0.15 respectively. For the dyes 14 and 50 the titration with fibrillar insulin was performed; the dependence of the dye fluorescence intensity on the concentration of the added fibrils allowed us to estimate the equilibrium constant of the dye to fibril binding. This estimation gave the values of the binding constant K = (2.70.9)×105 M-1 for the dye 14 and (2.51.4)×105 M-1 for the dye 50, being thus of the same order of magnitude. Spectral-luminescent properties of β-ketoenole dyes in the presence of the As for the fibrillar lysozyme, its presence leads to the long-wavelength shift of the excitation spectrum maximum wavelength up to 25 nm, except for the dyes 51, 52 and 88 that slightly shift their maxima to the short-wavelength shift region. At the same time, for the emission maximum the wavelength shift was predominantly to the short-wavelength region (up to 43 nm for the dye 54), exceptions being the dyes 14, 52 and 88 (long-wavelength shifts for up to 10 nm). As for the emission intensity, only for three dyes 14, 50 and 53 the fluorescence sensitivity to the fibrillar lysozyme overpasses that to native protein. The IF/IM values for these three dyes are not significant: the highest value is for the dye 53 (8.7 times), while for the dyes 14 and 50 it is about 3.7. Other compounds possess the fluorescent sensitivity to lysozyme aggregates equal to or lower than this for the native protein. It should be mentioned that the insignificant IF/IM value observed for the most insulin-sensitive dye 14, as well as for several other dyes in the presence of fibrillar lysozyme is partially connected with the fluorescent sensitivity of these dyes to the native lysozyme (IM value). The binding to native lysozyme leading to the fixation of the fluorescent conformation of the molecule and thus to the fluorescent response occurs for the majority of the studied dyes. That could be explained by the high content of the charges amino acids in the structure of this protein (8 negatively charged residues and 18 positively charged ones) and thus attributed to the electrostatic interaction. Generally the β-ketoenole molecules are uncharged, so its affinity to the charged amino acid residues would be higher in the case of the molecule’s transition to the zwitterionic tautomer form (Fig. 3). Fig. 3. Zwitterionic tautomer form of β-ketoenole dye Fig. 3. Zwitterionic tautomer form of β-ketoenole dye It was observed that some of β-ketoenoles are even more sensitive to the native lysozyme than to its well-structured beta-pleated aggregates that formed the binding places in the fibrillar grooves. Thus we could suggest that electrostatic interaction with charged groups mainly drive the interaction of the dye molecules to the lysozyme. The reorganization of the protein structure to beta-pleated aggregates does not lead to the essential increase in the number of tightly bound and thus fluorescent molecules, and can even decrease their number. linear detection range To examine the applicability of the dye 14 bearing 2-hydroxyethyl group as amyloid-sensitive probe for the quantification of amyloid insulin, we performed titration of the 2 µM dyes solutions with increasing amounts of the aggregated protein (Fig. 4). The lower limit for the fibrillar insulin detection by the dye 14 was determined as equal to the insulin concentration leading to the two times dye fluorescence intensity increase. The upper limit of the detection range was determined as the maximum concentration where the dependence of dye fluorescence intensity on the fibrils concentration is still linear. Thus the 2μM concentration of the dye 14 allows fluorescent detection of the insulin amyloid fibrils in the concentration range 0.2-9 μM (1-50 μg/ml) (R = 0,97703; Fig. 4); the upper limit of the fibrillar insulin detection by the dye 14 thus exceeds that for the amyloid-specific dye Thioflavin T (0.5-25 μg/ml) [17]. Fig. 4. Titration of 14 dye by fibrillar insulin. The linear concentration range 0.2-9 μM (1-50 μg/ml) (linear, R = 0,977). The 2 µM dye solution was used. Fig. 4. Titration of 14 dye by fibrillar insulin. The linear concentration range 0.2-9 μM (1-50 μg/ml) (linear, R = 0,977). The 2 µM dye solution was used. μM (1-50 μg/ml) (linear, R = 0,977). The 2 µM dye solution was used. It should be mentioned also that the large shifts between the excitation and emission maxima for the fibril-bound dye (about 100 nm), good fluorescence intensity increase, rather high quantum yield and sufficient detection range make the dye 14 promising as probe for fluorescent detection of the fibrillar aggregates. 4. CONCLUSION The series of β-ketoenole dyes was firstly synthesized and characterized, their fluorescent properties as potential probes for the sensing of amyloid aggregates of proteins were studied. These dyes are low fluorescent when free, but have the large shift between excitation and emission maxima reaching 190 nm. The majority of dyes possess the insignificant sensitivity to the native proteins insulin and bovine serum albumin, while their fluorescent response on the presence of native lysozyme is more pronounced (emission intensity increase up to 10 times was observed). This could be explained by the high content of the charged aminoacids in the protein and allows suggesting about high impact of the electrostatic interaction into this binding. The sensitivity of these dyes to fibrillar proteins depends strongly of the nature of their functional substitutens. The most intensive fluorescent response on the presence of amyloid fibrils of insulin is observed for dyes 13 and 14 that carry “short” propylamino and or 2-hydroxyethylamino groups (fluorescence intensity in the presence of fibrils exceeds that for the native proteins in 22 and 60 times respectively). For the dyes containing 2-hydroxyethylamino (14) and dimethylaminopropylamino groups (50), the values of equilibrium constant of binding to fibrillar insulin were found to be (2.70.9)×105 M-1 and (2.51.4)×105 M-1 respectively. At the same time, the studied dyes demonstrate significantly lower fluorescent sensitivity to fibrillar lysozyme as compared to fibrillar insulin. fluorescent sensitivity to fibrillar lysozyme as compared to fibrillar insulin. The dye 14 demonstrates the good fluorescent response and linear range (1-50 μg/ml) of amyloid fibril detection, large value of the shift between excitation and emission maxima (about 100 nm), its quantum yield in the complex with fibrillar insulin reaches 0.15. We suggest this dye as prospective fluorescent molecule for the sensing of β-pleated protein aggregates and investigation of the aggregation reaction. Thus we suggest the β-ketoenole dyes as prospective fluorescent molecules for the design on their base of the probes for the detection of the β-pleated protein aggregates and investigation of the aggregation reaction. This work was supported by the H2020-MSCA-RISE-2014-RISE-645628 - METCOPH project REFERENCES 1. Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools protein characterization. Pharm res 2008; 25(7):1487-99. doi: 10.1007/s11095-007-9516- 9. 2. Johnson J, Spence M. The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Life Technologies Inc, Eugene, 11th ed; 2010. DOI: 10.1134/S0006297911110101 3. Tokar V, Losytskyy M, Ohulchanskyy T, Kryvorotenko D, Kovalska V, Balanda A, Dmytruk I, Prokopets V, Yarmoluk S, Yashchuk V. Styryl dyes as two-photon excited fluorescent probes for DNA detection and two-photon laser scanning fluorescence microscopy of living cells. J Fluoresc 2010; 20(4): 865-72. DOI: 10.1007/s10895-010-0630-4 3. Tokar V, Losytskyy M, Ohulchanskyy T, Kryvorotenko D, Kovalska V, Balanda A, Dmytruk I, Prokopets V, Yarmoluk S, Yashchuk V. Styryl dyes as two-photon excited fluorescent probes for DNA detection and two-photon laser scanning fluorescence microscopy of living cells. J Fluoresc 2010; 20(4): 865-72. DOI: 10.1007/s10895-010-0630-4 4. Wolfbeis O, Demchenko A. Optimized Dyes for Protein and Nucleic Acid Detection Advanced Fluorescence Reporters in Chemistry and Biology. III Applications in Sensing and Imaging, Chapter: Optimized Dyes for Protein and Nucleic Acid Detection. Springer Berlin Heidelberg; 2014: 161-99. 5. Naiki H, Higuchi K, Hosokawa M, Takeda T. Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem 1989; 177: 244–49. DOI:10.1016/0003-2697(89)90046-8 6. Klunk W, Wang Y, Huang G, Debnath M, Holt D, Mathis C. Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and 6. Klunk W, Wang Y, Huang G, Debnath M, Holt D, Mathis C. Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain. Life Sci 2001; 69: 1471-84. DOI:10.1016/S0024- 3205(01)01232-2 readily enter the brain. Life Sci 2001; 69: 1471-84. DOI:10.1016/S0024- 3205(01)01232-2 7. Schmidt M, Schuck T, Sheridan S, Kung M, Kung H, Zhuang Z, Bergeron C, Lamarche J, Skovronsky D, Giasson B, Lee V, Trojanowski J. The fluorescent congo red derivative,trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4- hydroxy)styrylbenzene (BSB), labels diverse beta-pleated sheet structures in postmortem human neurodegenerative disease brains. Am J Pathol 2001; 159: 937-43. DOI: http://dx.doi.org/10.1016/S0002-9440(10)61769-5 8. Volkova K, Kovalska V, Inshin D, Slominskii Y, Tolmachev O, Yarmoluk S. Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay. Biotech Histochem 2011; 86(3): 188-91. DOI: 8. Volkova K, Kovalska V, Inshin D, Slominskii Y, Tolmachev O, Yarmoluk S. Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay. Biotech Histochem 2011; 86(3): 188-91. DOI: 9. REFERENCES Kovalska V, Losytskyy M, Tolmachev O, Slominskii Y, Segers-Nolten G, Subramaniam V, Yarmoluk S. Tri- and pentamethine cyanine dyes for fluorescent detection of α-synuclein oligomeric aggregates. J Fluoresc 2012; 22(6): 1441-48. DOI: 10.1007/s10895-012-1081-x 9. Kovalska V, Losytskyy M, Tolmachev O, Slominskii Y, Segers-Nolten G, Subramaniam V, Yarmoluk S. Tri- and pentamethine cyanine dyes for fluorescent detection of α-synuclein oligomeric aggregates. J Fluoresc 2012; 22(6): 1441-48. DOI: 10.1007/s10895-012-1081-x 10. Volkova K, Losytskyy M, Fal K, Derevyanko N, Slominskii Y, Tolmachov O, Yarmoluk S. Hydroxy and methoxy substituted thiacarbocyanines for fluorescent detection of amyloid formation. J Fluoresc 2011; 21(2): 775-84. DOI: 10.1007/s10895-010-0770-6 11. 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Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands. Bioorg Med Chem 2012; 20(1): 330-34. DOI:10.1016/j.bmc.2011.10.083 14. Aït-Baziz N, Rachedi Y, Hamdi M, Silva A, Balegroune F, Thierry R, Sellier N. 4-hydroxy-6-methyl-3-(5-phenyl-2E,4E-pentadien-1-oyl)-2H-pyran-2-one: Synthesis and reactivity with amines. J Heterocyclic Chem 2004; 41(4): 587-91. DOI: 10.1002/jhet.5570410417 15. Kubin R, Fletcher A. Fluorescence quantum yields of some rhodamine dyes. J Luminesc 1982; 27(4): 455-62. doi:10.1016/0022-2313(82)90045-X 16. Chergui D, Hamdi M, Baboulène M, Spéziale V, Lattes A. Réactivité des cinnamoyl-3 pyrones-2 vis à vis des amines primaires. J Heterocyclic Chem 1986; 23(6): 1721-24. DOI: 10.1002/jhet.5570230624 17. Kuperman M, Chernii S, Losytskyy M, Kryvorotenko D, Derevyanko N, Slominskii Y, Kovalska V, Yarmoluk S. Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: the effect of N,N’-substituents. 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A Two-Stage Evolutionary Game Model for Collaborative Emergency Management Between Local Governments and Enterprises
International Journal of Disaster Risk Science/International journal of disaster risk science
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ARTICLE ARTICLE ARTICLE A Two‑Stage Evolutionary Game Model for Collaborative Emergency Management Between Local Governments and Enterprises Yanqing Wang1 · Hong Chen2,3 · Xiao Gu1 Accepted: 17 December 2023 / Published online: 4 January 2024 © The Author(s) 2023, corrected publication 2024 Accepted: 17 December 2023 / Published online: 4 January 2024 © The Author(s) 2023, corrected publication 2024 Abstract Enterprises play a vital role in emergency management, but few studies have considered the strategy choices behind such participation or the collaborative relationship with the government. This study contended that enterprises have at least three strategies regarding emergency management: non-participation, short-term participation, and long-term participation. We constructed a two-stage evolutionary game model to explore the behavioral evolution rules and evolutionary stability strate- gies of the government and enterprises, and employed numerical simulation to analyze how various factors influence the strategy selection of the government and enterprises. The results show that if and only if the utility value of participation is greater than 0, an enterprise will participate in emergency management. The evolutionary game then enters the second stage, during which system stability is affected by a synergistic relationship between participation cost, reputation benefit, and gov- ernment subsidies, and by an incremental relationship between emergency management benefit, government subsidies, and emergency training cost. This study provides a new theoretical perspective for research on collaborative emergency manage- ment, and the results provide important references for promoting the performance of collaborative emergency management. Keywords  Collaborative relationship · Emergency management · Enterprise participation · Numerical simulation · Two- stage evolutionary game International Journal of Disaster Risk Science (2023) 14:1029–1043 https://doi.org/10.1007/s13753-023-00531-9 International Journal of Disaster Risk Science (2023) 14:1029–1043 https://doi.org/10.1007/s13753-023-00531-9 www.ijdrs.com www.springer.com/13753 * Hong Chen hongchenxz@163.com * Hong Chen hongchenxz@163.com 1 School of Economics and Management, China University of Mining and Technology, Xuzhou 221116, China 2 School of Business, Jiangnan University, Wuxi 214122, China 3 Research Institute of National Security and Green Development, Jiangnan University, Wuxi 214122, China 1  Introduction governments, social organizations, enterprises, and the resi- dents (Kong and Sun 2021; Fan et al. 2022). Collaborative emergency management (CEM) has been defined as joint activity of two or more stakeholders that work together in pursuit of greater public good. It aims to eliminate wasted resources and efforts via communication, coordination, and interoperability (Kapucu et al. 2010). With the complexity of emergencies continues to increase, the success of emergency management becomes increasingly depending on effective collaboration (Oh et al. 2014). The September 11 attacks in 2001 and Hurricane Katrina in 2005 have been followed by a significant increase in the role of social forces in emergency management (Lv 2017). In par- ticular, private enterprises have made great contributions by enhancing their advantages in capital, technology, and equipment. For example, Walmart and Home Depot imple- mented effective relief efforts in responding to Hurricane Katrina, combining proactivity with logistic expertise (Wang et al. 2014). During the Covid-19 pandemic, Meituan pro- vided people with many kinds of food, necessities, medi- cines, epidemic prevention and disinfection supplies, and other errand services, thereby deploying its professional The mounting frequency and intensity of various emergen- cies are placing increased pressure on emergency manage- ment (Bosomworth et al. 2017), and the traditional gov- ernment-centered system struggles to respond effectively. To improve the market mechanism and give full play to its efficiency, professionalism, and competitiveness, mar- ket participation in emergency management is imperative (Nan et al. 2022). In many countries, enterprises, non-profit organizations, and residents have played an important role in such work. The general trend of emergency management worldwide has been to advocate collaboration between * Hong Chen hongchenxz@163.com 1 School of Economics and Management, China University of Mining and Technology, Xuzhou 221116, China 3 Research Institute of National Security and Green Development, Jiangnan University, Wuxi 214122, China (012 3456789) Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 1030 and in different regions (vertical and horizontal) (Li 2017); and collaboration between different functional agencies at the same government level (Bennett 2018). Previous stud- ies focused mainly on the synergistic relationship between government emergency management departments and social organizations. For example, Tau et al. (2017) analyzed the CEM between different countries in the Southern African Development Community (SADC). 2  Literature Review This section introduces related works about CEM and evo- lutionary game theory, discusses gaps in the literature, and explains why we used evolutionary game method and system dynamics (SD) simulation in this study. 1  Introduction Olszewski and Siebe- neck (2021) discussed the nature of CEM between different levels of government in the United States, and proposed a framework visualizing collaboration as a trust-building and outcome cycle. Chen et al. (2019b) revealed the functions of social organizations in natural hazard-related disasters’ emergency relief, and analyzed their role orientation in the emergency management network. Elsewhere, the role of emerging technologies in promoting CEM capabilities has also attracted widespread scholarly attention. For example, Wang and Chen (2022) clarified the possibility of using blockchain technology to improve the efficiency of multi- agent CEM with respect to information sharing, supervision, and reward/punishment. Xiong and Xue (2023) analyzed the feasibility of using blockchain to improve the performance of emergency material collaborative transportation. Gupta et al. (2022) put forward an evidence-based framework that emphasizes the role of artificial intelligence and cloud-based collaboration platforms in emergency management. capabilities to provide necessary guarantees for people’s live (Zhang 2021). According to the 2020 Meituan Deliv- ery Action Report against COVID-19, Meituan’s order vol- ume reached 3.96 million during the lockdown of Wuhan, with 54.1% of Meituan riders delivering orders to hospitals. Meituan took the lead in launching a “contactless delivery” service, which played an important role in avoiding contact infection. Each enterprise has unique advantages, and the strong “joint force” they can form is increasingly important when dealing with emergencies, effectively mitigating inade- quacies of government provision in emergency management. The above illustrates that enterprises have become an important actor in emergency management. Government failures in emergency response and the greater efficiency of the private sector further reflect enterprises’ important functions (Linnenluecke and McKnight 2017). However, enterprises participating in emergency management con- tinue to self-organize, rather than being included in the gov- ernment response system (Zhang and Tong 2015). Previous studies have focused mostly on the emergency management capability of public organizations, such as the government and schools (Shi 2012; Shah et al. 2018), while giving little attention to private organizations. In particular, the greater efficiency of enterprises’ participation in emergency man- agement through the market mechanism has scarcely been explored. Therefore, this study aimed to analyze the strategy choices and game relationship between local governments and private enterprises, and to explore what incentives pro- mote CEM performance. Our findings will have important theoretical and practical significance for promoting the establishment of a sustainable, effective CEM system and improving capabilities. 2.1  Related Works on Collaborative Emergency Management (CEM) Due to the increasing frequency and severity of large-scale disasters worldwide, since the beginning of the twenty-first century, particularly following the September 11 attacks in 2001, multi-agent CEM for cross-border and major crises has become a hot topic among scholars (Ansell et al. 2010; Hart 2013). Multi-agent collaboration in emergency management has three main aspects: collaboration between the govern- ment, the market, and social organizations (Diehlmann et al. 2021); collaboration between governments at different levels 1  Introduction capabilities to provide necessary guarantees for people’s live (Zhang 2021). According to the 2020 Meituan Deliv- ery Action Report against COVID-19, Meituan’s order vol- ume reached 3.96 million during the lockdown of Wuhan, with 54.1% of Meituan riders delivering orders to hospitals. Meituan took the lead in launching a “contactless delivery” service, which played an important role in avoiding contact infection. Each enterprise has unique advantages, and the strong “joint force” they can form is increasingly important when dealing with emergencies, effectively mitigating inade- quacies of government provision in emergency management. q g p g y g The above illustrates that enterprises have become an important actor in emergency management. Government failures in emergency response and the greater efficiency of the private sector further reflect enterprises’ important functions (Linnenluecke and McKnight 2017). However, enterprises participating in emergency management con- tinue to self-organize, rather than being included in the gov- ernment response system (Zhang and Tong 2015). Previous studies have focused mostly on the emergency management capability of public organizations, such as the government and schools (Shi 2012; Shah et al. 2018), while giving little attention to private organizations. In particular, the greater efficiency of enterprises’ participation in emergency man- agement through the market mechanism has scarcely been explored. Therefore, this study aimed to analyze the strategy choices and game relationship between local governments and private enterprises, and to explore what incentives pro- mote CEM performance. Our findings will have important theoretical and practical significance for promoting the establishment of a sustainable, effective CEM system and improving capabilities. In terms of enterprises’ participation in emergency man- agement, previous studies have mainly focused on enter- prises’ own emergency management capability and role. For example, Li et al. (2021) explored the effect of personal initiative on enterprise emergency management capability, seeking an evidential basis for proposing effective measures to improve emergency management ability. Ai and Zhang (2019) studied the storage and distribution problem of mar- itime emergency supplies under the CEM of the govern- ment and enterprises using a two-stage optimization loca- tion model. Xie et al. (2022) studied the decision-making and cooperation problems for emergency medical supplies considering corporate social responsibility with the govern- ment, manufacturers, and retailers. However, few scholars have examined how enterprises can participate more effec- tively, or the CEM relationship between enterprises, govern- ments, and other participants such as social organizations and residents. 2.2  Evolutionary Game Theory Evolutionary games have occupied an increasing share of game theory literature in recent years. Evolutionary game is a combination of game theory and dynamic evolution- ary process analysis, originated from the study of biological evolutionary process in theory of evolution. In an evolution- ary game involving any form of strategic interaction, higher 1031 International Journal of Disaster Risk Science payoff strategies tend to replace lower payoff strategies over time, but there is some inertia, and players do not system- atically attempt to influence other players’ future decisions. Friedman (1998) indicated that like the traditional game, an evolutionary game model must have a game framework, including hypothesis, structure, and rules of the game, and the evolutionary game process is always carried out under such a framework. Grafica, Campari Group, Caracol, Ellamp, Distillerie Silvio Carta, and Mares) to the Covid-19 emergency; their find- ings show that with the ability to rapidly reconfigure pro- cess and innovation, these enterprises have made important contributions to early recovery from Covid-19 by providing needed supplies. Therefore, this study explored the strat- egy choices and influencing factors of enterprises and gov- ernments in CEM using the combination of evolutionary game theory and SD simulation. This method should better explain the interaction between collaborating governments and enterprises. Evolutionary game has become a classical method for studying the multi-agent collaborative governance mecha- nism, and is widely used in research on the formation and evolution process of social habits, rules, and institutions (Liu et al. 2015; Semasinghe et al. 2015). In the field of organizational cooperation, evolutionary game theory has been widely used to various problems, such as industry- university-research cooperation (Cao and Li 2020), market- supervision cooperation (Lu et al. 2018), environmental governance cooperation (Chen et al. 2019a), public goods game (Wang et al. 2013), and sustainable humanitarian sup- ply chains (Li et al. 2019). It is also widely applied in the research of CEM involving multiple stakeholders. For exam- ple, Du and Qian (2016) used evolutionary game theory to explore the interactions between government and non-profit organizations in emergency mobilization within China. Shao et al. (2022) established an evolutionary game model for the collaborative governance of construction waste with multi-agent participation, and integrated system dynamics (SD) to simulate and analyze the system’s strategy selection. Fan et al. 3  Methods Evolutionary game theory posits that players are subject to bounded rationality, and uses the concept of natural selection (rather than profit maximization) for economic analysis. At the beginning of the game, both players of the game can- not determine their optimal strategies, but find the optimal strategy combination through continuous imitation, trial, and error and learning, finally reach a stable state (Levine and Pesendorfer 2007). One of the key concepts in evolu- tionary game theory is evolutionary stable strategy (ESS), which is when the vast majority of individuals in the popu- lation choose a certain strategy; the group that chooses the mutation strategy cannot invade the group that contains the vast majority of individuals as it includes fewer individuals (Wang et al. 2022). When analyzing multi-variable nonlinear complex systems, SD can not only solve the modeling problem of complex systems, but also perform quantitative coordina- tion and optimization of the relationships among system elements under the overall framework. System dynamics model describes the causality and feedback relationship between system elements through a casual flow diagram (Fan et al. 2021), and carries out simulation experiments by establishing computer-based models. Specifically, based on evolutionary game modeling, this study used a SD model to simulate the evolutionary game process and the dynamic equilibrium results of CEM between governments and enter- prises, and analyze the effect of various factors on the ESS. Although the importance of enterprise participation in emergency management has been widely recognized, there is little research on how they effectively collaborate with the government. Most game studies on CEM have focused on the cooperation between local governments, or between governments and social organizations, while few have inves- tigated collaboration between governments and enterprises. Research on collaborative governance of emergencies between the government and enterprises mainly analyzed the challenges of enterprise participation in emergency management in a theoretical framework, or illustrated the importance of their participation through case studies. For example, Puliga and Ponta (2021) investigated the fast inno- vation reactions of different enterprises (including Isinnova, f Nearly all previous studies are based on a binary oppo- sition perspective, such as “(not) governance,” “(not) par- ticipation,” “(not) incentive,” and “(not) coordination.” In practice, however, enterprises have at least three strate- gies of non-participation, short-term participation, and long-term participation with respect to emergency man- agement, while governments also have at least three strat- egies of non-incentive, short-term incentive, and long- term incentive. 2.2  Evolutionary Game Theory (2021) analyzed interactions among the behavio- ral strategies of government, community, and citizens amid public health emergencies by combining evolutionary game method and SD. Liu et al. (2021) examined the cooperation relationship between government and social organizations in emergency management by adopting evolutionary game theory and simulation analysis. Evidently, as an effective method to study the dynamic problems of complex systems, SD simulation is often combined with evolutionary game theory to better explore the strategy evolution mechanisms of multiple stakeholders in the CEM system. 3  Methods In other words, the classical evolutionary game method in existing studies cannot appropriately describe enterprise strategy choice regarding emergency 1032 Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 4  Construction of a Two‑Stage Evolutionary Game Model management. Furthermore, in a traditional evolutionary game, the matrix is usually static, and all players select their respective strategies concurrently, making it difficult to explain some social and economic phenomena with successive decision making in real society. The two-stage game model is an effective method to analyze multi-agent interaction, multi-element collaboration, and multi-stage progression. In the emergency management process, the enterprises have priority in choosing whether or not they will participate; accordingly, the choice behavior of both players has the order of priority, but the game players can see one another’s choices before choosing their own strat- egies. Therefore, in the game of emergency management between the government and enterprises, it is more appro- priate to use a two-stage game method. Based on previous studies involving two-stage games (Petrosyan et al. 2016; Gao et al. 2017; Tang et al. 2021), we constructed a two- stage evolutionary game framework for emergency man- agement (Fig. 1), seeking to provide explanatory ideas for the evolution of government-enterprise CEM. This section details the construction of a two-stage evolu- tionary game model for CEM between the government and enterprises, calculates the system’s ESS, and tests the model stability. Fig. 1   Two-stage game matrix with two players 4.1  Game Scenario From the rational choice perspective, collaboration between the government and enterprises is premised on both sides recognizing the possibility of benefit. On the one hand, the government plays a key role in effectively promoting enter- prises’ participation in emergency management, whether by formulating preferential tax policies or providing subsidies (Luo et al. 2022). Its aims in this endeavor are to improve emergency management capability, quickly and effectively respond to various emergencies, reduce disaster losses, maximize the protection of people’s lives and property, and maintain social security and stability. On the other hand, enterprises’ participation in emergency management can provide enormous social benefit by reducing pressure on the government, while also enhancing their corporate image (Hamann et al. 2020). For example, Lüttenberg et al. (2022) indicated that enterprises’ involvement in emergency man- agement is general highly valued by consumers, leading to increase willingness to choose their products and services. In addition, active participation in emergency management is also an important part of corporate social responsibil- ity (Johnson et al. 2011). Studies have shown that fulfilling social responsibility obligations can also lead to growth and sustainability of an enterprise’s business. Li et al. (2023) indicated that during Covid-19, enterprises involved in emergency hospital construction and donations had higher excess return rates.f As shown in Fig. 1, during the process of two-stage evolutionary game, player 1 makes decisions first, i and j refer to two strategies of player 1 in the first stage. If player 1 adopts strategy i, the original benefit of player 1 and player 2 remains unchanged, which are Π1 and Π2 respectively, and then the game ends. If player 1 selects strategy j, the game enters the second stage. In this stage, player 1 has two strategies, p and q, player 2 has two strat- egies, m and n, and the evolutionary game system has four strategy combinations: (p, m), (p, n), (q, m), and (q, n); and the benefit of player 1 and player 2 in each situation is (Πp, Πm), (Πp, Πn), (Πq, Πm), and (Πq, Πn), respectively. In addition, the benefit of both sides is affected by the strat- egies of the other side, and they find their own optimal strategies in the process of constant gaming, and finally reach a stable state. 4.1  Game Scenario Enterprises’ participation strategies are also affected by participation cost and stakeholder behaviors (Zhang and Enterprises’ participation strategies are also affected by participation cost and stakeholder behaviors (Zhang and Fig. 1   Two-stage game matrix with two players The first stage The second stage Player 1 Player 2 (Π , Π ) (Π , Π ) (Π , Π ) (Π , Π ) Player 2 (Π1, Π2) The first stage 1033 International Journal of Disaster Risk Science Kong 2022).First, participating in emergency management inevitably involves extra cost, and as a limited rational sub- ject, if the cost of participating is greater than the benefit, the enterprise will not choose to participate. Second, the gov- ernment plays an important role in promoting enterprises’ participation in emergency management (Luo et al. 2022). Government subsidies can reduce the cost of participation, and thereby improve the willingness of enterprises to par- ticipate in emergency management. However, as the subject of bounded rationality, the government will choose differ- ent incentive strategies according to different participation strategies of enterprises to maximize the performance of emergency management. The differences in motivation and preference of the government and enterprises make it diffi- cult for them to construct long-term collaborative relation- ships in emergency management. the emergency rescue stage (Yang and Zhang 2018). Long-term participation first refers to the enterprises’ participation in the whole process of emergency man- agement, including prevention, preparation, response, and recovery. In addition, long-term participation means that enterprises formally become partners of the government and effectively integrate with the govern- ment’s emergency forces. Long-term participation of enterprises will contribute more to emergency manage- ment, but it also means more cost. Correspondingly, the government also has two strategies to encourage enter- prise participation in emergency management: “short- term incentive” and “long-term incentive.” Compared with short-term incentive, long-term incentive means that the government provides emergency training to enterprises participating in emergency management to improve their participation efficiency. In addition, long- term incentive aims to reduce the participation cost of enterprises through government subsidies, thereby enhance their willingness and capabilities to continue to participate. The parameters of this evolutionary game model are defined and described in Table 1. Furthermore, in the CEM of the government and enter- prises, the latter retain the right to choose whether to par- ticipate, and only if they do so can the CEM mechanism be produced. 4.1  Game Scenario In the process of collaboration, the evolution of the government and enterprise strategies has a mutually pro- moting mechanism, and both sides seek the optimal strategy combination through continuous evolution, imitation, and learning, eventually reaching a stable state. Therefore, in this study the collaborative relationships were explored based on the perspective of evolutionary game theory, aiming at seeking effective measures to improve the performance of CEM between the government and enterprises. 4.3  Construction and Analysis of the Two‑Stage Evolutionary Game Model According to the two-stage evolutionary game framework presented in Fig. 1 and the special nature of emergency man- agement, this study took the government and enterprises as the players in dynamic and static games between the two sides and constructed a pay-off matrix of two-stage evolu- tionary game with collaborative relationships in emergency management (Fig. 2). 4.2  Assumptions and Parameters Our proposed model is based on the following assumptions: (1) There are only two players in the evolutionary game system—local government and private enterprises (referred to as “government” and “enterprises”). (2) Both the government and enterprises are bounded rational. Due to information asymmetry, they have lim- ited abilities in rational cognition, analytical reasoning, and decision making. 4.3.1  Analysis of the First Stage 2   Pay-off matrix of the government and enterprises 1034 Table 1   Parameters and description Player Notation Description Enterprises (E) 퓍 The probability that the enterprise chooses “participation” in the first stage ( 0 ≤퓍≤1) 퓏 The probability that the enterprise chooses “long-term participation” in the second stage ( 0 ≤퓏≤1) CE1 The cost of the enterprise when choosing “short-term participation” in emergency management CE2 The cost of the enterprise when choosing “long-term participation” in emergency management ( CE2 > CE1) CE The fixed cost of the enterprise when choosing “non-participation” strategy RE The original benefit of the enterprise when choosing “non-participation” strategy 퓇 The reputation benefit of the enterprise when choosing “participation” strategy θ퓇 The incremental benefit of reputation for the enterprise when choosing “long-term participation” strategy ( θ > 0) ρ The benefit of emergency management when enterprises choose “short-term participation” strategy αρ The incremental benefit of emergency management when enterprises choose “long-term participation” strategy ( α > 0) Government (G) 퓎 The probability that the government chooses “long-term incentive” strategy ( 0 ≤퓎≤1) S The subsidies provided by the government for enterprises that choose “long-term participation” strategy; enter- prises that choose “short-term participation” strategy cannot receive these subsidies CG1 The collaboration cost input by the government when enterprises choose “short-term participation” strategy CG2 The collaboration cost input by the government when enterprises choose “long-term participation” strategy (CG2 > CG1) 풹 The emergency training cost paid for enterprises when the government chooses “long-term incentive” strategy κρ The incremental benefit of emergency management after the enterprises have been trained ( κ > 0) RG The benefit of emergency management when enterprises choose “not participation” strategy CG The cost of emergency management of the government when enterprises choose “not participation” strategy Fig. 2   Pay-off matrix of the government and enterprises Fig. 2   Pay-off matrix of the government and enterprises of limited rationality, enterprises do not inevitably partici- pate in emergency management even if their willingness to do so improves. The interaction mechanism of influencing factors on enterprises’ participation strategies is represented by the dotted line in Fig. 3. Zhang and Kong 2022). First, according to the theory of planned behavior (Luiza et al. 2020), some influencing fac- tors enhance enterprises’ willingness to participate, thus indirectly affecting their strategy choices regarding partici- pation. Specifically, influencing factors comprise internal enterprise factors and external environmental factors. 4.3.1  Analysis of the First Stage The interaction mechanism of influencing factors on enterprises’ participation strategies is represented b h d d li i Fi 3 Table 1   Parameters and description Player Notation Description Enterprises (E) 퓍 The probability that the enterprise chooses “participation” in the first stage ( 0 ≤퓍≤1) 퓏 The probability that the enterprise chooses “long-term participation” in the second stage ( 0 ≤퓏≤1) CE1 The cost of the enterprise when choosing “short-term participation” in emergency management CE2 The cost of the enterprise when choosing “long-term participation” in emergency management ( CE2 > CE1) CE The fixed cost of the enterprise when choosing “non-participation” strategy RE The original benefit of the enterprise when choosing “non-participation” strategy 퓇 The reputation benefit of the enterprise when choosing “participation” strategy θ퓇 The incremental benefit of reputation for the enterprise when choosing “long-term participation” strategy ( θ > 0) ρ The benefit of emergency management when enterprises choose “short-term participation” strategy αρ The incremental benefit of emergency management when enterprises choose “long-term participation” strategy ( α > 0) Government (G) 퓎 The probability that the government chooses “long-term incentive” strategy ( 0 ≤퓎≤1) S The subsidies provided by the government for enterprises that choose “long-term participation” strategy; enter- prises that choose “short-term participation” strategy cannot receive these subsidies CG1 The collaboration cost input by the government when enterprises choose “short-term participation” strategy CG2 The collaboration cost input by the government when enterprises choose “long-term participation” strategy (CG2 > CG1) 풹 The emergency training cost paid for enterprises when the government chooses “long-term incentive” strategy κρ The incremental benefit of emergency management after the enterprises have been trained ( κ > 0) RG The benefit of emergency management when enterprises choose “not participation” strategy CG The cost of emergency management of the government when enterprises choose “not participation” strategy Fig. 4.3.1  Analysis of the First Stage Whether enterprises should participate in emergency man- agement is no longer a question. The real question is what roles enterprises should play and how to improve emergency management capabilities by establishing effective public- private collaboration mechanisms. Therefore, we analyzed the motivation of enterprises to participate and its influenc- ing factors in the first stage of this evolutionary game model. In the process of emergency management, enterprises have the option of priority, the participation of enterprises can be regarded as event A, and the probability that event A occurs is x. Then P(A) = x, P ( A ) = 1 −x. (3) The government will always adopt active emergency management strategies toward social stability, security, and sustainable development. (4) Enterprises are free to choose whether or not to partici- pate in emergency management, respectively defined as “participation” and “non-participation.” The game will enter the second stage if and only if the enterprise chooses the “participation” strategy in the first stage. i (5) In the second stage, enterprises have two strategies: “short-term participation” and “long-term participa- tion.” Short-term participation refers to the tempo- rary and random participation behavior of enterprises in emergency management, which often occurs in Previous studies have shown that enterprise participation in emergency management results from many influencing and decisive factors (Lu and Zhu 2022; Luo et al. 2022; Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 1034 Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management Zhang and Kong 2022). First, according to the theory of planned behavior (Luiza et al. 2020), some influencing fac- tors enhance enterprises’ willingness to participate, thus indirectly affecting their strategy choices regarding partici- i S ifi ll i fl i f i i l of limited rationality, enterprises do not inevitably partici- pate in emergency management even if their willingness to do so improves. 4.3.2  Analysis of the Second Stage (4) Incentives from the government have a significant impact on enterprise participation in emergency management (Luo et al. 2022). They include a series of targeted poli- cies, providing participation conveniences, and benefit such as financial rewards and technical or policy support, which are conductive to enterprises’ own economic development. Incentives from the government have a significant impact on enterprise participation in emergency management (Luo et al. 2022). They include a series of targeted poli- cies, providing participation conveniences, and benefit such as financial rewards and technical or policy support, which are conductive to enterprises’ own economic development. Therefore, the second stage focuses mainly on sustainable collaboration between the government and enterprises in emergency management, considering the choice of govern- ment incentives and the choice of enterprises’ participation strategy, and exploring their behavior evolution rules and stability strategies. The benefit of the government choosing the long-term incentive strategy is EG1: The benefit of the government choosing the long-term incentive strategy is EG1: (5) EG1 =퓏[ (1 + α + κ)ρ −S −풹−CG2 ] + (1 −퓏) [ (1 + κ)ρ −풹−CG1 ] (5) Therefore, the second stage focuses mainly on sustainable collaboration between the government and enterprises in emergency management, considering the choice of govern- ment incentives and the choice of enterprises’ participation strategy, and exploring their behavior evolution rules and stability strategies. The benefit of the government choosing the short-term incentive strategy is EG2: (6) EG2 = 퓏[ (1 + α)ρ −CG2 ] + (1 −퓏) (ρ −CG1 ) (6) Then the average expected revenue is recorded as EG: Then the average expected revenue is recorded as EG: Under the premise of bounded rationality, the evolution- ary game of CEM between the government and enterprises is a process of mutual learning and dynamic self-adaptation. Therefore, we used the replicated dynamic equation to simu- late their decision-making process. The benefit of the enter- prises choosing the long-term participation strategy is EE1: (7) EG = 퓎EG1 + (1 −퓎)EG2 (7) The replicated dynamic equation of the government choosing the long-term incentive strategy is: The replicated dynamic equation of the government choosing the long-term incentive strategy is: Therefore, we used the replicated dynamic equation to simu- late their decision-making process. 4.3.1  Analysis of the First Stage The former includes emergency resources, experience, and capa- bilities possessed by enterprises themselves, while the latter are external, particularly participation policies, emergency culture, and other participants. As an economic stakeholder Second, decisive factors include extra benefit ( 휔 ) and cost (c) of participation for enterprises, which have a more significant and direct impact on their decision on whether to participate (Shi et al. 2023). Therefore, whether enter- prises participate in emergency management depends International Journal of Disaster Risk Science 1035 Decisive factors Participation willingness Participation behavior Emergency resources Emergency capabilities Emergency culture The second stage Influencing factors Emergency experience Other participants Participation policies Yes No No Yes End Fig. 3   Evolution stability in stage 1. U(ω, 풸) = ω −풸 refers to the utility function of enterprises in the first stage, ω refers to the extra benefit of enterprises’ participation in emergency management, 풸 refers to the extra cost of enterprises’ participation. Influencing factors Fig. 3   Evolution stability in stage 1. U(ω, 풸) = ω −풸 refers to the utility function of enterprises in the first stage, ω refers to the extra benefit of enterprises’ participation in emergency management, 풸 refers to the extra cost of enterprises’ participation. (2) EE2 = 퓎[ (1 + 휅)휌+ 퓇−CE1 ] + (1 −퓎) (휌+ 퓇−CE1 ) ultimately on the utility function of participation, which can be expressed as U(휔, c) = 휔−c . If and only if U(𝜔, c) = 𝜔−c > 0, then c = 1 , and the enterprise will choose participation strategy, with the evolutionary game then entering the second stage. Otherwise, x = 0, and the enter- prise chooses not to participate because the cost outweighs the benefit; correspondingly, the government will choose the non-incentive strategy (Fig. 3). (2) Then the average expected revenue is recorded as EE: Then the average expected revenue is recorded as EE: (3) EE = 퓏EE1 + (1 −퓏)EE2 (3) The replicated dynamic equation of enterprises choos- ing the long-term participation strategy is: (4) 풹퓏 풹퓉 =퓏퓉 ( EE1 −EE ) = 퓏퓉 ( 1 −퓏퓉 ) ( αρ + θ퓇+ CE1 −CE2 + S퓎퓉 ) 4.3.3  Model Stability Test (11) The replicated dynamic equation needs to meet stability requirements to ensure it will not significantly change due to slight shifts in parameters. We therefore tested stability before numerical analysis using the stability analysis method suggested by Wei et al. (2012) and Sun et al. (2023): the sen- sitivity index is calculated by changing 10% of key factors; and if the sensitivity index is less than 1, the model is stable. The sensitivity index is calculated as follows: The stability of the equilibrium point in the differential system can be calculated by analyzing the local stability of the Jacobi matrix: (12) J = [ (1 −2퓏퓉 )(훼휌+ 휃퓇+ CE1 −CE2 + S퓎퓉 ) S퓏퓉 (1 −퓏퓉 ) −S퓎퓉 (1 −퓎퓉 ) (1 −2퓎퓉 )(휅휌−풹−S퓏퓉 ) ] (12) Then the determinant of the Jacobi matrix can be calcu- lated as follows: The sensitivity index is calculated as follows: (15) SP = 1 n n ∑ i=1 |||| dPt Pt ⋅Ut dUt |||| (15) (13) Dℯ𝓉(J) =(1 −2𝓏𝓉 )(1 −2𝓎𝓉 )(훼휌+ 휃𝓇+ CE1 −CE2 + S𝓎𝓉 ) (κρ −𝒹−S𝓏𝓉 ) + 𝓏𝓉𝓎𝓉 (1 −𝓏𝓉 )(1 −𝓎𝓉 )S2 (13) where t denotes time; Sp refers to the sensitivity of system state P to parameter U; Pt represents the system state at time t; Ut is the value of the system parameter at time t; dPt and dUt respectively denote the values of changes in system state P and parameter U at time t; n is the number of sensitivity test points, and n = 2. We tested the sensitivity of some key model parameters (including 훼, 휃, CE1, CE2, κ, ρ, 풹, 퓇, andS ) by changing the value of each by +10% and −10% respec- tively. As shown by the results in Fig. 4, all the sensitivity indices are less than 1, which indicates that the model passed the stability test. Next, the trace of the Jacobi matrix can be calculated thus: (14) T퓇(J) =(1 −2퓏퓉 )(훼휌+ 휃퓇+ CE1 −CE2 + S퓎퓉 ) + (1 −2퓎퓉 )(휅휌−풹−S퓏퓉 ) (14) According to the evolutionary game theory, if Dℯ𝓉(J) > 0 and T퓇(J) < 0 are satisfied at equilibrium points of the replicated dynamic equations, they constitute ESS of the system. For each equilibrium point, the determi- nant and trace of the system are shown in Table 2. 4.3.3  Model Stability Test According to the above hypothesis, as well as the deter- minant and trace of equilibrium points in Table 2, the local stability of this evolutionary game system can be judged. Specifically: 4.3.2  Analysis of the Second Stage The benefit of the enter- prises choosing the long-term participation strategy is EE1: (8) 풹퓎 풹퓉 = 퓎퓉 (EG1 −EG ) = 퓎퓉 (1 −퓎퓉 )[(κρ −풹−S퓏퓉 ) (8) (1) EE1 =퓎[ (1 + 훼+ 휅)휌+ (1 + 휃)퓇+ S −CE2 ] + (1 −퓎) [ (1 + 훼)휌+ (1 + 휃)퓇−CE2 ] (1) Therefore, the two-dimensional dynamic system of the evolutionary game of CEM between the government and enterprise is: The benefit of the enterprises choosing the short-term participation strategy is EE2: 1036 Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management (2) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S < 0and𝜅𝜌−풹> 0 , then (1, 0) is ESS of the system. (2) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S < 0and𝜅𝜌−풹> 0 , then (1, 0) is ESS of the system. (9) ⎧ ⎪ ⎨ ⎪⎩ 풹퓏 풹퓉= 퓏퓉 EE1 −EE  = 퓏퓉 1 −퓏퓉 αρ + θ퓇+ CE1 −CE2 + S퓎퓉  풹퓎 풹퓉= 퓎퓉 EG1 −EG  = 퓎퓉 1 −퓎퓉 κρ −풹−S퓏퓉  (9) ) y (3) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 > 0 and 𝜅𝜌−풹−S < 0 , then (0, 1) is ESS of the system. Let dy /dt = 0, dz /dt = 0 then on the plane N = {(z, y); 0 ≤z, y ≤1} , this system has five equilibrium points: (0, 0), (0, 1), (1, 0), (1, 1), and ( y∗ t,z∗ t ). Among them: y (4) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S > 0 and 𝜅𝜌−풹−S > 0 , then (1, 1) is ESS of the system. y (4) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S > 0 and 𝜅𝜌−풹−S > 0 , then (1, 1) is ESS of the system. y (5) In any case, the equilibrium point ( 퓎∗ 퓉 ,퓏∗ 퓉 ) cannot be an ESS of the system; and if 퓏∗ 퓉퓎∗ 퓉 (1 −퓏∗ 퓉 )(1 −퓎∗ 퓉 )S2 < 0 , then ( 퓎∗ 퓉,퓏∗ 퓉 ) is saddle point of the system. (10) 퓎∗ 퓉= CE2 −CE1 −훼휌−휃퓇 S (10) 퓎∗ 퓉= CE2 −CE1 −훼휌−휃퓇 S (11) 퓏∗ 퓉= 휅휌−풹 S (10) (11) 퓏∗ 퓉= 휅휌−풹 S 5.1  Numerical Analysis Under Different Situations To show the dynamic evolution process of enterprise and government strategy selection more intuitively, we adopted numerical simulation in this study, using MATLAB to simulate the dynamic evolution trajectory from initial point to equilibrium point in several situations. The simulation results are shown in Fig. 5. g } Situation (c): When 𝛼𝜌+ 𝜃퓇> CE2 −CE1 , 𝜅𝜌< 풹+ S , and 훼= 0.4, 휃= 0.3, CE1 = 10, CE2 = 18, 휅= 0.7, 휌= 5, 풹= 2, 퓇= 10, S = 2, the evolution step of the system is shown in Fig. 5c. In this situation, the sum of emergency management benefit and reputation benefit exceeds the incremental cost of participation, leading enterprises to choose long-term participation strategy regardless of which incentive strategy is pursued by the govern- ment. For the government, however, the subsidies and emergency management training cost paid to enterprises pursuing long-term participation exceed the incremental benefit for emergency management brought by trained enterprises. Therefore, government will pursue the short- term incentive strategy, and the evolution result of the system finally converges to (1, 0). In this situation, the ESS is {long-term participation, short-term incentive}. Situation (d): When 𝛼𝜌+ 𝜃퓇+ S > CE2 −CE1 , 𝜅𝜌> 풹+ S , and 훼= 0.5, 휃= 0.6, CE1 = 10, CE2 = 16, 휅= 0.8, 휌= 5, 풹= 1, 퓇= 10, S = 1, the evolution step of the system is shown in Fig. 5d. This situation entails that the incremental benefit of enterprises’ participation in emergency manage- g Situation (c): When 𝛼𝜌+ 𝜃퓇> CE2 −CE1 , 𝜅𝜌< 풹+ S , and 훼= 0.4, 휃= 0.3, CE1 = 10, CE2 = 18, 휅= 0.7, 휌= 5, 풹= 2, 퓇= 10, S = 2, the evolution step of the system is shown in Fig. 5c. In this situation, the sum of emergency management benefit and reputation benefit exceeds the incremental cost of participation, leading enterprises to choose long-term participation strategy regardless of which incentive strategy is pursued by the govern- ment. For the government, however, the subsidies and emergency management training cost paid to enterprises pursuing long-term participation exceed the incremental benefit for emergency management brought by trained enterprises. Therefore, government will pursue the short- term incentive strategy, and the evolution result of the system finally converges to (1, 0). In this situation, the ESS is {long-term participation, short-term incentive}. 5  Numerical Analysis This section analyzes the system’s evolution path under different situations by adopting SD simulation methods, as well as the effects of cost difference, government subsidies, (1) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 < 0 and κρ −풹< 0 , then (0, 0) is ESS of the system. Table 2   Dℯ𝓉(J) and T퓇(J) in equilibrium points Equilibrium points Dℯ𝓉(J) T퓇(J) (0, 0) (훼휌+ 휃퓇+ CE1 −CE2 ) (κρ −풹) (훼휌+ 휃퓇+ CE1 −CE2 ) + (휅휌−풹) (1, 0) −(훼휌+ 휃퓇+ CE1 −CE2 + S) (κρ −풹) (훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹) (0, 1) −(훼휌+ 휃퓇+ CE1 −CE2 ) (κρ −풹−S) −(훼휌+ 휃퓇+ CE1 −CE2 ) + (휅휌−풹−S) (1, 1) (훼휌+ 휃퓇+ CE1 −CE2 + S) (κρ −풹−S) −(훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹−S) (퓏∗ 퓉, 퓎∗ 퓉) 퓏∗ 퓉퓎∗ 퓉 (1 −퓏∗ 퓉 )(1 −퓎∗ 퓉 )S2 0 Table 2   Dℯ𝓉(J) and T퓇(J) in equilibrium points Equilibrium points Dℯ𝓉(J) T퓇(J) (0, 0) (훼휌+ 휃퓇+ CE1 −CE2 ) (κρ −풹) (훼휌+ 휃퓇+ CE1 −CE2 ) + (휅휌−풹) (1, 0) −(훼휌+ 휃퓇+ CE1 −CE2 + S) (κρ −풹) (훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹) (0, 1) −(훼휌+ 휃퓇+ CE1 −CE2 ) (κρ −풹−S) −(훼휌+ 휃퓇+ CE1 −CE2 ) + (휅휌−풹−S) (1, 1) (훼휌+ 휃퓇+ CE1 −CE2 + S) (κρ −풹−S) −(훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹−S) (퓏∗ 퓉, 퓎∗ 퓉) 퓏∗ 퓉퓎∗ 퓉 (1 −퓏∗ 퓉 )(1 −퓎∗ 퓉 )S2 0 Table 2   Dℯ𝓉(J) and T퓇(J) in equilibrium points 1037 International Journal of Disaster Risk Science shown in Fig. 5b. Under this situation, the sum of the increase of emergency management benefit, reputation benefit, and government subsidies obtained by choosing long-term participation strategy is outweighed by the incremental cost of participation. Consequently, enter- prises will choose short-term participation strategy. For the government, the incremental benefit of enterprises supporting emergency management after training exceeds the training cost paid by the government. Therefore, the government will choose the long-term incentive strategy, and the evolution result of the system finally converges to (0, 1). In this situation, the ESS is {short-term participa- tion, long-term incentive}. reputation benefit, and training cost on the evolutionary game paths of long-term participation and short-term par- ticipation, thereby further exploring the collaboration mech- anism between enterprises and governments. Fig. 4   Parameter sensitivity index chart 5.1  Numerical Analysis Under Different Situations Situation (d): When 𝛼𝜌+ 𝜃퓇+ S > CE2 −CE1 , 𝜅𝜌> 풹+ S , and 훼= 0.5, 휃= 0.6, CE1 = 10, CE2 = 16, 휅= 0.8, 휌= 5, 풹= 1, 퓇= 10, S = 1, the evolution step of the system is shown in Fig. 5d. This situation entails that the incremental benefit of enterprises’ participation in emergency manage- Situation (c): When 𝛼𝜌+ 𝜃퓇> CE2 −CE1 , 𝜅𝜌< 풹+ S , and 훼= 0.4, 휃= 0.3, CE1 = 10, CE2 = 18, 휅= 0.7, 휌= 5, Situation (a): When 𝛼𝜌+ 𝜃퓇< CE2 −CE1 , κρ < 풹 , and 훼= 0.2, 휃= 0.3, CE1 = 10, CE2 = 16, 휅= 0.4, 휌= 5, 풹= 4, 퓇= 10, S = 1, the evolution step of the system is shown in Fig. 5a. In this situation, the incremental benefit for the government to pay for training an enterprise in emergency management is disproportionate to the train- ing cost when pursuing a long-term incentive strategy. Consequently, the government is unwilling to pay train- ing cost for enterprises and tends to pursue a short-term incentive strategy. Meanwhile, the sum of incremental emergency management benefit and reputation benefit for enterprises from long-term participation is outweighed by the extra cost paid. Therefore, as bounded rational stakeholders, enterprises will choose the short-term par- ticipation strategy to maximize self-interest. Finally, the evolution result of the system converges to (0, 0), and the ESS is {short-term participation, short-term incentive}. Situation (b): When 𝛼𝜌+ 𝜃퓇+ S < CE2 −CE1, 𝜅𝜌> 풹 , and 훼= 0.5, 휃= 0.5, CE1 = 10, CE2 = 17, 휅= 0.6, 휌= 5, 풹= 2, 퓇= 10, S = 2, the evolution step of the system is Situation (a): When 𝛼𝜌+ 𝜃퓇< CE2 −CE1 , κρ < 풹 , and 훼= 0.2, 휃= 0.3, CE1 = 10, CE2 = 16, 휅= 0.4, 휌= 5, Situation (a): When 𝛼𝜌+ 𝜃퓇< CE2 −CE1 , κρ < 풹 , and 훼= 0.2, 휃= 0.3, CE1 = 10, CE2 = 16, 휅= 0.4, 휌= 5, 풹= 4, 퓇= 10, S = 1, the evolution step of the system is shown in Fig. 5a. In this situation, the incremental benefit for the government to pay for training an enterprise in emergency management is disproportionate to the train- ing cost when pursuing a long-term incentive strategy. 5.2.1  Effect of Enterprise Participation Cost on Evolution Results ment exceeds the training cost and subsidies paid by the government. Consequently, whichever strategy enterprises choose, the government will pursue long-term incentive strategy. For enterprises, the sum of incremental emer- gency management benefit, reputation benefit, and gov- ernment subsidies exceeds the extra cost of pursuing long- term participation. Therefore, enterprises will choose the long-term participation strategy, and the evolution result of the system finally converges to (1, 1), the ESS under this situation is {long-term participation, long-term incentive}. Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, 풹= 6, 퓇= 10, S = 15. T h e i n i t i a l p r o b a b i l i t y i s 퓏0 = 0.5and퓎0 = 0.5 ; the incremental cost of enterprises choosing long-term versus short-term participation is Δ풸= CE2 −CE1 , varying between [0, 10]; and the evolution result of the system is shown in Fig. 6a. The result of the numerical analysis shows that if 0 ≤Δ풸≤6 , an enterprise’s ESS converges to long-term participation, and the evolution speed decreases as Δ풸 rises; if Δ풸≥8 , then the enterprises’ ESS converges to short-term participation, and the evolu- tion speed increases as Δ풸 rises. Compared with short-term participation, enterprises are willing to adopt long-term participation strategy when the cost increment of long-term participation is small. On the contrary, as the cost increment 5.1  Numerical Analysis Under Different Situations Consequently, the government is unwilling to pay train- ing cost for enterprises and tends to pursue a short-term incentive strategy. Meanwhile, the sum of incremental emergency management benefit and reputation benefit for enterprises from long-term participation is outweighed by the extra cost paid. Therefore, as bounded rational stakeholders, enterprises will choose the short-term par- ticipation strategy to maximize self-interest. Finally, the evolution result of the system converges to (0, 0), and the ESS is {short-term participation, short-term incentive}. Situation (b): When 𝛼𝜌+ 𝜃퓇+ S < CE2 −CE1, 𝜅𝜌> 풹 , and 훼= 0.5, 휃= 0.5, CE1 = 10, CE2 = 17, 휅= 0.6, 휌= 5, 풹= 2, 퓇= 10, S = 2, the evolution step of the system is Fig. 4   Parameter sensitivity index chart meter sensitivity Fig. 4   Parameter sensitivity index chart Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 1038 Fig. 5   Evolution steps of the system under different situations Fig. 5   Evolution steps of the system under different situations 5.2.1  Effect of Enterprise Participation Cost on Evolution Results 5.2  Numerical Analysis of Parameter Variation With the initial probability unchanged, we analyzed the effect of each parameter on the evolutionary game path. The results of the numerical analysis are shown in Fig. 6. International Journal of Disaster Risk Science 1039 1039 ational Journal of Disaster Risk Science ng-term participation increases gradually, enterprises adopt short-term participation strategy. 5.2.2  Effect of Reputation Benefit on Evolution Results Otherparametersare:훼= 0 6 휃= 0 4 κ = 0 6 ρ = 5 풹= 6  Effect of parameter variation on evolution results. Δ풸 refers to the incremental cost of enterprises choosing long-term versus short-term pation strategy. Fig. 6   Effect of parameter variation on evolution results. Δ풸 refers to the incremental cost of enterprises choosing long-term versus short- participation strategy. Fig. 6   Effect of parameter variation on evolution results. Δ풸 refers to the incremental cost of enterprises choosing long-term versus short-term participation strategy. of long-term participation increases gradually, enterprises will adopt short-term participation strategy. of long-term participation increases gradually, enterprises will adopt short-term participation strategy. 5.2.2  Effect of Reputation Benefit on Evolution Results Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, 풹= 6, CE2 = 20, CE1 = 10, S = 15 . The initial probability is Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, 풹= 6, CE2 = 20, CE1 = 10, S = 15 . The initial probability is 1040 Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 5.2.3  Effect of Government Subsidies on Evolution Results Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 15, 풹= 6, CE2 = 20, CE1 = 10, 퓇= 10 . The initial probability is 퓏0 = 0.5and퓎0 = 0.5 ; the value of government subsidies S varies within the range [0, 10]; and the evolution result of the system is shown in Fig. 6c. The result shows that if 0 ≤S ≤2 , the government’s ESS converges to long-term participation, and the evolution speed decreases as S rises; If S ≥4 , the government’s ESS converges to short-term par- ticipation, and the evolution speed increases as S rises. For enterprises, their ESS converges to long-term participation, and the evolution speed increase as S rises (Fig. 6d). The results indicate that as the government subsidies increase, the government tends toward pursuing the short-term incen- tive strategy. On the contrary, government subsidies have positive effect on enterprises’ participation strategy, and the more subsidies provided by the government, the less time it takes for enterprises to converge to long-term participation. The simulation results reveal that in the second stage, there are two situations in which the government will choose long-term incentive strategy: the first is that although the enterprises adopt short-term participation strategy, the incre- mental benefit of enterprises supporting emergency man- agement after training exceeds the training cost paid by the government; the second is that the enterprises adopt long- term participation strategy, and the incremental benefit of enterprise participation in emergency management exceeds the training cost and subsidies paid by the government. Correspondingly, there are also two situations in which the enterprises will choose long-term participation strategy: the first is that although the government adopt short-term incentive strategy, the sum of emergency management ben- efit and reputation benefit exceeds the incremental cost of long-term participation; the second is that the government adopt long-term incentive strategy, and the sum of incre- mental emergency management benefit, reputation benefit, and government subsidies exceeds the extra cost of pursuing long-term participation. 6  Conclusion and Future Prospects 퓏0 = 0.5and퓎0 = 0.5 ; the value of the reputation benefit 퓇 obtained by enterprises for participating in emergency man- agement varies within the range [0, 20], and the evolution result of the system is shown in Fig. 6b. The result shows that if 0 ≤퓇≤15 , the enterprise’s ESS converges to short- term participation, and the evolution speed decreases as 퓇 rises; If 퓇≥20 , the enterprise’s ESS converges to long-term participation, and the evolution speed increases as 퓇 rises. This indicates that increasing reputation benefit incentiv- ize enterprises to pursue long-term participation; otherwise, they will adopt the short-term participation strategy. In other words, reputation benefit has a positive impact on enter- prises’ strategy choice. With the complexity and impact of various emergencies worldwide continuing to increase, enterprise participation has become an indispensable part of effective response to emergencies. However, the performance of enterprises par- ticipating in emergency management needs to be further improved. Considering the challenges of enterprise partici- pation and government incentives, this study constructed a two-stage evolutionary game model based on stakeholders’ bounded rationality to analyze the behavioral evolution char- acteristics of both participants in the emergency manage- ment system, and analyzed the system’s evolution path under different situations using SD simulation, thereby exploring the collaboration mechanism between the government and enterprises. We found that although enterprise participation in emergency management is affected by both influencing and decisive factors, their final decision depends on the util- ity function U(휔, 풸) : if and only if U(𝜔, 풸) = 𝜔−풸> 0, an enterprise will choose the participation strategy and enter the emergency management system, initiating the second stage of the evolutionary game. 5.2.4  Effect of Training Cost on Evolution Results Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, CE2 = 20, CE1 = 10, 퓇= 10, S = 15. The initial probability is 퓏0 = 0.5 and 퓎0 = 0.5 ; training cost 풹 paid by the govern- ment varies within the range [0.5, 3]; and the evolution result of the system is shown in Fig. 6e. Some cases in this situ- ation converge slowly or fail to converge to a stable state. Therefore, to show the long-term trend, evolution time 퓉 is extended to [0, 50], and the evolution result of the system is again shown in Fig. 6e. The results indicate that if 풹≥2 , the government’s ESS converges to short-term incentive strategy; If 0.5 ≤풹≤1.5 , there is less stability in whether the government chooses to pay the emergency management training cost for enterprises. In addition, if other factors remain unchanged and the cost increment of long-term participation is low, the enter- prises’ ESS converges to long-term participation, whereas as emergency management cost increases, enterprises tend to opt for short-term participation. With the increase of reputation benefit, enterprises will turn their ESS from short-term participation into long-term participation. In other words, reputation benefit has a positive impact on enterprises’ strategy choice. Similarly, government subsidies such as tax reduction and preferential policies 1041 International Journal of Disaster Risk Science provide important references for government measures pro- moting CEM performance through enterprise participation. However, this study also has some limitations that future research should address. First, while this study considers only enterprises and governments, CEM in practice also includes social organizations, citizens, media, and other stakeholders. Therefore, future research needs to compre- hensively analyze collaboration relationship among multiple agents in the emergency management process. Second, as a complex system, the government-enterprise collabora- tive relationship in emergency management is affected by many factors that were given little attention in this study. Some influencing factors, such as relevant regulations and polices need to be further discussed in future research. In particular, the introduction of information and communica- tion technology such as blockchain, 5G, and big data offers great potential to improve CEM capability with multi-agent participation. More studies are needed to further explore how the application of these technologies affects the game strategy choice of enterprises and governments for emer- gency management. incentivize enterprises to pursue a long-term participation strategy. 5.2.4  Effect of Training Cost on Evolution Results Overall, if and only if the ESS of the system con- verges to {long-term participation, long-term incentive}, the benefit of CEM for both the government and enter- prises outweigh the extra cost they paid. Therefore, the most important thing is that the government should adopt effective measures to promote the continuous participa- tion of enterprises in emergency management. First, the government can provide continuous financial subsidies and promotion incentive for enterprises’ participation. Funding  This work was supported by the Major Project of National Social Science Foundation of China (Grant No. 21&ZD166), the National Social Science Foundation of China (Grant No. 22VRC200), and the China Scholarship Council (CSC, Grant No. 202206420064). 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. 5.2.4  Effect of Training Cost on Evolution Results If other factors remain unchanged, the increase in government subsidies will increase the evolution speed of enterprises’ ESS converging to long-term participation, but the ESS of the government simultaneously converges to short-term incentive. Finally, when the cost of emer- gency management training is low, there is less stability in whether the government chooses to pay the training cost for enterprises, but as the training cost rises, the ESS of the government quickly converges to short-term incentive. y gency management training is low, there is less stability in whether the government chooses to pay the training cost for enterprises, but as the training cost rises, the ESS of the government quickly converges to short-term incentive. Overall, if and only if the ESS of the system con- verges to {long-term participation, long-term incentive}, the benefit of CEM for both the government and enter- prises outweigh the extra cost they paid. Therefore, the most important thing is that the government should adopt effective measures to promote the continuous participa- tion of enterprises in emergency management. First, the government can provide continuous financial subsidies and promotion incentive for enterprises’ participation. Financial subsidies such as tax breaks can directly reduce the cost of enterprise participation, promotion incentive such as honor system can improve the reputation benefit of enterprise participation, and both financial subsidies and promotion incentive can promote continuous participation behavior of enterprises in emergency management. Sec- ond, the government also needs to optimize the commu- nication mechanisms between itself and enterprises, and provide diversified participation channels and information support for enterprises. This can not only reduce the cost of coordination for both the government and enterprises in CEM, but also improve the benefit of CEM between the government and enterprises, so as to establish long-term collaborative relationships in emergency management. 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Impact of corporate social responsibility on sustainable competitive
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Sonstige Dermatosen. l~Ialoney, William J. Uber die Beziehungen der chemi- schen Zusammensetzung der Muttermilch zur Diarrhoe u nd zum Ek zero d cr S~uglinge. The Practitioner. Febr. 1911. M a 1 on e y gelangt dureh seine Untersuchungen zu der Anschauung, daft wohl Uberern~hrung des S~iuglings, allzu reicher Zuckergehal~ der Milch oder fehlerhafte Zusammensetzung derselben infolge ~a]seher Er- n~hrung dcr Mutter als Ursachc fiir das Zustandekommen der S~uglings- ekzeme in Betracht komme; der Umstand jedocb, daft solche aueh bei Feh|en dieser Momente beobachtct werden, spricht dafiir, daf noch andere, mit der Ern~hrung nicht im Zusammenhange stehende Faktoren als Ur- sachen anzusehen sind. Wilhelm B alb an (Wien). M a 1 on e y gelangt dureh seine Untersuchungen zu der Anschauung, daft wohl Uberern~hrung des S~iuglings, allzu reicher Zuckergehal~ der Milch oder fehlerhafte Zusammensetzung derselben infolge ~a]seher Er- n~hrung dcr Mutter als Ursachc fiir das Zustandekommen der S~uglings- ekzeme in Betracht komme; der Umstand jedocb, daft solche aueh bei Feh|en dieser Momente beobachtct werden, spricht dafiir, daf noch andere, mit der Ern~hrung nicht im Zusammenhange stehende Faktoren als Ur- sachen anzusehen sind. Wilhelm B alb an (Wien). Poltowitsch, K. Ein Fall yon chronischem Ekzem. (Zur Frage des Ursprunges, des klinischen Bildes und der B h dl di K kh it ) R k W t h 1910 N 14 15 Poltowitsch, K. Ein Fall yon chronischem Ekzem. (Zur Frage des Ursprunges, des klinischen Bildes und der Behandlung dieser Krankheit.) Russky Wratsch. 1910. Nr. 14u. 15. Nichts Neues. P.A. Welikanow (Petersburg). Behandlung dieser Krankheit.) Russky Wratsch. 1910. Nr. 14u. 15. Nichts Neues. P.A. Welikanow (Petersburg). Roeaz. S~uglingsekzcm. Archives de Mcdecine des Enfants. T. XV. 1911. p. 81. Die Atiologie der Ekzeme ist bisher noch nlcht gekl~rt; man nimmt eine Intoxikation als Ursache an, ohne fiber die Natur der Intoxi- kation etwas Sicheres zu wissen. In Betracht kommen aliment~re Seh~digungen, die besonders bci kiinstlich gen~hrten Kindcrn die Ursache der Ekzeme sein k6nnen. Dcr Verf. meint, daft besonders grol~e Flfissig- keitsmengen die Intoxikation begfinstigen. Auch h~lt Verf. es ffir mSg- lich, dab durch einc unzweckm~fige Ern~hrung der Amine Toxine ent- stehen k6nnen, die dureh die Milch den Kindern zugefiihrt werden und zu Ekzemen ffihrcn k6nncn. Bei eincr Reihe yon Kindern kommt cs zu Ekzemen, ohnc daf irgend einc Ern~hrungsst6rung vorliegt; in solchen FMlen soll der Arthritismus der Eltern yon ~itiologischer Bedeutung sein. ]n manchen F~l|en yon Ekzem sollcn nach Ansicht des Yeff. Sonstige Dermatosen. St6rungen der Dri~sen mit innerer Sekretion vorliegcn, was aus den thcrapeutischen 36* Bericht fiber die Leistungen auf dera Gebiete 564 Erfolgen, z. B. Behandlung tier Ekzeme mit Schildd~fisenpr~paraten zu erschliel~en sei. Den Ubergang von den Ekzemen zu den Erythemen bildet eine jfingst yon Moussous studierte Hautaffektion, der er den •amen 6ry- th~me seborrheique gegeben hat. Das Wesen der Dermatose ist eine von der Gefiil~gegend ausgehende, bisweilen universelle R6te der I-Iaut mit Seborrhoe des Kopfes. C. L e i n e r. NIoro. Uber dieStellung derErythrodermia de squama- tiva ~Leiner) im Kranktieitssystem, Mfinchener mediz. Woehen- schrift. 1911. Nr. 10. Die yon L ein er mit dem Namen ,Erythrodermia desquamativa" bezeichnete eigenartige Dermatose tier S~uglinge hat More ia den letzten 3 Jahren in 11 F~llen beoloachtet und behandelt. Er h~ilt die Sonder- stellung, die Leine r der Dermatose einr~umt, fiir zu weitgehend and faint die Erkrankung als eine universelle Dermatitis ex intertrigine bei jungen S~uglingen mit ausgesprochenem status seborrhoious auf exsuda- flyer Grund]age auf. Oskar Mii IIe r (Reeklinghausen). •oro, Uber Dermatitis fibrinosa faciei. Miinehener mediz. Wochenschr. 1911. I~r. 6. More beschreibt eine Hauterkrankung, die bei Kindern an Kinn, Oberlippe und am Nasene~ngang vorkommt und ein Analogen zu den Stomatitis aphthosa bildet. Die Effloreszenzen zeigen die gleiehen Eigen- tiimliehkeiten ~vie die Mundaphthen und bestehen in der Hauptsache aus Fibrin und wenig Leukozyten. g y Verf. sehl~gt fi~r die noch nieht besehriebene Af[ektion den Namen ,Dermatitis fibrinosa" vor. Oskar M ii 1 l e r (Recklinghausen). Haslund, Poul. Studien fiber die Histologie and die Pathogenesis der Psoriasis. Kopenhagen 1911. p. 150. Yerf. hat yon 25 Patienten mit klinlseh typischer Psoriasis din versehiedenen Stadien der Effloreszenz untersuebt, um feststetlen zu k6nnen, we der prim~ire Sitz der Krankheit ist and auch am das mikroskopisehe Bild der versehiedenen Stadien genau zu zeiehnen. Zu diesem Zweeke hat er 6000 meistens in Serien gesehnittene Pr~parate uutersueht und gezeigt, da$ die in dem ersten Stadium der Krankheit nur im Mikroskope sichtbare u der Haut eine Oberhautentzfindung ist und zwar dadurch entsteht, dal~ sieh ein kleiner ttaufe yon emigrierten Leukozyten gerade unter dem Stratum corneum bfldet, cloch sieht man auch nicht selten einen wirklichen kleinen ,Abszel~ ~. Die gleiehzeitig gefundenen Yer~nderungen im Papilt~rkfrper and im Korium sind deutlieh vorhanden, sic sind aber wahrseheinlich yon sekundirer Bedeutung. Spiter kommt das allgemein bekannte Bild yon typischer Psoriasis. y yp Yerf. schliefit sieh denn ziemlich nahe an die Ansehauungen, die erst v. Sonstige Dermatosen. Munro und Sabouraud ausgesprochen haben, doch hat er hie die yon diesen Forsehern gefundene primire Erosion gesehen und VerL meint auch, daft das Bild sich nicht durch eine prim~re Erosion erkl~rea der Hautkrankheiten. 565 'l~Bt. Auch hat er --im Oegensatze zu den erw~hntea Autoren -- ge~ funden, dab eine ,Exoserose" ziemlieh h~ufig zu sehen ist. 'l~Bt. Auch hat er --im Oegensatze zu den erw~hntea Autoren -- ge~ funden, dab eine ,Exoserose" ziemlieh h~ufig zu sehen ist. g Die Arbeit ist wegen der guten detaillierten Beschreibung des mikroskopisehen Bildes besonders erw~hnenswert. g Die Arbeit ist wegen der guten detaillierten Beschreibung des mikroskopisehen Bildes besonders erw~hnenswert. Henrik Bang (Kopenhagen). Henrik Bang (Kopenhagen). Henrik Bang (Kopenhagen). g p g Mayer~ A. Normales Schwangersehaftsserum als Heil- mittel gegen Schwangerschaftsdermatosen im besonderen und Schwangerschaftstoxikosen iiberhaupt. Zentralbl. f. d. ges. Med. 1911. Nr. 9. Mayer~ A. Normales Schwangersehaftsserum als Heil- mittel gegen Schwangerschaftsdermatosen im besonderen und Schwangerschaftstoxikosen iiberhaupt. Zentralbl. f. d. ges. Med. 1911. Nr. 9. ~Das Wesen der unter dem l~amen Sehwangerschaftstoxikosen zuo sammengefaflten Krankheitsformen erblieken wir, u sehreibt Mayer, ,ganz allgemein gesagt darin, da~ gewisse durch den waehsenden Keim in dem miitterliehen Organismus normaliter gebildete Giftstoffe diesem in pathologischeu F~llen seh~idlieh werden kSnnen. Nach dieser Voro stellung ist es flit den normalen Ab]auf der + Gravidit~t n5tig, da~ jene Oift~toffe durch Gegengifte unseh~dlich gemaeht werden. Gesehieht das nicht, dann entstehen blutfremde Stoffe im miitterliehen Blut, die zu autotoxischeu Erkrankungen ffihren. Man kann also sagen~ dal} elne an einer Gravldit~tstoxikose erkrankte Frau an einem D efizit yon ~egen- giften leidet, die eine gesunde Schwangere besitzt." Durch diese Er- ~v~gung wurde M a y e r veranla~t, L i n s e r die Behandlungsmethode vor- zuschlagen, die dieser erfolgreich inaugurierte. ~Das Wesen der unter dem l~amen Sehwangerschaftstoxikosen zuo sammengefaflten Krankheitsformen erblieken wir, u sehreibt Mayer, ,ganz allgemein gesagt darin, da~ gewisse durch den waehsenden Keim in dem miitterliehen Organismus normaliter gebildete Giftstoffe diesem in pathologischeu F~llen seh~idlieh werden kSnnen. Nach dieser Voro stellung ist es flit den normalen Ab]auf der + Gravidit~t n5tig, da~ jene Oift~toffe durch Gegengifte unseh~dlich gemaeht werden. Gesehieht das nicht, dann entstehen blutfremde Stoffe im miitterliehen Blut, die zu autotoxischeu Erkrankungen ffihren. Sonstige Dermatosen. Man kann also sagen~ dal} elne an einer Gravldit~tstoxikose erkrankte Frau an einem D efizit yon ~egen- giften leidet, die eine gesunde Schwangere besitzt." Durch diese Er- ~v~gung wurde M a y e r veranla~t, L i n s e r die Behandlungsmethode vor- zuschlagen, die dieser erfolgreich inaugurierte. g Felix Miinchheimer (Wiesbaden). Mfiller, Rudolf. Lichen ruber aeuminatus atypieus eaplitis. Ikonographia dermatologica, Fuse. V. Tab. XL. Miil 1 e r besehreibt einen Fall yon Lichen rub. aeum. der Kopf- haut, der sich yon den sonst beobachteten dadureh wesentlich unter- scheidet, dab auch dab Kapillitium ,deutliche hyperkeratotisehe isolierte KnStchen aufwies, wie sie ffir die fibrige Lokalisation des Lichen charakteristiseh sind% w~hrend sonst stets ,das prim~ir infiltrierte Horn- kuStchen vollst~indig fehlte und nur eine diffuse Pityriasis der Kopfhaut beobaehtet wurde". Eiu 38j~ihr. Patient~ der a]s Kind an Kopfekzem gelitten hatte, er- krankte vor 7 Monaten an einer Hautaffektion~ die sich fiber Kopf, Brust und Vorderarme ausbreitete. Die Untersuchung des Kopfes ergab neben einer zentralen Alopeeia pityroides das Vorhandensein verschieden gro~er sehuppender Plaques, die sich am Rande in zahlreiche einzelne KnStchen und Gruppen yon solchen aufl6sten. Die kleinsten KnStchen bestehen aus einem Hornkege], der einem gelblichroten Infiltrate aufsitzt und tragen z, T. eiu erhaltenes, z. T. ein abgebrochenes Haar; an anderen KnStehen fehlen die Haare, an deren Stelle ein griibehenfSrmiger Defekt siehtbar ist. Stellenweise sind die FoIlikelmfindungen erweitert oder yon einem Schfippchen bedeckt. Vorderarme, Handrficken und Brust tragen Kn6tchen, die z. T. die charakteristischen, an den Follikel- mtindungen sitzenden Hornkegel zeigen. Unter energischer Arsenbehand- lung heilte die Affektion ab. 566 Berieht fiber die Leistungen auf dem Gebiete Der histologische Befund zeigte eine starke Hyperkeratose der Follikel und zirkumskripte kleinzellige Infiltration um den Follikelboden. Ferner f'~llt die dutch den Druck der hyperkeratotischen Schichten zu- stande gekommene Yer~nderung der Epithelzellen auf im Sinne des AuG treteas flacher Zellformen bet senkrechtem und yon Zylinderzellen bei seitlichem Drueke. Wilhelm B a 1 b a n (Wien). Der histologische Befund zeigte eine starke Hyperkeratose der Follikel und zirkumskripte kleinzellige Infiltration um den Follikelboden. Ferner f'~llt die dutch den Druck der hyperkeratotischen Schichten zu- stande gekommene Yer~nderung der Epithelzellen auf im Sinne des AuG treteas flacher Zellformen bet senkrechtem und yon Zylinderzellen bei seitlichem Drueke. Wilhelm B a 1 b a n (Wien). Nobl. Zur Kenntnis des Herpes zoster generalisatus Wiener klinische Wochensehr. 1911. Nr. 1. Sonstige Dermatosen. An der Hand eines einschl~gigen Falles erfrtert N obl unsere Kenntnisse fiber den Herpes zoster. Der engere pathogenetisehe Zu- sammenhang der 5rtlichen systemisierten und der a]lgemeinen Bl~ischen- eruption wird verst~ndlicher, wenn man die in allen Beobachtungen ver- folgte Abh/ingigkeit der Ern~hrungsstfrung im Bereiche der disseminierten Aussaat yon den intensiven Seh~digungen im Projektionsfeld eines Spinalganglions in Berficksichtigung zieht. Man kann den Herpes zoster als vasomotorisehes Ph~nomen betrachten, das ~ihnlich der neurotiscben Hautgangriin auf sp~treflektorischem Wege zustandekommt. Viktor Bandler (Prag). Viktor Bandler (Prag). Kanera, Franz. Herpes zoster und l~ierenkolik. Dtsch. reed. Woch. Nr. 14. 1911. Kanera h~lt das Zusammentreffen dieser beiden Erkrankungen nieht fiir so selten, wie Bitt orf gelegentlieh der Verfffentliehung eines ~hnlichen Falles annimmt und beriehtet seinerseits fiber sine 56j~hrige ~atientin, die bet sonst gutem Kr~ftezustand wegen starker Sehmerzen im r. Hypoehondrium seine Hilfe suehte. Verf. konsiatierte eine reehte Wanderniere, der Harn war normal, der Stuhl aber oft verhalten. Eine Woehe sparer entwickelte sich, you 2 roten Fleeken am Rfieken aus- gehend, ein typiseher Herpes zoster, der unter entsprechender Behand- lung bald heilte, wfihrend auch die Nierengeschwuls~ sieh in die Tiefe verlor und nur noch als dumpfe Resistenz unter dem Rippenbogen zu ffihlen war und die Schmerzen verschwanden. Kanera h~lt das Zusammentreffen dieser beiden Erkrankungen nieht fiir so selten, wie Bitt orf gelegentlieh der Verfffentliehung eines ~hnlichen Falles annimmt und beriehtet seinerseits fiber sine 56j~hrige ~atientin, die bet sonst gutem Kr~ftezustand wegen starker Sehmerzen im r. Hypoehondrium seine Hilfe suehte. Verf. konsiatierte eine reehte Wanderniere, der Harn war normal, der Stuhl aber oft verhalten. Eine Woehe sparer entwickelte sich, you 2 roten Fleeken am Rfieken aus- gehend, ein typiseher Herpes zoster, der unter entsprechender Behand- lung bald heilte, wfihrend auch die Nierengeschwuls~ sieh in die Tiefe verlor und nur noch als dumpfe Resistenz unter dem Rippenbogen zu ffihlen war und die Schmerzen verschwanden. Max Joseph (Berlin). Max Joseph (Berlin). Max Joseph (Berlin). Bertarelli, E. und Parauhos, U. ~tiologiaehe Unter- suehungen fiber den Pemphigus der Tropengegenden. Zentralbl. f. Bakt. Bd. LVII. Heft 6. pag. 493. Uber den Tropenpemphigus liegen sehr wenige Angaben in der Literatur vor. Verff. fassen die wiehtigsten kllnisehen Charaktere der Krankheit kurz, wie folgt, zusammen: ,Der Tropenpemphigus ist eine Hautkrankheit, charakterisiert dureh die Entstehung yon Blasen oder Bl~isehen versehiedener Grfl]e, welehe ohne Reaktionserscheinungen von- ~eiten der umgebenden Haut auftreten und keine Gesehwfire und somit keine Narben erzeugen. Sonstige Dermatosen. Die L~sion beginnt mit kleinen erythemat6sen, stark juckenden Flecken, an deren Stelle naeh kurzer Zeit eharakteristisehe Blasen erscheinen. Diese sind anfangs durehscheinend, gespannt, gl~nzend; dann wird ihr serfser Inhalt trfibe, die W~inde erseMaffen und die Blasen 5ffnen sieh spontan oder beim geringsten Druek. Diese Urn- Bertarelli, E. und Parauhos, U. ~tiologiaehe Unter- suehungen fiber den Pemphigus der Tropengegenden. Zentralbl. f. Bakt. Bd. LVII. Heft 6. pag. 493. Uber den Tropenpemphigus liegen sehr wenige Angaben in der Literatur vor. Verff. fassen die wiehtigsten kllnisehen Charaktere der Krankheit kurz, wie folgt, zusammen: ,Der Tropenpemphigus ist eine Hautkrankheit, charakterisiert dureh die Entstehung yon Blasen oder Bl~isehen versehiedener Grfl]e, welehe ohne Reaktionserscheinungen von- ~eiten der umgebenden Haut auftreten und keine Gesehwfire und somit keine Narben erzeugen. Die L~sion beginnt mit kleinen erythemat6sen, stark juckenden Flecken, an deren Stelle naeh kurzer Zeit eharakteristisehe Blasen erscheinen. Diese sind anfangs durehscheinend, gespannt, gl~nzend; dann wird ihr serfser Inhalt trfibe, die W~inde erseMaffen und die Blasen 5ffnen sieh spontan oder beim geringsten Druek. Diese Urn- der Hautkrankheiten. 567 wandlung erfolgt durehschnittlich im Laufe yon 6--7 Tagen. Danach bliittert die Epidermis auf Streeken yon einigen cm do und die IIaut er- scbeint an den betreffenden Ste]len rosig und wie lacklert, wRhrend sich feine IIautl~ppeben ablSsen. ~ Die Dermatose ~ritt schubwe~se auf und verl~uft fieberlos; ihre ~_tiologie ist noch unklar. Sodann wird die Krankheitsgeschiehte eines Falles mitgeteilt, aus dessen Material ver- ,sehiedene Untersuchungen ausgefiihrt wurden. Morphologische Unter- suchungen der Blasenflfissigkeit, Krusten, Schuppen verliefen ergebnislos. Tierversuehe an der Haut einer Coebus-Art~ Meersehweinchen, Hund, Kanlnchen waren negativ. Bei den Kaninchen kam es nach Inokulation in die vordere Augenkammer und Infektion der Kornea nach 8 Tagen zu IIyper~mie, Bindehautkatarrh, Keratitis und tIypopyon, in welehem Staphylokokken naehgewiesen wurden, w~ihrend die bistologiscbe Unter- suchung der IIornhautsehnitte keinen besonderen Befund ergaben. Komplement-Ablenkungsversuche verliefen negativ. Alfred Kr au s (Prag). g Feinberg, A. Ein Fall yon purpura variolosa. Wratschebn. Gazeta. 1910. Nr. 5. Beschreibung eines Falles bei einem 13j~ihrigen Knaben. Beschreibung eines Falles bei einem 13j~ihrigen Knaben. P A W lik (P t b ) Beschreibung eines Falles bei einem 13j~ihrigen Knaben. P. A. Welikanow (Petersburg). Wolkowitseh, Nieolai. Zur Statistik und dem Vor- kommen des Skleroms (Rhinoskleroms) in Rutland. Monats- Wolkowitseh, Nieolai. Zur Statistik und dem Vor- kommen des Skleroms (Rhinoskleroms) in Rutland. Monats- sehrift f. Ohrenheilk. und Laryngo-Rhinologie. XLV. Jahrg. IIeft 1. Statistisehe Arbeit. Zum Referate nicht geeignet. Wilhelm B a i b a n (Wien). Sonstige Dermatosen. sehrift f. Ohrenheilk. und Laryngo-Rhinologie. XLV. Jahrg. IIeft 1. Statistisehe Arbeit. Zum Referate nicht geeignet. Wilhelm B a i b a n (Wien). Kahler. Zahnfleisehsklerom. (Demonstration in tier Wr. laryngol. Gesellsehaft.) Monatssehr. f. Obrenheilk. und Lar.-Rhinologie. XLV. Jabrg. 3. Heft. Krankendemonstration. Wilhelm B a 1 b a n (Wicn). K Kono, Michiyo. Beitrag zur Kasuistik der Sklerodermie. Diss. GSttingen 1910. (Mediz. Klinik Hirsch.) Fall yon Sklerodermie ohne weiteres Interesse. Weiler (Leipzig). Hartzell, B. Ein Fall yon Pellagra, der seinen Ursprung in Pennsylvania hatte. Philadelphia County Medical Society. 191l. 8. Februar. The Journal of the American Medical Association. 1911. 1. April. pag. 998. Bericht fiber einen Fall yon Pellagra mit ansehlielSender Diskussion fiber Pellagra. Fritz J u li u s b e r g (Posen). Nice, Charles, Me Lester, James, Torrance, Gaston. Pell agr a mit Salvarsan behandelt. The Journal of the American Medical Association. 1911. 25. M~irz. pag. 896, p g Nice, Mc Lester und Torrance haben bei drei F~llen yon Pellagra Salvarsan intraven6s angewendet und elne Besserung erzielt. Fritz Juliusberg (Posen). Nice, Mc Lester und Torrance haben bei drei F~llen yon Pellagra Salvarsan intraven6s angewendet und elne Besserung erzielt. Bericht fiber die Leistungen auf dem Gebiete 568 Cole, M. P. Transfusion bet Pellagra. Ein Bericht fiber 20 Y~lle. The Journal of the American Medical Association. 1911. 25. Februar. pag. 584. Cole, M. P. Transfusion bet Pellagra. Ein Bericht fiber g 20 Y~lle. The Journal of the American Medical Association. 1911. 25. Februar. pag. 584. g 20 Y~lle. The Journal of the American Medical Association. 1911. p g Cole kc, mmt zu folgenden Schliissen: Mit der Transfusion bet 20 F~llen yon Pel]a'gra baben wir hie direkt dutch die Operation schlechie Resullate bet den Paiienien erlebt. Wir dfirfen zu der Transfusion greifea bet schweren F~llen, die unter den erprobten Mitteln st~indige Ver- schlechterung aufweisen. Es ist ganz gleich, ob man das Blur yon einem Patienten nimmt, der Pellagra fiberstanden hat oder yon einem, der nie Pellagra hatte. Heilungen gab es bet dem schweren Typus der Krank- heir durch die Transfusion in 600/0, w~ihrend die sonstigen Behandlungs- methoden nur einen Prozentsatz yon 10 bis 20 Heilungen fatten. Die Anwenduug der Transfusion im Endstatium der Pellagra muB mit vollem Bewu~tse~n der Schwierlgkeilen und der Gefahren der Methode vorge- nommen werden. Nur sorgf~ltige Auswahl der F~ille und unvorein- genommene Schliisse k5nnen das Verfahren vor eiuer Diskreditierung bewabren, die es nicht verdient. Fritz Juliusberg (Posen). Abbe beriehtet fiber zwei Fglle yon Ainhum. Die FMle spreehen dafi~r, dal~ das Primgre der Erkrankung eine Trophoneurose ist. F it J li b (P ) De Azfia, Juan. Akuter 5demat5ser and erythematfser Jodismus der kranken Zonen bei einem Fall von erythe- Sonstige Dermatosen. Caccilli, A. Die Pellagra, wie wir sie in Italien sehen; a 1 t e u n d n e u e T h e o r i e n. Bericht fiber F/ille aus New-York. Medical Record. I911. ii. M~rz. pag. 421. Die Arbeit Caecinis enth~ilt eine eingehende Darstellung der Symptomatologie und der Ansichten fiber die Atiologie der Pellagra. Im Anschlu~ werden s~cben in New-York beobaehtete F~lle besehrieben. Der Autcr kommt zu fo]genden Schlfissen: 1. Die Hauptursache der Pellagra ist noch unbekannt. 9. Die Pellagra hat in den Vereinigten Staaten in sporadischen F~llen schon lange Zeit bestanden, abet erst in letzter Zeit hat sie sieh ausgebreitet and eincn epidemisehen Charakter angenommen. 3. In den Vereinigten Staaten nimmt die Krankheit einen akuieren Verlauf als in Europa und speziell in Italien, wo sie sehr h~ufig einen ehronischen Charakter trfigt und das Erythem nie den schweren Charakter aufweist, wie in den Vereinigiea Staaten. 4. In Amerika ist die Kranl~heit relativ hfiufig unter den gut situierten Klassen, wahrend sie in Europa auf die armen Kreise beschriinkt ist. Fritz J u 1 i u s b e r g (Posen). Wile, Udo. Uber Granuloma pyogenicum (Pseudo- B o t r y o m y k o s i s). U n n a s Dermatologlsehe Studien (Unna-Festschrif~ Bd. I), Bd. XX. pag. 333. Naoh ausfiihrlicher Darstellung zweier einschl~gigerF~lle kommt W i i e zu folgendenSchliissen: 1. Dieganze Gruppevon sogenannterPseudo-Botryo- mykosis, Granuloma simplex, Granuloma teleangiectodes und Granuloma pyogenicum mag in eine Klasse zusammengefal~t und als Granulationsgewebe betrachtet werden, we]e]~es aus vor]~ufig unbestimmten Grfinden eine ungewehnliche Form angenommen hat. 2. Als iitiologischer Faktor tritt bier nicbt ein Fungus, sondern wahrscheinlich der Staphylococcus aureus auf, mSglicherweise auf ungfinstigem Nfihrboden oder in abgeschw~ichter Form. S. Die histologischen Ver/inderungen sind wesentlich die~elben der Hautkrankheiten. 569 ~in allen Formen ; der einzige Unterschied, der konstatiert werden kann, ist ein quantitativer Unterschied in Vasku]aritiit und Entzfindung. 4. In keinem Falle 1M~t sieh ein Zusammenhang zwischen Tumor und Schwei•- drfisen nachweisen. 5. 0bgleich hie b6sartig, hat doeh die Geschwulst eine Neigung zu rezidivieren, wenn bei der Enffernung der Stumpf nicht kaaterisiert worden ist. 6. Granaloma pyogenicum ist nuch des Autors Ansicht anter allen ffir dieses Krankheitsbild vorgeschlagenen Named der passendste. Fritz Juliusberg (Posen). ~in allen Formen ; der einzige Unterschied, der konstatiert werden kann, ist ein quantitativer Unterschied in Vasku]aritiit und Entzfindung. 4. Sonstige Dermatosen. In keinem Falle 1M~t sieh ein Zusammenhang zwischen Tumor und Schwei•- drfisen nachweisen. 5. 0bgleich hie b6sartig, hat doeh die Geschwulst eine Neigung zu rezidivieren, wenn bei der Enffernung der Stumpf nicht kaaterisiert worden ist. 6. Granaloma pyogenicum ist nuch des Autors Ansicht anter allen ffir dieses Krankheitsbild vorgeschlagenen Named der passendste. Fritz Juliusberg (Posen). Gjorgjevi6. Beitrag zur Kenntnis der Streptothrix- erkrankungen des Menschen. Wiener klinische Wochenschrift. 1911. Nr. 6. Der Autor berichtet in extenso fiber 4 beobachtete Fiille. Die einze]nen Organe liel~en entsprechend dem makroskopischen Knftchen- befand mikroskopiseh das Bild yon Eiterherden erkennen; erwiihnenswert erscheint der Befund yon Riesenzellen. Wie in gef~rbten Priiparaten so auch im Mngenden Tropfen crkennt man echte u der Stature tier Fiiden hat die gleicbe Dicke wie die abgehenden Zweige, welche an de~ Enden die Andeutung einer leiehten ko|bigen Anschwel]ung zcigen kfnnen. Die Pilze erweisen sich als siiarefest, aber nieht als alkoholfest. Was die kultare]len Eigensehaften anbe]angt, so unter- seheidet sich die Pilzart yon den meisten his jetzt bekannten dureh das tkkultativ an~irobe Waehstum; die meiste ubereinstimmung zeigt sich mit den yon Eppinger, Aoyama and Miyamoto, A. Horst , Mac Collum und A. Schabad beschriebenen Stihnmen, Yiktor Bundler (Prag). Yiktor Bundler (Prag). Yiktor Bundler (Prag). Sutton, Richard. Cheilitis glandularis apostematosa. (Eine klinische und histopathologische Studie.) Unnas Dermatologische Studien (Unna-Festsehrift Bd. I)~ Bd. XX. pug. 612. Sutton kommt in seinen Untersuchangen fiber die zuerst yon V o I km a n n beschriebene Cheilitis glandularls apostematosa zu folgenden Sehlfissen: Die Cheilitis glandularis apostematosa ist eino adenomatfse Yeriinderung der Sehleimdriisen der Lippe, die histologlseh char~kterisiert ist durch eine enorme Dilatation nnd Hyperfrophie der Driisengiinge, wclche gewfhnlich begleitet ist yon einer starken Zunahme des Drfisen- gewebes. Es ist sehr wahrscheinlich, da~ die u ursprfinglich eine kongenitale ist, der dann degenerative Ver~nderungen und Zunahme des elastischen Gewebes in derumgebenden Haut folgen. Diebefriedigendst0 Art der Behandlung ist die Exzision unter loka]er oder allgemeiner Aniisthesie. Fritz J u I i u s b e r g (Posen). g Abbe, Truman. Zwel F~lle yon Ainhum. Medical Record. 1911. 18. Miirz. long. 478. Abbe, Truman. Zwel F~lle yon Ainhum. Medical Record. 1911. 18. Miirz. long. 478. Abbe beriehtet fiber zwei Fglle yon Ainhum. Die FMle spreehen dafi~r, dal~ das Primgre der Erkrankung eine Trophoneurose ist. De Azfia, Juan. Akuter 5demat5ser and erythematfser Jodismus der kranken Zonen bei einem Fall von erythe- Berieht fiber die Lelstungen auf dem Gebiete 570 mat68er Anetodermie Jadassohn. Sammelreferat, in dem yon Hauterschelnungen Pigmenta~ionen~ Pruritus~ Tumoren~ erythematSse, exsudative Effloreszenzen und Gesehw[ire erw~hnt werden. F. L e w a n d o w s k y (Hamburg). Sonstige Dermatosen. Unnas Dermatologisehe Studien (Unna-Festschrift Bd. I)~ Bd. XX. pag. 216. ( ) p g D e Az5a berichtet fiber eine 28i~hrige Patientin, die seit drei Jahren an einer Hautaffektion leidet, die folgendermal]en verlfiuft: Zu- n{iehst erscheinen runde und ovale Herde yon dunkelroter Farbe, einer vermehrten Konsistenz, wie oberfi~chliche Papeln mit glatter Oberfl{iche. 8ie sind absolut schmerzlos. Nach Wochen oder Monaten verlieren sie ihren papulSsen Charakter, um dann ira Niveau der Haut zu sitzen. Dann wird ihre Farbe bl~sser, die Epidermis verdfinnt sich und man fiihlt eine Resistenzverminderung. Dieser Zustand wird immer aus- gesproehener und als Endstadium finder sich eine scharf begrenzte Depression~ innerhalb der der Finger in eln tiefes Loeh eindringt. Diese Effloreszenzen haben dieselbe Ausdehnung wie der urspr[iugliche Herd. Der Autor stellte die Diagnose: Atrophie maeuleuse en plaques consg- cutives ~ un processus chronique ind6termin6 de type inflammatoire 6rythemateux. Der Fall entspricht der Anet~dermia erythematosa Jadassohn. Der Autor gab Jod 0"5. Einige Stunden darauf trat eine Sehwellung der kranken Partien auf und eine starke RStung derselben. Drei Tage naehher war wieder der Status quo antea eingetreten. Eine erneute Joddarreichung hatte dieselben Fo]gen. Bernhardt, 1%. Lupus erythematosus linearis. Gazeta lek, 1911. Nr. 5. Siehe dieses Archiv Bd CVIIL pag 55 Bernhardt, 1%. Lupus erythematosus linearis. Gazeta lek, 1911. Nr. 5. Siehe dieses Archiv Bd CVIIL pag 55 lek, 1911. Nr. 5. Siehe dieses Archiv Bd. CVIIL pag. 55. Brinitzer. Fall yon hoehgradiger Akne. Altonaer iirztl. Verein. Sffzung v. 22. Februar 1911. 94j~hriger Mann seit 10 Jahren an Akne vulgarls pustulosa mit perifollikul~ren Abszessen leidend. Ausgedehnte Narbenbildungen; an manchen Stellen des Riiekens kaum noch gesunde Hautstellen im er- krankten Gebiet. F. L e w a n d o w s k y (Hamburg). Fabian, E. Die Lymphogranulomatosis (Paltauf~ Sternberg). Zentralbl. f. allg. PathoL u. pathol. Anat. Bd. XXII. pag. 145. 1911. Sammelreferat, in dem yon Hauterschelnungen Pigmenta~ionen~ Pruritus~ Tumoren~ erythematSse, exsudative Effloreszenzen und Gesehw[ire erw~hnt werden. F. L e w a n d o w s k y (Hamburg).
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Shifting Desulfurization Equilibria in Ionic Liquid–Oil Mixtures
Energy & fuels
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Email: jalil.hussein@epu.edu.krd; Email: jalil.hussein@epu.edu.krd; Shifting desulfurisation equilibria in ionic liquid-oil mixtures 1Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK. E-mail: apa1@le.ac.uk; 2Petroleum Technology Department, Erbil Technology Institute, Erbil Polytechnic University, Erbil, Iraq. 1Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK. E-mail: apa1@le.ac.uk; 2Petroleum Technology Department, Erbil Technology Institute, Erbil Polytechnic University, Erbil, Iraq. Abstract Ionic liquids (ILs) and deep eutectic solvents have been used for the extraction of molecules, particularly natural products. An often studied system is that for thiophene removal from oil. In the current study, ILs have been used for the removal of thiophene (Th), benzothiophene (BT) and dibenzothiophene (DBT) by liquid-liquid extraction. The equilibrium can be shifted by polymerising the thiophenic compounds both electrochemically and chemically. A 1:1 mixture of 1-butyl-3- methylimidazolium chloride (Bmim)Cl and FeCl3 was used to extract Th and BT from alkane layers using electropolymerisation to shift the position of the equilibrium. While the process could be carried out, the kinetics of polymer formation were too slow to make this a viable process. The final part of the study used a 1:2 (Bmim)Cl: FeCl3 mixture to chemically remove sulfur containing compounds from alkane layer. It was shown that the rate of the chemical polymerisation reaction of Th is around 1500 times faster than electrochemical polymerisation. Issues associated with the regeneration of the liquid are discussed. Key words: Thiophene, liquid-liquid extraction, electrochemical polymerisation, desulfurization, imidazolium ionic liquids. 1 Experimental All materials and reagents employed in this work: ferric chloride, 1-Butyl-3-methylimidazolium chloride, n-decane, thiophene and dibenzothiophene (all Sigma-Aldrich ≥99 %), benzothiophene (Alfa Aeser > 98%), were all used as received.(Bmim)FeCl4 eutectic mixture has been made according to the literature procedure.7 In the extraction experiments, different solutions of Th was prepared in n-decane as a model oil and then stirred and extracted with that of each of the mixture of ILs addition at different conditions of ILs. The same conditions and experiments were repeated with BT and DBT. The S-content with respect to Th, BT, and DBT in decane was measured by GC-FID with a fused silica capillary column (PE Elite-5, 29.45 m long, 0.25 mm in diameter) connected to gas chromatograph (Perkin Elmer Autosystem XL) using the Totalchrom software. The operational temperature of the FID was 320 ºC, and that of the injector was 310 ºC. For the first three minutes, the temperature of column was set at 50 ºC, increased to 300 ºC at a rate of 15 ºC min-1, kept at 300 C for 2 min. Helium was the carrier gas at a flow rate of 1ml min-1. The quantities of S-compounds in a model fuel were determined from peak areas corresponding to these sulfur species on the gas chromatograph. According to the GC setup, the retention time chromatograms for Th, BT and DBT were around 3.14, 9.88 and 14.70 min respectively. The amount extracted was determined using a calibration plots. Good linear correlations were achieved for analytical technique, with an R2 value of more than 0.99 for GC-FID. The error bars for most of the data are within the size of the plot symbols showing that replicate results are accurate. All electrochemical experiments were carried out using a three electrode set up (Pt working electrode (WE), a Ag wire pseudo-reference electrode (RE) and Pt mesh counter electrode (CE). CVs were carried out at a polished 0.5 mm diameter (1.963 x 10-3 cm2) Pt disc working electrode immersed in a (Bmim)Cl FeCl3 mixture as a function of sweep rate. Before each experiment, the WE was cleaned and washed with deionised water, polished with alumina paste and after sonicating in deionised water for 5 minutes; it was dried with N2 gas. The WE acts as a substrate for electro-deposition of polythiophene. Introduction Ionic liquids and deep eutectic solvents (DESs) have been proposed as solvents for extraction although one obvious drawback is that the solutes are difficult to remove from the ionic phases as they have low vapour pressures.1 Liquid-liquid extraction has been extensively studied for the removal of metals from aqueous solutions using hydrophobic ILs and the removal of sulfur containing compounds from oils using hydrophilic ILs.2 While numerous solutes have been extracted, relatively little is known about the specificity and thermodynamics of extraction. A recent study on the extraction of thiophenic compounds from alkane phases into DESs showed that extraction was limited by enthalpic change and driven by entropic change. In most cases the enthalpy of extraction was endothermic limited by the energy required to create a hole in which to put the solute. The highest partition coefficients were found with the liquids with the lowest surface tensions and therefore the lowest cohesive energy densities.3 The extraction of thiophene and its polyaromatic analogues has been studied by several groups in different DESs and while efficient extraction has been observed little is known about the process. Lewis acidic tetrachloroferrate based ILs, particularly those containing imidazolium cations, are very efficient at chemically removing sulfur containing compounds from alkane based liquids. The advantage of using an iron-based catalyst is naturally that it is sustainable and hence has a low cost. Dharaskar and coworkers used a 1:1(BmimCl): FeCl3 misture and found that 59.2% of Th was extracted.4 The authors proposed a specific interaction between thiophene and the FeIII-based anion. Li et al.5 and Yang et al.,6 studied the adsorption of thiophene using a Ag+ or Cu+-Y zeolite. They found that the metals interacted with the thiophene π orbitals with a donation into the vacant σ orbitals of the metal and a back donation of electron charge to the π* orbital of thiophene from the d orbitals of metals. The authors believed that the same mechanism occur with the Lewis acidic FeCl3 based- triethyl ammonium chloride. In the current study, the extraction and polymerisation of thiophene (Th), benzothiophene (BT) and dibenzothiophene (DBT) was studied from alkanes. Initially, these compounds are extracted and then electropolymerised from decane in an equimolar (Bmim)Cl- FeCl3 mixture. In the second part, the extraction and chemical polymerisation was carried out in 1:2 (Bmim)Cl: FeCl3 to compare and determine the rate of their reactions. 2 Experimental Since the polymeric film was deposited by an oxidative process, an inert electrode (Pt) was requested so as not to oxidise concurrently with the aromatic thiophene monomer.8 3 3 Results and Discussion Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 The extraction of Th, BT and DBT has previously been shown to be possible into a range of DESs. Th showed the best extraction efficiency followed by BT and then DBT and this was found to be related to the size of the solute (the latter requiring a larger hole).3 The equilibrium can be driven by making a solid product from the thiophenic compounds by polymerisation of thiophene to form polythiophene (PTh). Previous work has converted the thiophene to a solid thiophene sulfoxide catalysed by DESs in the presence of H2O2 as an oxidising agent.9 The issue with this approach is that H2O is introduced as an aqueous solution as a by-product, contaminating the oil. In the current study it is proposed that Th could be converted into a solid by polymerising it to polythiophene (PTh). This is an interesting material which has been used in numerous electronic devices as a semiconductor and electrochromic material.8, 10 Attempts to drive the equilibrium by electropolymerising the thiophenic molecules in non-metallic DES were unsuccessful. Electropolymerisation of several monomers has successfully been achieved in a variety of ionic liquids although polymer formation can be sensitive to the medium. This topic has been reviewed in more depth by Pringle.11 Csihony et al.,12 showed that thiophene can be successfully partitioned into an ionic liquid composed of a 1:1 (Bmim)Cl: FeCl3 mixture from a decane layer. We propose that the efficiency of this partitioning occurs because (C4mim)FeCl4 has a relatively low surface tension (46.4 mN m-1) meaning that the enthalpy of hole formation is decreased by adding ferric chloride to the liquid. In this study, a DES composed of 1:2 molar mixture of choline chloride: ethylene glycol (1:2 ChCl: EG) has been used to extract and electro-polymerise thiophene by cyclic voltammetry (CV). 1:2 ChCl: EG was used with and without FeCl3, using Pt working and counter electrodes and a Ag wire reference electrode. The cyclic voltammetric response of thiophene in 1:2 ChCl: EG is shown in Figure 1a. 4 Figure 1: CVs for polymerisation of (0.15 mol/L) thiophene in (a) in 1:2 ChCl: EG, (b) 1:2 ChCl: EG with (0.077 mol/L) of FeCl3,, (c) 0.5 mol/L Th in 1:1 FeCl3/(Bmim)Cl and (d) as (c) but with 0.4 mol/L (0.8mmol) Th in decane. All 40 cycles at 25 ºC, scan rate: 50 mV s-1. -0.4 0.0 0.4 0.8 1.2 1.6 2. Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 5 5 The 1:1 (Bmim)Cl: FeCl3 mixture should be sufficiently aprotic to enable electropolymerisation of Th. Figure 1c shows a cyclic voltammogram of 0.5 mol/L Th in a 1:1 (Bmim)Cl: FeCl3 mixture and the characteristic shape is indicative of the polymerisation of Th on the electrode surface. Figure 1d shows the same experiment run using Th in decane (0.4 mol/L). A similar voltammogram is obtained to the one where Th was in the ionic liquid phase. This shows that the Th is effectively extracted from the decane layer. The addition of decane should dilute the total concentration of thiophene in the system, so the charge observed should decrease. The observation that the charges with and without decane are similar shows that the partition coefficient must be > 1. The kinetics of polymerisation can be estimated from the charge data. A charge equivalent to 2.25 electrons per molecule will lead to the addition of one molecule of thiophene and 0.25 doping anions (Cl-).14 Consequently, the electropolymerisation charge, QTh, 14-15 can be written as: 𝑄𝑇𝑛𝑛𝑛 The 1:1 (Bmim)Cl: FeCl3 mixture should be sufficiently aprotic to enable electropolymerisation of Th. Figure 1c shows a cyclic voltammogram of 0.5 mol/L Th in a 1:1 (Bmim)Cl: FeCl3 mixture and the characteristic shape is indicative of the polymerisation of Th on the electrode surface. Figure 1d shows the same experiment run using Th in decane (0.4 mol/L). A similar voltammogram is obtained to the one where Th was in the ionic liquid phase. This shows that the Th is effectively extracted from the decane layer. The addition of decane should dilute the total concentration of thiophene in the system, so the charge observed should decrease. The observation that the charges with and without decane are similar shows that the partition coefficient must be > 1. The kinetics of polymerisation can be estimated from the charge data. A charge equivalent to 2.25 electrons per molecule will lead to the addition of one molecule of thiophene and 0.25 doping anions (Cl-).14 Consequently, the electropolymerisation charge, QTh, 14-15 can be written as: 𝑄𝑛𝑛𝑛 (1) 𝑄𝑄𝑇𝑇ℎ= 𝑛𝑛𝑛𝑛𝑛𝑛 where, n is the number of electron reduced or oxidised, N is the number of moles of thiophene formed and F is the Faraday constant. where, n is the number of electron reduced or oxidised, N is the number of moles of thiophene formed and F is the Faraday constant. Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 -9 -6 -3 0 3 6 9 12 15 Current (mA) x10 -1 Potential (V) c) 20 cycles 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -1 0 1 2 3 4 5 Current (mA) x10 -2 Potential (V) B 0.2 0.4 0.6 0.8 1.0 1.2 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Current (mA) x10 -3 Potential (V) A -0.4 0.0 0.4 0.8 1.2 1.6 2 -9 -6 -3 0 3 6 9 12 15 Current (mA)X10 -1 Potential (V) 20 cycles d) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -1 0 1 2 3 4 5 Current (mA) x10 -2 Potential (V) B 0.2 0.4 0.6 0.8 1.0 1.2 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Current (mA) x10 -3 Potential (V) A B Current (mA)X10 -1 Potential (V) Figure 1: CVs for polymerisation of (0.15 mol/L) thiophene in (a) in 1:2 ChCl: EG, (b) 1:2 ChCl: EG with (0.077 mol/L) of FeCl3,, (c) 0.5 mol/L Th in 1:1 FeCl3/(Bmim)Cl and (d) as (c) but with 0.4 mol/L (0.8mmol) Th in decane. All 40 cycles at 25 ºC, scan rate: 50 mV s-1. FeCl3/(Bmim)Cl and (d) as (c) but with 0.4 mol/L (0.8mmol) Th in decane. All 40 cycles at 25 ºC, scan rate: 50 mV s-1. Figure 1a shows the clear oxidation and reduction of the thiophene with a half-wave potential of approximately 0.6 V. Scans to 1.2 V do not show the further oxidation to form the polymer growing on the electrode surface. The main reason for this is probably because 1:2 ChCl: EG is a protic solvent containing a hydroxyl group and it could also possibly contains small amounts of water. A large number of studies have shown that the presence of traces of water in the medium precludes the electropolymerisation of thiophene through reaction of the water molecule with thiophene radical cations which make a passivating layer on the working electrode.13 The same experiment was repeated using ferric chloride as an electrocatalyst. Ferric chloride is known to be a good chemical catalyst for the polymerisation of thiophene. A redox process is again observed in Figure 1b, but this time the redox half wave potential is at a lower over-potential (0.4 V) suggesting that it is the FeII/III redox couple. Again no signal for the polymerisation of Th was observed which again is probably due to the protic nature of 1:2 ChCl: EG. Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 So as to compare the amount of Th converted to polymer in Figure 1c and d, the reaction rate has been calculated.16 The electropolymerisation rates of Th in (Bmim)FeCl4 were calculated from Figure 1c and d and are shown in Table 1. The rate of the reaction of consumed Th should be equal to the reaction rate of produced PTh. However, owing to the biphasic nature of the system, it was difficult to estimate the amount of Th in the IL phase after 20 cycles from reaction of polymerisation, therefore, reaction rates were calculated in terms of the products. The data from Table 1 show that the rate of PTh formation in monophasic system is only marginally higher than that from the biphasic system. Given that an equal volume of decane to (Bmim)FeCl4 was used the charge should decrease by half, but the observation that it decreases by less than 10% shows that the partition coefficient must be significantly greater than 1. Table 1: Reaction rate of electropolymerisation of thiophene. Scan rate 50mVs-, 20 scans, potential window:-0.4 – 1.9V. Q (C) N of PTh (mmol) from R (mmol/min) Fig. Qox Qred Ox. Red. Ox. Red. c 0.0183 0.0188 7.6x10-4 8.70x10-5 2.48x10-5 2.84x10-6 d 0.0162 0.0186 6.7x10-4 8.55x10-5 2.2x10-5 2.79x10-6 Table 1: Reaction rate of electropolymerisation of thiophene. Scan rate 50mVs-, 20 scans, potential window:-0.4 – 1.9V. On the other hand, as can be seen in Figure 1(c) and 1(d), as the film thickness of the polymer increases, the voltammetric wave for the reduction of the polymer shifts to more negative potentials and for the oxidation peak shifts to more positive values with consecutive scans which is characteristic 6 of polymer growth. This is possibly due to the polymer conductivity which increases the resistivity of the film on the electrode surface due to slows counter-ion mobility and electron transfer kinetics.17 The Fe-based ionic liquids cannot chemically polymerise thiophene until the molar ratio of FeCl3 and (Bmim)Cl is above 1:1. Above this molar ration the liquid becomes more Lewis acidic and coincidentally less viscous. The concentration of free Cl- is significantly decreased as FeCl4- dominates. This also prevents the chloride ion reacting with the radical cation causing polymer termination. Optimisation of electrocatalytic polymerisation Optimisation of electrocatalytic polymerisation Optimisation of electrocatalytic polymerisation 7 It is interesting to note that while the current increased in 1:1 volume ratio, there was no significant difference in the amount of PTh deposited when compared with that obtained from 1:7 volume ratio which suggests that polymerisation is rate limiting rather than the solubility of Th in the DES. The reaction rates shown in Table 2 are relatively slow which is probably because electropolymerisation is a heterogeneous process.18 In most homogeneous solution polymerisation reactions, the kinetics of addition reactions are first order in monomer.19 Assuming the charge is proportional to concentration and the sweep rate can be related to the time of polymerisation then a pseudo first order kinetic plot could be approximated. 𝑅𝑑𝑑𝑑𝑑𝑘𝐴 𝑅𝑅= − 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑= 𝑘𝑘[𝐴𝐴] (2) 𝐴 𝑅𝑅= − 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑= 𝑘𝑘[𝐴𝐴] (2) where, k is the rate constant of the polymer reaction and [𝐴𝐴] is the initial concentration of monomer. It can be seen from Figure 3 that a plot of ln [Th] vs t gives an approximately straight line correlation suggesting that the process is pseudo first order with a rate constant of 1.4 x 10-5 min-1. where, k is the rate constant of the polymer reaction and [𝐴𝐴] is the initial concentration of monomer. It can be seen from Figure 3 that a plot of ln [Th] vs t gives an approximately straight line correlation suggesting that the process is pseudo first order with a rate constant of 1.4 x 10-5 min-1. Figure 3: Plot of a) deposition charge (from reduction peak) against scan rate, b) ln(unpolymerised Th) vs time for the electrochemical polymerisation of thiophene into 6.87 mmoles of 1:1 (Bmim)Cl: FeCl3, with 0.25 mmoles Th in 2ml decane, at 30 ºC, 15 cycles. Optimisation of electrocatalytic polymerisation Equi-volume amounts of IL and oil are clearly impractical and pre-concentration of Th into the IL are clearly required. This was investigated using different rations of decane to IL. Figure 2 shows the cyclic voltammograms of two different IL: decane ratios. The number of moles of Th converted to polymer in Figure 2, was calculated from the charge under the voltammogram using equation 1 and the data are summarised in Table 2. Figure 2: CVs of a) 1:7 v/v of DES: decane: 0.3 ml includes1.43 mmoles of FeCl3 into 1-1 Fe-DES, 2ml of Th -containing decane (0.21mol/L), b) 1:1 v/v of DES: decane, Th in 2ml of decane is 0.21 mol/L, 25 cycles, mV s-1, 25 ºC. -0.4 0.0 0.4 0.8 1.2 1.6 2. -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Current (mA) Potential (V) 25 cycles b) -0.4 0.0 0.4 0.8 1.2 1.6 2. -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Current (mA) Potential (V) 25 cycles a) -0.4 0.0 0.4 0.8 1.2 1.6 2. -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Current (mA) Potential (V) 25 cycles a) -0.4 0.0 0.4 0.8 1.2 1.6 2. -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Current (mA) Potential (V) 25 cycles b) Current (mA) Figure 2: CVs of a) 1:7 v/v of DES: decane: 0.3 ml includes1.43 mmoles of FeCl3 into 1-1 Fe-DES, 2ml of Th -containing decane (0.21mol/L), b) 1:1 v/v of DES: decane, Th in 2ml of decane is 0.21 mol/L, 25 cycles, mV s-1, 25 ºC. Table 2: Stoichiometry effect on the rate of the electropolymerisation reaction of Th after extraction from decane. Scan rate 50 mV s-1, 25 scans, potential window:-0.4 – 1.9V. IL : Decane (v/v) Q (C) N of Th oxidised (mmol) R (mmol/min) Qox Qred Ox. Red. Ox. Red. 1:7 0.020 0.022 8.09x10-4 1.01x10-4 2.11x10-5 2.62x10-6 1:1 0.023 0.025 9.36x10-4 1.15x10-4 2.44x10-5 3.0x10-6 Table 2: Stoichiometry effect on the rate of the electropolymerisation reaction of Th after extraction from decane. Scan rate 50 mV s-1, 25 scans, potential window:-0.4 – 1.9V. Table 2: Stoichiometry effect on the rate of the electropolymerisation reaction of Th after extraction from decane. Scan rate 50 mV s-1, 25 scans, potential window:-0.4 – 1.9V. Extractive chemical polymerisation of Th and its analogues in 1:2 (Bmim)Cl: FeCl3 For several decades, the lowest sulfur content achieved by HDS of fuels was around 500 mg/kg (around 6 mmoles/L). 20 To demonstrate the efficacy of the DESs to chemically remove aromatic sulfur compounds mixtures of Th, BT and DBT were prepared in decane. Previously, the process could be estimated to be a dynamic equilibrium between the two phases, however with the Lewis acidic liquids, the Th in the IL phase is constantly being oxidised to form PTh and driving the equilibrium towards to IL phase. By analysing the effect of some factors on the pre-equilibrium, the rate of extraction polymer formation can be determined. Optimisation of electrocatalytic polymerisation 20 40 60 80 100 0.01 0.02 0.03 0.04 Charge (C) Scan rate (mV.s -1) a) 10 20 30 40 50 60 4.8274 4.8275 4.8276 4.8277 4.8278 4.8279 4.8280 4.8281 4.8282 Ln(unpolymerised) Time (min) b) K= 1.4X10 -5 min -1 10 20 30 40 50 60 4.8274 4.8275 4.8276 4.8277 4.8278 4.8279 4.8280 4.8281 4.8282 Ln(unpolymerised) Time (min) b) K= 1.4X10 -5 min -1 20 40 60 80 100 0.01 0.02 0.03 0.04 Charge (C) Scan rate (mV.s -1) a) Figure 3: Plot of a) deposition charge (from reduction peak) against scan rate, b) ln(unpolymerised Th) vs time for the electrochemical polymerisation of thiophene into 6.87 mmoles of 1:1 (Bmim)Cl: FeCl3, with 0.25 mmoles Th in 2ml decane, at 30 ºC, 15 cycles. To determine the amount of Th that could be turned over in a given time, the rate of oxidation from Table 2 (1:7) was used with the initial monomer concentration (0.21M) to calculate the rate constant. This was found to be 1.0 x 10-7 moles min-1. Taking a 1 L sample of decane containing 1wt % of Th and 10 x10 cm WE, the time to remove 75 % of this Th from 1wt % in 1 L fuel using electropolymerisation would be 4.5 hrs. This is clearly far too slow to be practically viable. 8 Removal of BT from decane into IL mixture followed by electropolymerisation was also attempted and no polymer could be formed with this monomer which was thought to be due to the high reactivity 8 of radical BT cations, which can undergo fast reaction with the chloride anions or with the imidazolium cation. Electropolymerisation of DBT, was also found to be impossible in this medium. Effect of temperature and stirring Table 3 shows the ability of 1:2 (Bmim)Cl: FeCl3 to extract Th, BT and DBT. It can be seen that the amount of each component extracted after 5 min decreases when the temperature is raised. This shows that the extraction must be an exothermic process as the position of the equilibrium is shifted toward the reagents when the temperature is increased as per Le Chateliers’s principle. It is not possible to calculate the thermodynamics of phase transfer as the system is not at equilibrium, but it is possible to contrast the data with those for extraction in different DESs where the enthalpy of extraction was endothermic (10 – 70 kJ mol-1). 3 Spontaneous polymerisation processes are always strongly exothermic and it could be this driving the extraction of the monomer. Table 3: Extractive desulfurisation of 35 mmol L-1 of each one of Th, BT and DBT into 1:2 (Bmim)Cl: FeCl3 mixture, as a function to time, 500 rpm, fuel/IL mass ratio: 2:1. Table 3: Extractive desulfurisation of 35 mmol L-1 of each one of Th, BT and DBT into 1:2 (Bmim)Cl: FeCl3 mixture, as a function to time, 500 rpm, fuel/IL mass ratio: 2:1. Table 3: Extractive desulfurisation of 35 mmol L-1 of each one of Th, BT and DBT into 1:2 (Bmim)Cl: FeCl3 mixture, as a function to time, 500 rpm, fuel/IL mass ratio: 2:1. Temperature (oC) 25 40 Time (min) 5 10 5 10 20 Extracting (%) Th 85.7 ± 5 ˃99.9 76.3 ± 2 95 ± 4 ˃99.9 BT 67.5 ± 3 ˃99.9 75.3 ± 3 92.8 ± 4 ˃99.9 DBT 58 ± 6 ˃99.9 65.8 ± 3.8 90.6 ± 2 ˃99.9 Transfer of BT and DBT are quite similar once experimental errors are taken into account but either way neither are significantly affected by temperature. This could suggest that the equilibrium is governed by the rate at which the monomer is converted to the polymer (oligomer). After 10 min, the extraction at 25 oC is all but complete whereas those at 40 ºC have still not quite gone to completion. This further suggests that the transfer is slightly exothermic. Contrasting these data with 9 those obtained for the DESs where after 1 h, a significant proportion of Th was still in the alkane phase. It can be seen that converting Th to PTh helps in extracting the Th from the decane phase. Effect of temperature and stirring The previous study of thiophene extraction in DESs showed that the surface tension gives a measure of the cohesive energy density in ionic liquids and this in turn controls the viscosity and conductivity of the liquid. The lower the surface tension the larger the void volume and the lower the viscosity. 3 Measuring the surface tension of two different molar ratios of the 2:1 and 1:1 FeCl3: (Bmim)Cl it was found that they were both 46.4 mN m-1. Previously it was found that the best solvent for extracting of Th, BT and DBT was a mixture of pentylene glycol and choline chloride (4:1) which had a surface tension of 46 mN m-1. The ferric chloride decreases the surface tension of BmimCl (71 mN m-1) by delocalising the charge and it also has a stronger interaction with Th than occurs in a non-metallic DES and these two factors are sufficient to change the enthalpy of transfer from endothermic to exothermic which makes the partition coefficient larger. those obtained for the DESs where after 1 h, a significant proportion of Th was still in the alkane phase. It can be seen that converting Th to PTh helps in extracting the Th from the decane phase. In the previous studies, the reactivity of organosulfur rings in HDS processes has been reported by some researchers and it was investigated that the reactivity follows the order: Th˃ BT ˃ DBT.21 This could be due to two factors, increasing the size of the solute molecule increase the size of the hole required to accommodate the solute, the interaction between the iron species and the heterocyclic sulfur atom is probably precluded due to steric factors. This also explains why BT and DBT are slower to partition into the ionic liquid since polymerisation of these monomers will be slower due to steric hindrance and deactivation. The influence of the stirring speed on the sulfur compound extractions is statistically significant as seen in Figure 4. Doubling the rotation speed of the stirrer in the ionic liquid phase ensured that all the S-containing species were extracted from the decane at 25 ºC within 5 min which took about double the time with a slower stirring rate. Figure 4: Effect of stirring speed on the extraction of sulfur compounds, IL: fuel mole proportion 1:2. Extraction time was 5 min. Effect of temperature and stirring 500 1000 0 20 40 60 80 100 S-removal (%) Mixing speed (rpm) Th BT DBT Figure 4: Effect of stirring speed on the extraction of sulfur compounds, IL: fuel mole proportion 1:2. Extraction time was 5 min. 10 Effect of phase ratio Effect of phase ratio To investigate the effect of the mass ratio of the organic and DES phases on the extraction efficiency, different mass ratios were employed and the results are shown in Figure 5. All were mechanically agitated (500 rpm) with a magnetic stirrer for 5 min and then left to settle for 5 min to ensure complete thermodynamic equilibrium at 25 ºC. Figure 5: Desulfurisation of a mixture of sulfur compounds from decane containing 35 mmol L-1 of Th, BT and DBT into (Bmim)FeCl4. 1:1 1:2 1:3 1:4 0 20 40 60 80 100 S-removal (%100) Mass ratio of IL:fuel Th BT DBT Figure 5: Desulfurisation of a mixture of sulfur compounds from decane containing 35 mmol L-1 of Th, BT and DBT into (Bmim)FeCl4. It can clearly be seen that the amount of (Bmim)FeCl4 was an important factor in EDS. Since the iron chloride is still in vast excess to thiophene it is probably a mass transport effect which is still controlling the conversion rate. It can clearly be seen that the amount of (Bmim)FeCl4 was an important factor in EDS. Since the iron chloride is still in vast excess to thiophene it is probably a mass transport effect which is still controlling the conversion rate. Effect of time A mixtures of Th, BT and DBT were extracted with different oil to IL ratios, all in one mixture tried to elucidate the rate limiting factor. Figure 6a shows the efficiency of extracting 35 mmol L-1 of Th, BT and DBT from the decane phase into 2:1 FeCl3: (Bmim)Cl ionic liquids in an unstirred system. This was done at an IL: oil mass ratio of 1:1. 11 Figure 6: Extractive desulfurisation of Th and its derivatives as a mixture from decane into 2:1 FeCl3: (Bmim)Cl at 25 ºC, with equimass ratio of IL to fuel: a) extracting S-compound (%), b) plot of ln (monomer) vs time. 6min 12min 1/2h 1h 2h 4h 8h 1day 0 20 40 60 80 100 Th BT DBT Extracting amount (%) Time a) 0 100 200 300 400 500 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Th (K= 2.13X10 -2 min -1) BT (K= 5.7X10 -3 min -1) DBT (K= 1.3X10 -3 min -1) Ln(unreacted) Time (min) b) 6min 12min 1/2h 1h 2h 4h 8h 1day 0 20 40 60 80 100 Th BT DBT Extracting amount (%) Time a) y 0 100 200 300 400 500 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Th (K= 2.13X10 -2 min -1) BT (K= 5.7X10 -3 min -1) DBT (K= 1.3X10 -3 min -1) Ln(unreacted) Time (min) b) Figure 6: Extractive desulfurisation of Th and its derivatives as a mixture from decane into 2:1 FeCl3: (Bmim)Cl at 25 ºC, with equimass ratio of IL to fuel: a) extracting S-compound (%), b) plot of ln (monomer) vs time. After 6 min, 38 % of Th had been extracted and it required about 4 hours to extract all the Th (Figure 6a). Comparing this with the stirred system in Figure 5 it can be seen that mass transport is a very important factor governing extraction and conversion to the polymer. With respect to BT, after 1 h, only 35 % had been extracted and it only reached 100 % after 8 hrs. Removal of DBT was the slowest process with only 34 % removed after 4 hrs. This fits in with the earlier observation that the polymerisation of Th is the fastest followed by BT and DBT. Strong interaction between the Lewis acid and S-species made the extraction easier even when the reaction takes place in the absence of stirring. Effect of time The overall removal of the sulfur containing species is given by; Th(decane) ↔ Th(DES) Th(DES) →PTh(s) Th(decane) ↔ Th(DES) Th(DES) →PTh(s) The reaction could either be limited by the transfer of species into the DES or by the activation of the monomer. If it is the latter of these then the reaction could be thought of as a pseudo-first order reaction since the catalyst is present in excess. The rate equation for this type of reaction should therefore approximate to: 𝑇𝑘𝑘 [𝑇𝑇ℎ] [𝑇𝑇ℎ]0 = −𝑘𝑘𝑘𝑘 (5) [𝑇𝑇ℎ] [𝑇𝑇ℎ]0 = −𝑘𝑘𝑘𝑘 (5) A plot of ln [S-compound] should give a straight line plot. The data in Figure 6a were tested in this way and the results are shown in Figure 6b. It can be seen that the extraction of all of the sulfur 12 containing compounds follow this pseudo first order plot. However, it should be stressed that this is by no means proof that this is the definite mechanism for the extraction process. The data do not, however fit well to a second order plot or to a half order plot as may be appropriate if diffusion was the limiting factor. It can therefore only be suggested that a pseudo first order polymerisation may be an appropriate model that fits the observed data relatively well. Finally, it is important to note the comparative rate constants for the electrochemical and chemical rates of polymer production. Comparing the data for Th from Figure 6b with those presented in Figure 3b, it can be seen that the rate for the chemical production of PTh is approximately 1500 times faster than the electrochemical process. This would be expected given that the chemical process is a 3-D rather than a 2-D reaction as would be the case for the electrocatalytic process. It should also be noted that the concentration of FeCl3 is significantly higher for the chemical process. The main difficulty of using ILs to extract thiophenic compounds from oils is that the polymerisation is a stoichiometric reaction with iron and to make it a catalytic process the Fe(II) produced would have to be reoxidised. If all the Fe(III) is reduced then the IL becomes more viscous and eventually solidifies. It is difficult to extract the polymer from the ionic liquid due to the high viscosity. Conclusion This study has shown that the main factor controlling the partitioning of species between an oil and an ionic liquid is the enthalpy of solvation of the solute and the enthalpy of hole formation in the DES. These are controlled by the relative solvent-solvent and solvent-solute interactions. Using a mixture of (Bmim)Cl and FeCl3 causes more thiophene to partition into the ionic phase than when non-metallic DESs are used. This is thought to be due to the low surface tension but also the specific interaction between FeCl3 and thiophene. It is shown for the first time that electropolymerisation of thiophene can be carried out in the in the 1:1 (Bmim)Cl: FeCl3 phase by cyclic voltammetry. This can drive the equilibrium to pull the Th from the alkane phase as it is polymerised in the IL phase. This study has shown that the main factor controlling the partitioning of species between an oil and an ionic liquid is the enthalpy of solvation of the solute and the enthalpy of hole formation in the DES. These are controlled by the relative solvent-solvent and solvent-solute interactions. Using a mixture of (Bmim)Cl and FeCl3 causes more thiophene to partition into the ionic phase than when non-metallic DESs are used. This is thought to be due to the low surface tension but also the specific interaction between FeCl3 and thiophene. It is shown for the first time that electropolymerisation of thiophene can be carried out in the in the 1:1 (Bmim)Cl: FeCl3 phase by cyclic voltammetry. This can drive the equilibrium to pull the Th from the alkane phase as it is polymerised in the IL phase. While electrochemical polymerisation of Th is possible it is not practically viable due to the slow reaction kinetics. A 1:2 (Bmim)Cl: FeCl3 mixture was successfully used to polymerise Th, BT and DBT. The extraction Th into 1:2 (Bmim)Cl: FeCl3 is exothermic and occurs 1500 times faster than electropolymerisation in 1:1 (Bmim)Cl: FeCl3. While electrochemical polymerisation of Th is possible it is not practically viable due to the slow reaction kinetics. A 1:2 (Bmim)Cl: FeCl3 mixture was successfully used to polymerise Th, BT and DBT. The extraction Th into 1:2 (Bmim)Cl: FeCl3 is exothermic and occurs 1500 times faster than electropolymerisation in 1:1 (Bmim)Cl: FeCl3. Regeneration of used IL mixture was difficult because polythiophene does not separate from the ionic liquid (due to similar densities and high viscosity). Conclusion The polymer causes the ionic liquid to gel which decreases mass transport and stops the polymerisation reaction. To enable ionic liquids to be used for ultra-desulfurisation of fuel the phase behaviour of the catalyst needs to be addressed and a facile method of reoxidation needs to be found. Effect of time Regeneration of FeCl3 were attempted using solvent extraction and an electrochemical set up using a liquid-liquid junction to enable electron transfer.22 Both of these approaches were ultimately unsuccessful and the ionic liquid solidified with extended use. To improve the viability of ionic liquids for preparing ultra-low sulfur fuels it is important to focus on the reoxidation of the ionic liquid. The main issue behind this is maintaining liquid phase behaviour for the ionic liquid and establishing a catalytic method of reoxidation. Nie and coworkers investigated the EDS process using ILs on commercial diesel samples.23 The authors suggested that S-removal from commercial fuel is much harder than desulfurisation of model fuel without giving the reasons.24 Similarly, Eßer et al, 25 reported the Kp for S-removal of commercial diesel is less than Kp of a model oil. Similarly, Xu et al. and Yu et al. also found that in addition the relatively high moisture content in commercial diesel fuel necessitates additives which have a negative impact on the removal of sulfur compounds.26 For practical applications the other components in fuels need to be taken into account when preparing a sulfur extraction process. Commercial diesel was used but it was spiked with Th, BT, and DBT each at a concentration of 35 mmol L-1. The concentrations of the thiophenic compounds were determined by GC at the same operational reaction conditions as those described above. During the process, the sulfur levels in the upper phase of each one of Th, BT and DBT could be extracted to (27.8, 18.8 and 16.3 mmol L-1) respectively through the optimisation of reaction conditions; which means 79.3, 53.8 13 and 46.5 % of Th, BT and DBT respectively were removed just after 5 min. This compared with 85.7, 67.5 and 58% respectively for the corresponding extraction from decane. As expected, the extraction data for removing Th, BT and DBT from commercial diesels are lower than from decane. This may be attributed to the coexistence of many aromatics, oxygen and nitrogen additives which compete with the partition of S-compounds in the (C4mim)FeCl4 and saturate the ionic liquid phase.27 References g , ( ), (2) (a) Visser, A. E.; Swatloski, R. P.; Reichert, W. M.; Mayton, R.; Sheff, S.; Wierzbicki, A.; Davis Jr, J. H.; Rogers, R. D., Task-specific ionic liquids for the extraction of metal ions from aqueous solutions. Chem. 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Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel
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Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel Erven, Jolijn; Çakirlar, Canan; Raemaekers, Daan; Madsen, Ole; Bradley, Daniel Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel Erven, Jolijn; Çakirlar, Canan; Raemaekers, Daan; Madsen, Ole; Bradley, Daniel Published in: Frontiers in Genetics DOI: 10.3389/fgene.2022.872486 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2022 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Erven, J., Çakirlar, C., Raemaekers, D., Madsen, O., & Bradley, D. (2022). Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel. Frontiers in Genetics, 13, Article 872486. https://doi.org/10.3389/fgene.2022.872486 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. 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. 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. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. ORIGINAL RESEARCH published: 12 July 2022 doi: 10.3389/fgene.2022.872486 Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel J. A. M. Erven 1*, C. Çakirlar 1, D. G. Bradley 2, D. C. M. Raemaekers 1 and O. Madsen 3 1Groningen Institute of Archaeology, University of Groningen, Groningen, Netherlands, 2Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland, 3Animal Breeding and Genomics, Wageningen University and Research, Wageningen, Netherlands Sequencing ancient DNA to high coverage is often limited by sample quality and cost. Imputing missing genotypes can potentially increase information content and quality of ancient data, but requires different computational approaches than modern DNA imputation. Ancient imputation beyond humans has not been investigated. In this study we report results of a systematic evaluation of imputation of three whole genome ancient Sus scrofa samples from the Early and Late Neolithic (~7,100–4,500 BP), to test the utility of imputation. We show how issues like genetic architecture and, reference panel divergence, composition and size affect imputation accuracy. We evaluate a variety of imputation methods, including Beagle5, GLIMPSE, and Impute5 with varying filters, pipelines, and variant calling methods. We achieved genotype concordance in most cases reaching above 90%; with the highest being 98% with ~2,000,000 variants recovered using GLIMPSE. Despite this high concordance the sources of diversity present in the genotypes called in the original high coverage genomes were not equally imputed leading to biases in downstream analyses; a trend toward genotypes most common in the reference panel is observed. This demonstrates that the current reference panel does not possess the full diversity needed for accurate imputation of ancient Sus, due to missing variations from Near Eastern and Mesolithic wild boar. Imputation of ancient Sus scrofa holds potential but should be approached with caution due to these biases, and suggests that there is no universal approach for imputation of non-human ancient species. Edited by: Xiangdong Ding, China Agricultural University, China Reviewed by: Luca Ermini, Luxembourg Institute of Health, Luxembourg Huashui Ai, Jiangxi Agricultural University, China *Correspondence: J. A. M. Erven jolijn_erven@hotmail.com Edited by: Xiangdong Ding, China Agricultural University, China Reviewed by: Luca Ermini, Luxembourg Institute of Health, Luxembourg Huashui Ai, Jiangxi Agricultural University, China *Correspondence: J. A. M. Erven jolijn_erven@hotmail.com Specialty section: This article was submitted to Livestock Genomics, a section of the journal Frontiers in Genetics Keywords: imputation, ancient DNA (aDNA), Sus scrofa, animal husbandry, Neolithic Received: 09 February 2022 Accepted: 20 May 2022 Published: 12 July 2022 Keywords: imputation, ancient DNA (aDNA), Sus scrofa, animal husbandry, Neolithic 2.1 Data Description and Preparation 2.1.1 Ancient Samples A number of factors are known to influence imputation ranging from reference panel characteristics to demographic history; assessing the potential and limitations of imputation of species beyond model species like humans is valuable to aid our understanding of not only imputation performance but also recent evolutionary events. This paper assesses the power of imputation to increase the quality and information potential of low coverage aDNA samples, using Sus scrofa as a case study. This species is an intensively studied livestock species in terms of aDNA, particularly in the context of expansion of animal husbandry into Europe and significantly enhancing our understanding of how farming started in Europe (Larson et al., 2007; Ottoni et al., 2013; Frantz et al., 2019). Investigations have indicated that ancient Near Eastern domestic pigs lost their Near Eastern genomic signatures after their introduction to Europe (Larson et al., 2007; Frantz et al., 2019). Obtaining HQ samples to pinpoint the pace and nature of this turnover in different regions and shorter timescales in relation to larger societal and economic developments is necessary, but it remains a challenge due to poor preservation and contamination. To address this challenge, a systematic evaluation of different imputation methods was performed on whole genome ancient Sus scrofa DNA using data from a recent study consisting of ancient whole genomes of pigs sequenced to an appropriate depth for imputation (Frantz et al., 2019). Imputation achieved high genotype concordance but this is paired with biases toward a fraction of the reference panel. These biases might be related to the size and diversity of the reference panel, the reference genome, or the genetic architecture of pigs, and they impose limitations on the interpretive power of imputed data in terms of the proposed genomic turnover of this species in particular and in general the evolution of animal husbandry in Neolithic Europe. Contamination from prokaryotes and humans was assessed by calculating percent identity score and coverage per read with BLAST + Blastn Megablast v2.10.1 on prokaryotes, human and Sus scrofa databases (Camacho et al., 2008). Reads were considered contaminants if the percent identity (E-value) and coverage of the contaminants (prokaryotes and humans) was higher than the percent identity and coverage of Sus scrofa. Contaminated reads were removed from the BAM file with a custom-made python script. 2.1 Data Description and Preparation 2.1.1 Ancient Samples 2009; Prüfer et al., 2010; Ginolhac et al., 2011; Sánchez-Quinto et al., 2012; Parks and Lambert, 2015; Kistler et al., 2017). One way to counter these problems is imputation, which is a powerful way to improve the quality of data and can potentially maximize the power of analysis that require dense genotypes such as runs of homozygosity (ROH), in depth admixture and trait association analyses (Gamba et al., 2014; Martiniano et al., 2017). Imputation is widely employed in studies of modern data (Van den Berg et al., 2019; Ye, et al., 2019), targeting allele frequencies from a set of reference individuals to infer allele frequencies at unknown or missing sites (Browning and Browning, 2007; Ausmees, 2019). Seven archaeological samples with high-coverage data and four archaeological samples with moderate coverage from Frantz et al. (2019) were used (Table 1; Supplementary Table S1). Raw FASTQ reads were downloaded from the ENA (accession numbers see Supplementary Table S1). Raw reads were trimmed using cutadapt v2.10 (Martin, 2011) for quality (<20), length (<20) and adapters used in the library preparation (Meyer and Kircher, 2010). FastQC v0.11.9 quality reports were made for the raw and trimmed data (Andrews, 2010). The trimmed reads were aligned applying the Burrows- Wheeler algorithm (BWA) aln v0.7.17 (Li and Durbin, 2009) to the Sus scrofa 11.1 reference genome (Warr et al., 2020), with default parameters apart from disabling the seed option (−l 1024), increasing the maximum number of gap opens (−o 2) and changing the maximum edit distance (−n 0.01). Duplicates were removed with Picard MarkDuplicates v2.18.17 (http:// broadinstitute.github.io/picard) and BAM files from different sequencing lanes were merged using SAMtools merge v0.1.19 (Li et al., 2009). Duplicates were removed with FilterUniqueSamCons.py for the merged BAM files (Kircher, 2012). Indels were realigned with GATK 3.8 RealignerTargetCreator and IndelRealigner with default parameters (Van der Auwera et al., 2013). Depth of coverage and quality were computed using Qualimap v2.2.1 (Okonechnikov et al., 2015). Molecular damage was assessed using MapDamage2.0 using default parameters (Jónsson et al., 2013). In aDNA studies, imputation has been applied on human genomes and achieved high levels of concordance between imputed genotypes and their high-quality (HQ) counterparts (>99%) (Gamba et al., 2014; Martiniano et al., 2017; Ausmees et al., 2021). Imputation of aDNA beyond humans is lacking; livestock aDNA is critical to understand pivotal moments in recent evolution such as domestication and pose an excellent case study. Citation: Recent advances in sequencing techniques led to a dramatic increase in the amount of retrievable ancient DNA (aDNA) from archaeological remains (Kircher, 2012), providing new insights into recent evolutionary history (Slatkin and Recimo, 2016; MacHugh et al., 2017; Brunson and Reich, 2019; McHugo et al., 2019). Poor preservation and contamination of exogenous DNA restricts sequence quality, reliability, and coverage of aDNA from archaeological bones (Pääbo et al., 2004; Prüfer et al., 2010). Furthermore, the damaged nature of aDNA poses computational challenges and introduces biases to the analysis of aDNA (Höss et al., 1996; Brotherton et al., 2007; Briggs et al., Erven JM, Çakirlar C, Bradley DG, Raemaekers DCM and Madsen O (2022) Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel. Front. Genet. 13:872486. doi: 10.3389/fgene.2022.872486 July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 1 Imputation of Ancient Sus scrofa Erven et al. 2.1 Data Description and Preparation 2.1.1 Ancient Samples Imputation was assessed by comparing imputed genotypes to their corresponding HQ genotypes, similar to previous studies (Gamba et al., 2014; Martiniano et al., 2017; Ausmees et al., 2021). Three of the seven samples with high-coverage data (KD033, KD037, and VEM185) were downsampled with Picard v2.18.17 (http://broadinstitute.github.io/picard), to create low coverage samples for imputation ranging from 0.5 to 2× with steps of 0. 5×. Three methods were used to assess the accuracy of imputation: Method 1, imputation with variant sites; Method 2, imputation with all confident sites; and Method 3, added to achieve higher genotype concordance which called only genotypes present in the reference panel. HQ genotypes were created from the high-coverage samples to create a golden standard. Genotype likelihoods were called with the Genome Analysis Toolkit (GATK) UnifiedGenotyper v3.8.0 (Van der Auwera et al., 2013) using either each alignment data of the ancient samples individually or by joined SNP calling. Genotype likelihoods were called with a minimum quality of 25, with output mode EMIT_VARIANTS_ONLY for Method 1, EMIT_ALL_ 2 MATERIALS AND METHODS Evaluating imputation of Sus scrofa aDNA by comparing three tools, two pipelines, and three variant calling methods. July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 2 Imputation of Ancient Sus scrofa Erven et al. TABLE 1 | Sample information. ID, origin, period and ancestry taken from Frantz et al. (2019). ID Origin Period Ancestry Genome coverage KD033 Germany-Herxheim Neolithic ~46% European, ~54% Near Eastern 6.9 KD037 Germany-Herxheim Neolithic ~91% European, ~9 Near Eastern 21.6 VEM185 England-Durrington Walls Neolithic/Bronze Transition ~90% European, ~10 Near Eastern 21.7 TABLE 2 | Reference panels with their respective number of individuals/population. Reference panel Number of individuals Main references Dutch wild boar-European wild boar (EUW) 12 Italian wild boar-European wild boar (EUW) 6 French wild boar-European wild boar (EUW) 1 Pig breeds-European Domestic (EUD) 25 Greek wild boar (BLW) 4 Near Eastern + Turkish wild boar- Near Eastern wild boar (NEW) 3 Total 51 Main + ancient references Main reference 51 Near Eastern-Ancients (ANC) 5 European-Ancients (ANC) 3 Total 59 TABLE 1 | Sample information. ID, origin, period and ancestry taken from Frantz et al. (2019). ID Origin Period Ancestry Genome coverage KD033 Germany-Herxheim Neolithic ~46% European, ~54% Near Eastern 6.9 KD037 Germany-Herxheim Neolithic ~91% European, ~9 Near Eastern 21.6 VEM185 England-Durrington Walls Neolithic/Bronze Transition ~90% European, ~10 Near Eastern 21.7 TABLE 1 | Sample information. ID, origin, period and ancestry taken from Frantz et al. (2019). TABLE 2 | Reference panels with their respective number of individuals/population. Reference panel Number of individuals Main references Dutch wild boar-European wild boar (EUW) 12 Italian wild boar-European wild boar (EUW) 6 French wild boar-European wild boar (EUW) 1 Pig breeds-European Domestic (EUD) 25 Greek wild boar (BLW) 4 Near Eastern + Turkish wild boar- Near Eastern wild boar (NEW) 3 Total 51 Main + ancient references Main reference 51 Near Eastern-Ancients (ANC) 5 European-Ancients (ANC) 3 Total 59 TABLE 2 | Reference panels with their respective number of individuals/population. Number of individuals effect of the reference composition on the imputation, multiple reference panels were considered. The main reference panel consists of modern pig breeds, European wild boar, and Near Eastern wild boar (51 individuals, 12,737,362 variants—Table 2; Supplementary Table S2). 2 MATERIALS AND METHODS In order to avoid introducing a possible bias from nucleotide misincorporations due to post-mortem damage, the generated VCF (Variant Call Format) files were filtered to exclude all sites where the most likely genotype could have been inferred from a deaminated allele with a custom-made python script. For C→T deaminations, C↔T SNPs were excluded from further analyses if the most likely genotype contained a T allele, and for G→A deaminations, G↔A SNPs were excluded from further analyses if the most likely genotype contained an A allele. Genotypes were not filtered in Method 3 when using GLIMPSE, because this software only imputes genotypes present in the target VCF, they were instead kept as no calls (./.). 2.1.2 Reference Panel The reference material used for imputation consisted of the wild boar and pig breeds collection of Wageningen University and two Iberian samples from Ramírez et al. (2015) (Supplementary Table S1). Pig breeds that have no known introgression with Asian breeds were selected to avoid potential bias. Genotype likelihoods were called with the Genome Analysis Toolkit (GATK) UnifiedGenotyper v3.8.0 (Van der Auwera et al., 2013), with a minimum quality of 15, calling SNPs, with the mode EMIT_VARIANTS_ONLY for Method 1 and EMIT_ALL_CONFIDENT_SITES for Method 2. The reference panel was filtered to only include autosomal biallelic SNPs, a minimum quality of 30, and a minimum depth of 4, a call rate of 0.8, and removal of repetitive elements. In order to evaluate the 2 MATERIALS AND METHODS To deduce the effect of ancient samples on imputation, eight ancient individuals were added to the main reference panel, consisting of two Near Eastern samples, three ancient Near Eastern, and three ancient European samples (59 individuals, 10,823,257 variants—Table 2, Supplementary Table S2), called Main + ancient reference. Moreover, the main reference panel was divided into several subsets to pinpoint the effect of reference bias on imputation (See Supplementary Material-Subsets of reference panel). Additionally, to deduce the effect of Asian haplotypes on imputation, Asian wild boars, Asian domestic pigs and South-East Asian Sus were added to the reference panel (See Supplementary Material-Including Asian samples). Different filters and combinations of filters were used on the reference panel to optimize the imputation workflow and deduce the effects of these filters on imputation. These filters consisted of removing 1) transversions, 2) transitions, 3) filtering for minor allele frequency (MAF) bins {<0.05, 0.05–0.1, 0.1–0.3, >0.05, >0.3, No MAF}, and their various combinations. Results of all combinations can be found in Supplementary Table S2. The reference panels were phased with Beagle5 (Browning et al., 2018), using default parameters apart from changing the effective population size (Ne) to 20,000 (Groenen et al., 2012). CONFIDENT_SITES for Method 2 and output mode EMIT_ ALL_SITES and genotyping mode GENOTYPE_GIVEN_ ALLELES for Method 3, with -alleles genotypes from the reference panel. Variants were filtered to keep only autosomal, biallelic SNPs, and a minimum quality of 30. In order to avoid introducing a possible bias from nucleotide misincorporations due to post-mortem damage, the generated VCF (Variant Call Format) files were filtered to exclude all sites where the most likely genotype could have been inferred from a deaminated allele with a custom-made python script. For C→T deaminations, C↔T SNPs were excluded from further analyses if the most likely genotype contained a T allele, and for G→A deaminations, G↔A SNPs were excluded from further analyses if the most likely genotype contained an A allele. Genotypes were not filtered in Method 3 when using GLIMPSE, because this software only imputes genotypes present in the target VCF, they were instead kept as no calls (./.). CONFIDENT_SITES for Method 2 and output mode EMIT_ ALL_SITES and genotyping mode GENOTYPE_GIVEN_ ALLELES for Method 3, with -alleles genotypes from the reference panel. Variants were filtered to keep only autosomal, biallelic SNPs, and a minimum quality of 30. 2.2 Genetic Map A genetic map was created using the recombination frequencies that Johnsson et al. (2020) estimated based on nine genotyped pedigrees on the Sus scrofa 11.1 reference genome. These July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 3 Erven et al. Imputation of Ancient Sus scrofa calculated by dividing the amount of imputed genotypes to the total amount of HQ genotypes. recombination frequencies were converted to cM using the Haldane formula (Haldane’s Mapping Function, 2008). Genetic maps were made for each chromosome in the plink format with bins of 1 MB (Supplementary Table S3). 2.6 Reference Affinity Correct and incorrect imputed genotypes were compared to their HQ counterpart to assess whether imputed genotypes show a systematic bias toward the reference genome. Reference bias was measured as the presence/absence of different ancestral/origin groups between the correct and incorrect imputed genotypes and their HQ counterpart. 2.3 Imputation p For Methods 1–3 imputation was performed using Impute5 and Beagle5, using default parameters, with a phased reference panel (Supplementary Table S2), with a Ne of 20,000 and, --div-select and –out-gp-field parameters for Impute5 (Rubinacci et al., 2020) and window = 40, overlap = 4 and gp = true parameters for Beagle5 (Browning et al., 2018). Imputation was performed for chromosome 1–18, individually and using sliding windows (See Supplementary Material—Chromosomal imputation). The effect of including multiple ancient samples on imputation was evaluated by imputing joint ancient samples and was compared to individual imputation (Supplementary Material—Joined Imputation). The focus was on individual imputation. Imputation was performed using two different imputation pipelines: 1) the original one-step pipeline used in Ausmees et al. (2021) and 2) the two-step pipeline used for low coverage samples in Hui et al. (2020). The two-step pipeline adds another filtering step prior to imputation that accounts for genotype probability. Beagle 4.1 was used to calculate genotype probabilities for the target downsampled VCF using default parameters, with the same phased reference panel that was used for imputation (Supplementary Table S2), with a Ne of 20,000 and gprobs = true parameters. Variants with a genotype probability (GP) < 0.99 were removed from the target downsampled VCF, leaving only confident genotype calls. The imputed genotypes were filtered for an imputation score of 1 (highest imputation accuracy). For Method 3, GLIMPSE v1.1.1 (Rubinacci, et al., 2021) was also used with similar settings as applied in ancient human imputation and the pipeline proposed by Rubinacci, et al. (2021), with default parameters, and a phased reference panel (Supplementary Table S2). Variants with an imputation score of <1 were removed from the target downsampled VCF, leaving only confident imputed genotype calls. GLIMPSE v1.1.1 was only tested with Method 3 because of the incompatibility with the other two methods/pipelines. 2.5 Downstream Analysis Downstream analyses were performed to investigate the difference and/or similarity between imputed and HQ genotypes. Data were pruned with PLINK 1.9 (Purcell et al., 2007) with the parameters—geno 0.10. A principal component analysis (PCA) was performed on diploid genotypes consisting of the reference panel, the HQ samples and the imputed samples using PLINK 1.9 pca on autosomes only. Eigenvalues and vectors were plotted with the use of Mathplotlib (Hunter, 2007) and Seaborn (https://zenodo.org/record/883859#.XSdFFugza01). An admixture analysis was performed using the same dataset as the PCA analysis, however separately for downsampled, imputed and HQ genotypes. ADMIXTURE v1.3.0 (David et al., 2009) was used with standard parameters and K ranging from 2 till 5. Furthermore, bootstrapping was performed using the parameter -B. Identical By Descent (IBD) analysis was conducted on the same dataset as the admixture analysis. IBDseq v2.0 with standard parameters was used to calculate IBD segments between samples (Browning and Browning, 2013). A regions of homozygosity analysis was performed using the same dataset as the admixture analysis using plink –homozyg with the parameters –homozyg-kb 10, --homozyg-gap 10, --homozyg-snp 100, --homozyg-window-het 2, --homozyg- window-snp 100 --homozyg-window-missing 1. DetectRUNS (https://cran.r-project.org/package=detectRUNS) was used to visualize and calculate ROH statistics. 2.4 Genotype Concordance Genotype concordance was calculated for three imputation tools, two pipelines and three variant calling methods to test the best method to approach imputation in Sus scrofa. Downstream analyses were performed to assess the accuracy and power of imputation. 2.4 Genotype Concordance Imputation accuracy was assessed by genotype concordance defined as the fraction of genotypes that were imputed correctly. This was measured by dividing the incorrectly imputed SNPs with all imputed SNPs and was measured separately for each sample. The correctly and incorrectly imputed SNPs were derived from comparing the imputed SNPs to their HQ counterpart similar to the approach of Picard GenotypeConcordance. The HQ genotypes used are pre-deamination filtered, to keep confident transitions (transitions that also occurred in the reference panel), and not transitions arising from deamination. The incorrectly imputed SNPs were classified into incorrect positions (positions not occurring in HQ) and incorrect genotypes (genotypes different from HQ genotypes). Information content, that is, the amount of gained genotypes, was Frontiers in Genetics | www.frontiersin.org 3.1 Genotype Concordance 3.1.1 Tools: Beagle5 Versus Impute5 g p Genotype concordance was higher for Beagle5 compared to Impute5 for KD037 and VEM185 but lower for KD033 (Figure 1). For both tools, KD037 and VEM185 performed better than KD033, this being more pronounced for Beagle5. The amount of correctly imputed variants differed greatly between the tools, with Beagle5 being systematically lower (Figures 1IC,D). Impute5 imputed 25%–34% of the total amount of HQ genotypes, while Beagle5 imputed around 5%. July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 4 Erven et al. Imputation of Ancient Sus scrofa FIGURE 1 | Genotype concordance and fraction of high-quality (HQ) genotypes (portrayed in decimals, total HQ genotypes: VEM185: 4,701,683, KD037: 4,531,126, KD033: 3,887,848) using different imputation tools and reference panels. Imputed from 1× downsampled coverage genomes of VEM185, KD037, and KD033. I) Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5 (C), and Beagle5 (D). II) Genotype concordance and Fraction of HQ genotypes of various MAF bins of Main + 2-step + Anc + all reference panel. Genotype concordance for Impute 5 (A), Beagle5, (B) Fractions of HQ genotypes covered by imputation in Impute5 (C), Beagle5 (D) and, Genotype concordance of only heterozygotic variants of various MAF bins imputed with Impute5 (E) III) Filters on target downsampled vcf Genotype concordance for Impute 5 (A) Beagle5 (B) Fractions of HQ genotypes covered by imputation in p FIGURE 1 | Genotype concordance and fraction of high-quality (HQ) genotypes (portrayed in decimals, total HQ genotypes: VEM185: 4,701,683, KD037: 4,531,126, KD033: 3,887,848) using different imputation tools and reference panels. Imputed from 1× downsampled coverage genomes of VEM185, KD037, and KD033. I) Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5 (C), and Beagle5 (D). II) Genotype concordance and Fraction of HQ genotypes of various MAF bins of Main + 2-step + Anc + all reference panel. Genotype concordance for Impute 5 (A), Beagle5, (B) Fractions of HQ genotypes covered by imputation in Impute5 (C), Beagle5 (D) and, Genotype concordance of only heterozygotic variants of various MAF bins imputed with Impute5 (E). III) Filters on target downsampled vcf. Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5, (C) Beagle5 (D). 3.1 Genotype Concordance IV) Genotype concordance (A) and fraction of HQ genotypes (B) from GLIMPSE, Impute5 and Beagle5 for Method 3. * Genotype concordance of Method 3 was done on all sites and cannot be directly compared to the other methods. Main, original one-step pipeline on main reference panel; 2-step, two-step pipeline with an extra filtering step; Anc, Ancient samples included in the reference panel; TV, transversions. FIGURE 1 | Genotype concordance and fraction of high-quality (HQ) genotypes (portrayed in decimals, total HQ genotypes: VEM185: 4,701,683, KD037: 4,531,126, KD033: 3,887,848) using different imputation tools and reference panels. Imputed from 1× downsampled coverage genomes of VEM185, KD037, and KD033. I) Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5 (C), and Beagle5 (D). II) Genotype concordance and Fraction of HQ genotypes of various MAF bins of Main + 2-step + Anc + all reference panel. Genotype concordance for Impute 5 (A), Beagle5, (B) Fractions of HQ genotypes covered by imputation in Impute5 (C), Beagle5 (D) and, Genotype concordance of only heterozygotic variants of various MAF bins imputed with Impute5 (E). III) Filters on target downsampled vcf. Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5, (C) Beagle5 (D). IV) Genotype concordance (A) and fraction of HQ genotypes (B) from GLIMPSE, Impute5 and Beagle5 for Method 3. * Genotype concordance of Method 3 was done on all sites and cannot be directly compared to the other methods. Main, original one-step pipeline on main reference panel; 2-step, two-step pipeline with an extra filtering step; Anc, Ancient samples included in the reference panel; TV, transversions. Beagle5 achieved the highest genotype concordances in KD037 and VEM185, but produced less correctly imputed variants. Impute5, on the other hand, achieved the highest genotype concordance in KD033 and produced more correctly imputed variants. Furthermore, genotype concordance between chromosomes was more uniform in Beagle5 compared to Impute5 (Supplementary Table S4). These results are based on the default one-step pipeline. The main one-step pipeline was extended to the two-step pipeline to test various settings for both tools that could influence imputation accuracy changing one element at a time. 3.1.3 Reference Panel: With and Without Ancient Samples Genotype concordance was ~5% higher in the two-step pipeline when using the reference panel including ancient samples for Impute5, whereas the inclusion of ancient samples only provided a 0.5% different genotype concordance for Beagle5 (Figures 1IA,B; Supplementary Table S4). Similarly, genotype concordance between chromosomes showed more uniformity with ancient samples than without ancient samples for Impute5, but not for Beagle5 (Supplementary Table S4). The amount of correctly imputed variants with respect to the inclusion of ancient samples had no effect for Impute5 but decreased for Beagle5 (Figures 1IC,D). this analysis were IMP1 and IMP2. Filtering for transitions pre- imputation, thus only keeping transversions, had the most favorable effect on genotype concordance. The highest genotype concordance for Beagle5 was filtering for transitions pre- and post-imputation, whereas the highest genotype concordance for Impute5 was filtering for transitions pre- imputation (Figures 1IIIA,B). The amount of correctly imputed variants of the pre- and post-imputation filters decreased drastically compared to using no filter on the target VCF for both tools (Figures 1IIIC,D). The imputed variants were further filtered for well-known positions, namely the 50 k porcine SNP-Chip (Yang et al., 2017), the transversions only, and main SNP-sets from the study of Frantz et al. (2019). These filters did not improve genotype concordance and decreased the amount of correctly imputed variants (Supplementary Figure S3 — Methods Known genomic positions). 3.1.5 Reference Panel: Pre-Imputation Filters Versus Standard Reference panels filtered for transversions and transitions showed similar genotype concordance, with transversions only having the lowest genotype concordance (Supplementary Figures 2A,B). The amount of correctly imputed variants decreased drastically for only transitions and only transversions with roughly 50%, in both tools (Supplementary Figures 2C,D). The uniformity of concordance between chromosomes was equal for all filters (Supplementary Table S4). Reference panels filtered for MAF showed variation in genotype concordance, where MAF bins <0.05 and 0.05–0.10 had the highest and MAF >0.3 the lowest genotype concordance for Impute5 (Figure 1IIA). This contrasted with Beagle5, where MAF <0.05 had the lowest genotype concordance and MAF >0.3 the highest (Figure 2B). Filtering for MAF (Beside the common variant >0.05 filter) drastically decreased the amount of correctly imputed variants in both tools (Figures 1IIC,D). Genotype concordance of heterozygotes did not show the same trend as all variant genotype concordance for Impute5 (Figure 1IIE), ~30% of the total imputed genotypes were heterozygotes (Supplementary Table S4). Genotype concordance of MAF bin <0.05 decreased drastically, while the other MAF bins decreased more modestly. 3.1.2 Pipeline: One-Step Versus Two-Step Genotype concordance in the two-step pipeline was higher compared to the one-step pipeline for both tools (Figures 1IA,B). The amount of correctlyimputedvariants increased for Beagle5 whereastheamountof correctly imputed variants for Impute5 stagnated (Figures 1IC,D). Genotype concordance between chromosomes was more uniform in July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 5 Imputation of Ancient Sus scrofa Erven et al. | Principal component analysis comparing high-quality, imputed genotypes and downsampled data together with samples from the reference panel. MP4 (B), and IMP5 (C). onent analysis comparing high-quality, imputed genotypes and downsampled data together with samples from the reference panel. P5 (C). FIGURE 2 | Principal component analysis comparing high-quality, imputed genotypes and downsampled data together with samples from the reference panel. IMP1 (A), IMP4 (B), and IMP5 (C). July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 6 Imputation of Ancient Sus scrofa Erven et al. TABLE 3 | Reference panel abbreviations. ID Reference panel IMP1 Main + 2-step + Ancients + All confident sites, Impute5 IMP2 Main + 2-step + Ancients + All confident sites, Beagle5 IMP3 Main + 2-step + Ancients + All confident sites + MAF<0.05, Impute5 IMP4 Main + 2-step + Ancients + All confident sites + MAF>0.3, Beagle5 IMP5 Main + 2-step + Ancients + All, GLIMPSE the two-step pipeline compared to the one-step pipeline (Supplementary Table S4) (variation of filter combinations used for the comparisons can be found in Supplementary Table S2). 3.1.4 Reference Panel: Variant Sites Versus All Confident Sites Category (All) Using the all confident sites category, method 2, slightly increased genotype concordance for both tools (Supplementary Figures S1A,B). The amount of correctly imputed variants was larger in the all confident sites category compared to the variant sites category, method 1, for both tools (Supplementary Figures S1C,D). The uniformity of genotype concordance between chromosomes was more equal in the all confident sites category compared to the variant sites category for Beagle5, except for KD037 (Supplementary Table S4). 3.1.7 Target VCF: All Sites (Sites Present in Reference Panel) A method that has been shown to achieve high genotype concordance in ancient human imputation (Martiniano et al., 2017; Hui, et al., 2020; Method 3) was also applied. This method was tested on three imputation tools, Beagle5, Impute5, and GLIMPSE. GLIMPSE achieved the highest genotype concordance, reaching 98% in KD037 and VEM185 and 96% in KD033 (Figure 1IVA). Genotype concordance for Beagle5 stayed constant for VEM185 and KD037 but increased for KD033 compared to the two-step pipeline. Genotype concordance for Impute5 decreased for all samples compared to the two-step pipeline. The amount of correctly imputed variants increased for all samples and all tools, reaching roughly 50% (Figure 1IVB). Furthermore, the genotype concordance between chromosomes was more constant for this method compared to the two-step pipeline (Supplementary Table S4). 3.2 Downstream Analysis Downstream analyses were performed on the imputed genotypes IMP1, 2, 3, 4, and 5 (Full descriptions found in Table 3). PCA were performed to pinpoint and compare the genetic affinities of imputed, HQ and downsampled samples. Variation captured by the first two principal components of IMP1 shows that the imputed genotypes of KD033, KD037, and VEM185 cluster closer to their HQ counterparts in the first principal component (PC1, 9.5% variation) but tend to have a bias toward European wild boar in the second principal component (PC2, 4.1% variation) (Figure 2A). This bias is greater for KD033 compared to KD037 and VEM185. PCA of IMP2 shows that the downsampled genotypes and the HQ 3.1.6 Target VCF: Filters Versus No Filters The target VCF was filtered pre- and post-imputation to deduce the effect on genotype concordance. The reference panels used for 3.1.6 Target VCF: Filters Versus No Filters The target VCF was filtered pre- and post-imputation to deduce the effect on genotype concordance. The reference panels used for 3.1.6 Target VCF: Filters Versus No Filters 3.1.6 Target VCF: Filters Versus No Filters The target VCF was filtered pre- and post-imputation to deduce the effect on genotype concordance. The reference panels used for The target VCF was filtered pre- and post-imputation to deduce the effect on genotype concordance. The reference panels used for July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 7 Imputation of Ancient Sus scrofa Erven et al. FIGURE 3 | Admixture analysis with K = 3, comparing high-quality and imputed data from IMP1-5 together with samples from the reference panel. NE, Near East; EU, European. The colors represent the three ancestral groups, where blue is European domestic pig, red is European wild boar, and yellow is Near Eastern. FIGURE 3 | Admixture analysis with K = 3, comparing high-quality and imputed data from IMP1-5 together with samples from the reference panel. NE, Near East; EU, European. The colors represent the three ancestral groups, where blue is European domestic pig, red is European wild boar, and yellow is Near Eastern. genotypes cluster closer to each other than to the imputed genotypes, the latter showing a bias toward the domestic pigs in the first principal component (PC1, 9% variation) (Supplementary Figure S4). PCA of IMP3 shows that rare alleles have a bias toward the domestic cluster in the first principal component (PC1, 9% variation) and a domestic and European wild boar bias in the second principal component (PC2, 3.8% variation) (Supplementary Figure S5). The PCA for IMP4 has the same trend as IMP2, where the downsampled genotypes and the HQ genotypes cluster closer to each other than to the imputed genotypes. However, the imputed genotypes of KD037 and VEM185 show a decreased bias toward the domestic pigs on the second principal component (PC2, 4.2% variation) (Figure 2B). The PCA for IMP5 shows a bias toward the European wild boar cluster for KD033 and VEM185 in both principal components, where the imputed genotypes of VEM185 cluster closer with the HQ genotypes than the downsampled genotypes (Figure 2C). This trend is not observed in KD033. The imputed genotypes of KD037 cluster closely toward the HQ counterpart, showing a slight bias on PC2 toward the European wild boar cluster. However, there seems to be a slight bias introduced in the HQ and downsampled genotypes, which is more evident for KD037 and VEM185. Frontiers in Genetics | www.frontiersin.org 3.1.6 Target VCF: Filters Versus No Filters They are clustering more toward the European domestic cluster than in their previous PCA (IMP1–4). Beagle5 showed a similar trend as GLIMPSE but with an elevated bias toward the downsampled genotypes (Supplementary Figure 6SA). Impute5 showed an increased amount of bias toward the European wild boar cluster (Supplementary Figure 6SB). and two ancestral groups in KD033. The ancestral groups of KD037 and VEM185 are similar, with VEM185 having a slightly larger “European domestic pig” component, whereas KD033 consists of a larger Near Eastern and smaller European component. Admixture analysis of IMP1 shows an increase in the European component, and a decrease of the other components of all imputed samples, highlighting a bias toward European samples. Admixture analysis of IMP2 shows a decrease of the European component in KD037 and VEM185 and an increase in KD033. The most noticeable difference between IMP2 and HQ is the increased component of “European domestic pigs” in all three imputed samples, again highlighting a potential bias toward “European domestic pigs.” KD033 showed most bias losing almost half of its Near Eastern component and increasement of both European wild boar and domestic pig’s components. Admixture analysis of IMP3 shows only one ancestral component, namely, the Near Eastern component (Figure 3). This potential bias toward the Near Eastern component could have arisen from a low amount of variants present in the imputed genotypes. Admixture analysis of IMP4 is similar to IMP2, with a slight decrease in Near Eastern ancestral component and an increase in European ancestral component, showing a bias toward the “European domestic pigs” component in all samples and a bias toward the European wild boar component in KD033. Admixture analysis for Method 3 shows a deviation between the two HQ, where the HQ in Method 3 shows a decrease in Near Eastern components and an increase in European wild and domestic components. Therefore, IMP5 was compared to the HQ of Method 3. Admixture analysis of IMP5 shows an increase in the European wild boar component in all samples and a decrease of “European domestic pig” and Near Eastern component in Admixture analysis of three ancestral groups (K = 3) shows the genetic ancestry of reference panel and HQ samples and indicates a presence of all three ancestral groups in KD037 and VEM185, July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 8 Imputation of Ancient Sus scrofa Erven et al. 3.1.6 Target VCF: Filters Versus No Filters TABLE 4 | ROH statistics of the IMP1 reference panel for all autosomes shown per class of 0–0.5 mb, 0.5–1 mb, and >1 mb. 0–0.5 0.5–1 >1 Count Sum kb Froh Count Sum kb Froh Count Sum kb Froh KD033_HQ 4,496 232,157 0.1025 1 516 0.0002 NA NA NA KD033_Imp1 3,287 343,670 0.1517 17 13,152 0.0058 2 2,377 0.0010 KD037_HQ 4,434 193,928 0.0856 NA NA NA NA NA NA KD037_Imp1 2,743 285,325 0.1259 12 8,626 0.0038 1 1404 0.0006 VEM185_HQ 3,071 124,173 0.0548 NA NA NA NA NA NA VEM185_Imp1 2,585 266,071 0.1174 14 9,909 0.0044 1 1600 0.0007 Each class has three statistics, ROH count, total sum of kb and Froh. TABLE 4 | ROH statistics of the IMP1 reference panel for all autosomes shown per class of 0–0.5 mb, 0.5–1 mb, and >1 mb. FIGURE 4 | Genotype concordance and fraction of high-quality (HQ) genotypes (total HQ genotypes: VEM185:4,701,683, KD037: 4,531,126, KD033, 3,887,848) of imputed samples for different levels of coverage for Impute5 and Beagle5. Imputation was performed using IMP1 and IMP2. Bars are standard errors. Genotype concordance (A), Fraction of HQ genotypes (B). FIGURE 4 | Genotype concordance and fraction of high-quality (HQ) genotypes (total HQ genotypes: VEM185:4,701,683, KD037: 4,531,126, KD033, 3,887,848) of imputed samples for different levels of coverage for Impute5 and Beagle5. Imputation was performed using IMP1 and IMP2. Bars are standard errors. Genotype FIGURE 4 | Genotype concordance and fraction of high-quality (HQ) genotypes (total HQ genotypes: VEM185:4,701,683, KD037: 4,531,126, KD033, 3,887,848) of imputed samples for different levels of coverage for Impute5 and Beagle5. Imputation was performed using IMP1 and IMP2. Bars are standard errors. Genotype concordance (A), Fraction of HQ genotypes (B). KD037 and VEM185, whereas KD033 only has a decrease in the Near Eastern component. IBD segments. IMP3 and 4 did not have enough depth to perform a proper IBD analysis. ROH analysis shows a similar but less drastic trend. The amount of ROHs was smaller in the imputed samples but they consisted of longer stretches (Table 4). The imputed samples had considerably larger ROHs, some larger than 1 MB, while the HQ samples had smaller fragmented ROHs. The elongated ROH stretches in the imputed samples attributed to a higher Froh compared to the HQ samples (Table 4). 4 DISCUSSION The analyses revealed that for imputation of Sus scrofa aDNA data: 1) genotype concordance is relatively high, similar to modern imputation, with a minor increase in information content (fraction of gained genotypes in relation to HQ) in imputed genotypes and 2) imputation performance showed inaccuracies in downstream analyses. These results have a variety of implications for our understanding of the potency of imputation of non-human ancient DNA in terms of its performance and limitations. 3.4 Effects of Reference Bias The HQ genotypes were considered a baseline of the true genotypes that overlap with the groups in the reference panel (Supplementary Figure S7). For Impute5, the correctly imputed genotypes showed a bias toward the genotype that is most common in the reference panel and occurs across all groups (Ancients, EUW, EUD, and NEW), while the incorrectly imputed genotypes showed a bias toward the EUD; EUW and EUW groups (Supplementary Figures S8,9). For Beagle5, the correctly imputed genotypes showed a bias toward ANC; EUD; EUW, EUD; EUW, EUD; EUW; NEW, and, ANC; EUD, which is similar to the bias shown in the incorrectly imputed genotypes (Supplementary Figures S10,11). The incorrectly imputed genotypes were randomly divided throughout the chromosomes (Supplementary Figure S12). When only looking at genotype concordance the imputation performance of imputation of Sus scrofa aDNA could be deemed sufficient. However, there are potential shortcomings. High genotype concordance obtained in the imputed genotypes does not result in an equal representation of genotypes from the original high coverage genome and consists of only a subset, covering roughly 5%–50%. Moreover, imputed genotypes showed greater affinity with populations that are overrepresented in the reference panel as seen in downstream analyses (e.g., PCA, Admixture). One example of the unequal representation of genotypes from the original HQ genome is apparent from genotype concordances on different MAF bins. Genotype concordance in rare alleles (MAF < 0.05) reached 97% but resulted in a bias toward main components in the reference panel in downstream analyses. This is potentially due to the representation of only ~5% of the original HQ genotypes in the imputed genotypes It is therefore essential to look beyond genotype concordance and focus on multiple aspects like fraction of HQ genotypes obtained by imputation and potential biases in downstream analysis. 3.3 Effects of Coverage on Imputation Coverage levels vary in genotype concordance, reaching 0.94 for KD037 and VEM185, and 0.75 for KD033 using Beagle5, where 2× reached the highest genotype concordance (Figure 4A). This trend is opposite for Impute5, which reached a genotype concordance of 0.92 for KD037, 0.91 for VEM185, and 0.88 for KD033, with the lowest coverage 0.5×. Imputed genotypes increased with increasing coverage (Figure 4B) All tools achieved high genotype concordance but differed in amount of information gained. Moreover, Beagle5 showed less variation in imputation of repeated downsampled VCFs compared to Impute5, showing that Beagle5 might be less affected by the randomness of downsampling. Genotype concordance increased with the two-step imputation pipeline. This was specifically designed for genomes with low coverage (Hui et al., 2020). Our results indicate that non-model species and species without an extensive reference panel could also benefit from this approach. Furthermore, genotype concordance increased when ancient samples were added to the reference panel, adding to the number and diversity of individuals. Finally, genotype concordance and number of correctly imputed genotypes increased when using all confident sites but this increased computational time and memory significantly. GLIMPSE achieved the highest genotype concordance with Method 3, that consisted of reference panel called genotypes in the three target downsampled samples. However, this method did not improve the genotype concordance for Impute5 and Beagle5, but did improve amount of genotypes recovered in all tools. 3.1.6 Target VCF: Filters Versus No Filters However, the ROHs in the imputed samples overlap with the HQ samples Identity-By-Descent (IBD) analysis shows that the imputed genotypes of IMP1 share large IBD segments with each other, covering whole chromosomes. These IBD segments are not present in their HQ or downsampled counterparts. IMP2 showed more variation with the imputed genotypes resulting in more fragmented IBD segments when compared to IMP1. However, most of the IBD segments do not overlap with the HQ July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 9 Imputation of Ancient Sus scrofa Erven et al. downsampled to a similar coverage, excluding this possibility (Supplementary Methods- Downsampling KD037 and VEM185). Moreover, KD033 showed most deviation when downsampled multiple times, showing that the ancestry components of this sample seem to be a factor in the level of accuracy in imputation. but consists of longer stretches (Supplementary Appendix ROH). The ROH analysis was only performed for IMP1, because Beagle5 had a low amount of variants. 3.3 Effects of Coverage on Imputation Coverage levels vary in genotype concordance, reaching 0.94 for KD037 and VEM185, and 0.75 for KD033 using Beagle5, where 2× reached the highest genotype concordance (Figure 4A). This trend is opposite for Impute5, which reached a genotype concordance of 0.92 for KD037, 0.91 for VEM185, and 0.88 for KD033, with the lowest coverage 0.5×. Imputed genotypes increased with increasing coverage (Figure 4B) 4.1 Imputation Performance The relatively high genotype concordance of 0.95 for Beagle5, 0.925 for Impute5, and 0.98 for GLIMPSE at 1× coverage on ancients is along the lines of genotype concordance in imputation of modern breeds (see Song et al., 2019; Ye et al., 2019; Wang et al., 2021). The higher genotype concordance in KD037 and VEM185 compared to KD033 might be explained by their difference in ancestry. Frantz et al. (2019) have shown that KD033 possessed ~54% Near Eastern ancestry, while KD037 and VEM185 possessed only ~10% Near Eastern ancestry (Frantz et al., 2019). The larger component of Near Eastern ancestry in KD033 may have caused the lower performance due to the reference panel being skewed toward European individuals. Another explanation could be the difference in coverage between the samples, KD037 and VEM185 both have coverages >20×, whereas KD033 has a coverage of ~7x. However, KD037 and VEM185 had the same genotype concordance when Downstream analyses can identify how imputed genotypes act in comparison to their HQ counterpart. The PCA of IMP1 resulted in accurate clustering of imputed and HQ genotypes with only a slight bias toward the European wild boar component. This same analysis for Beagle5 resulted in a stronger bias toward the European domestic pig component. This illustrated that a high genotype concordance does not necessary lead to accurate downstream analyses. The imputed genotypes are correct, but introduce bias in subsequent downstream analyses because they are from specific regions of the genome and are not informative enough to detect genetic variation among samples. This trend is also apparent in the admixture analysis, where imputed genotypes have biases toward European wild boar and domestic pig components. IMP3 is an exception that might be July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 10 Imputation of Ancient Sus scrofa Erven et al. European wild boar, Near Eastern wild boar and domestic pigs to the reference panel. attributed to the high amount of missing genotypes pulling it toward the Near Eastern component that featured missingness, due to low coverage and ancient individuals. IMP5 achieved the highest genotype concordance and resulted in the most accurate clustering for KD037 in the PCA, suggesting that imputation of ancient Sus is feasible. 5 CONCLUSION The use of imputation of ancient low-coverage Sus scrofa data resulted in relatively high genotype concordance and a moderate increase in information content. However, the imputed genotypes represented only a fraction, roughly 5%–50%, of all genotypes called in the HQ ancient genomes and featured biases toward the main population components in the reference panel. Our analysis indicated that these can lead to misidentifications or overrepresentation of ancestry components and selective traits in imputed genotypes. This is especially significant considering the weight archaeological debates place on ancestral relationships and admixture patterns of domesticated animals to understand the mechanisms of emergence and dispersal of early animal husbandry throughout the Neolithic across Europe and the Near East. This further highlights the measures needed to interpret the results and biases introduced by imputation and difficulty of imputation of admixed individuals. A more diverse reference panel is one of the most important priorities in ancient Sus imputation and particularly, introducing diversity present in ancient Sus could elevate accuracy and limit bias. 4.2 Factors Limiting the Power of Imputation of Sus scrofa aDNA One of the limitations is size of the reference panel (59 individuals), but more specifically diversity in the reference panel. Studies on both humans and pigs showed that a larger and more diverse reference panel increase imputation accuracy (Jostins et al., 2011; Pistis et al., 2015; Van Den Berg et al., 2019; Ausmees et al., 2021; Wang et al., 2021). Ancient human imputation studies had a minimum of ~250 individuals to perform successful imputation (Ausmees, 2019). Adding individuals to the reference panel, that do not add genetic diversity to target samples does not increase genotype concordance, as seen from the results when adding Asian samples to the reference panel. The current reference panel lacks diversity, as the main groups in the reference panel consisted of European wild boar, European domestic pigs and Near Eastern wild boar, with (Dutch) European wild boar and European domestic pig dominating. A study on ancient human imputation observed a lower genotype concordance and similarity in their PCA for hunter-gatherer genomes of which ancestry is more or less absent in the reference panel (Ausmees et al., 2021). This was also found in imputation of pig breeds where rarer pig breeds had a lower genotype concordance and dosage score than breeds that were common in the reference panel (Wang et al., 2021). In this study genotype concordance improved when adding five ancient samples with Near Eastern ancestry to the reference panel. Improving and mitigating current biases of the reference panel should aid imputation. This could be achieved by including Mesolithic wild boar, Iberian, British, Scandinavian and East 4.1 Imputation Performance However, VEM185 had a similar genotype concordance as KD037 but showed the most bias in downstream analyses for this specific method, implying that high genotype concordance does not preclude bias across samples. Another potential limitation that is associated with the reference panel is the available reference genome. The Sus scrofa 11.1 reference genome, is from a Duroc individual, with known Asian introgression. Moreover, the nature of the reference genome could potentially increase the rate of false genotyping leading to errors in haplotypes and LD structure, which could result in decreasing imputation accuracy. p p The IBD analysis shows that imputed genotypes of different samples from Impute5, share large IBD segments, sometimes even stretching chromosome wide. This could be a result of: 1) samples which lost their individual variation and became more similar due to imputation and/or 2) imputed genotypes that did not have enough depth for IBD analysis. The second explanation is unlikely, as imputed genotypes for Beagle5, did not show these large IBD segments. The ROH analysis shows that there are longer homozygous stretches throughout the genome in imputed genotypes compared to their HQ counterparts. Causes for this could be that the imputed genotypes were predominantly homozygous with little representation of heterozygotes, contributing to long ROHs and that the imputed genotypes have less markers than the HQ counterparts, resulting in an unequal density of markers. Thus, interpretation of ROHs in imputed ancient Sus should be taken with caution as it can be a result of the increase in homozygosity for Impute5. Overall, these downstream analyses highlight that there are biases and limitations toward imputation of Sus scrofa aDNA. One final potential limitation is the genetic architecture of pigs. Accuracy of imputation is dependent on LD, recombination, genetic distance, and MAF (Stephens and Scheet, 2005; Browning et al., 2018; Ye et al., 2019). These factors are different in pigs compared to humans and even other livestock species, where average heterozygosity is lower and, LD and genetic distance are significantly greater (Zhang and Plastow, 2011). The recombination rate used in this study was based on nine breeding lines, all having introgression with Asian domestic pigs. This potentially introduced inaccuracies but is mitigated as the recombination was divided into bins of 1 MB, which might not be at a size resolution to introduce inaccuracies between wild and domestic pigs. DATA AVAILABILITY STATEMENT Publicly available datasets were analyzed in this study. These data can be found here: https://www.ebi.ac.uk/ena/browser/view/ PRJEB30282?show=reads Accession number PRJEB30282. Frontiers in Genetics | www.frontiersin.org REFERENCES Ginolhac, A., Rasmussen, M., Gilbert, M. T. P., Willerslev, E., and Orlando, L. (2011). mapDamage: Testing for Damage Patterns in Ancient DNA Sequences. Bioinforma. Appl. 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JE, DB, and OM analyzed July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 11 Erven et al. Imputation of Ancient Sus scrofa ACKNOWLEDGMENTS the data. JE wrote the manuscript and JE, CÇ, OM, DB, and DR contributed to reviewing and editing the manuscript. DR and CÇ contributed to project oversight. The authors would like to thank the Smurfit Institute of Genetics and the EDAN project for their helpful advice during discussions. The authors would also like to thank the reviewers for their comments and efforts towards improving our manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2022.872486/ full#supplementary-material AncestralWeave). SUPPLEMENTARY MATERIAL This study is supported by the Dutch Research Council Open Competition (Grant No. 406.18.HW.026) and the European Research Council award to DB under the European Union’s Horizon 2020 research and innovation programme (885729- AncestralWeave). This study is supported by the Dutch Research Council Open Competition (Grant No. 406.18.HW.026) and the European Research Council award to DB under the European Union’s Horizon 2020 research and innovation programme (885729- AncestralWeave). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2022.872486/ full#supplementary-material REFERENCES Taming the Past: Ancient DNA and the Study of Animal Domestication. Annu. Rev. Anim. Biosci. 5, 329–351. doi:10.1146/annurev-animal-022516-022747 Gamba, C., Jones, E. R., Teasdale, M. D., McLaughlin, R. L., Gonzalez-Fortes, G., Mattiangeli, V., et al. (2014). 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An Improved Pig Reference Genome Sequence to Enable Pig Genetics and Genomics Research. Gigascience 9 (6), giaa051. doi:10.1093/gigascience/ giaa051 Pääbo, S., Poinar, H., Serre, D., Jaenicke-Després, V., Hebler, J., Rohland, N., et al. (2004). Genetic Analyses from Ancient DNA. Annu. Rev. Genet. 38, 645–679. doi:10.1146/annurev.genet.37.110801.143214 Yang, B., Cui, L., Perez-Enciso, M., Traspov, A., Crooijmans, R. P. M. A., Zinovieva, N., et al. (2017). Genome-wide SNP Data Unveils the Globalization of Domesticated Pigs. Genet. Sel. Evol. 49 (71). doi:10.1186/s12711-017-0345-y Parks, M., and Lambert, D. (2015). Impacts of Low Coverage Depths and Post- mortem DNA Damage on Variant Calling: a Simulation Study. BMC Genomics 16 (9). doi:10.1186/s12864-015-1219-8 Ye, S., Gao, N., Zheng, R., Chen, Z., Teng, J., Yuan, X., et al. (2019). Strategies for Obtaining and Pruning Imputed Whole-Genome Sequence Data for Genomic Prediction. Front. 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(2007). PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am. J. Hum. Genet. 81 (3), 559–575. doi:10.1086/519795 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Ramírez, O., Burgos-Paz, W., Casas, E., Ballester, M., Bianco, E., Olalde, I., et al. (2015). Frontiers in Genetics | www.frontiersin.org July 2022 | Volume 13 | Article 872486 REFERENCES Genome Data from a Sixteenth Century Pig Illuminate Modern Breed Relationships. Hered. (Edinb) 114, 175–184. doi:10.1038/hdy.2014.81 Rubinacci, S., Delaneau, O., and Marchini, J. (2020). Genotype Imputation Using the Positional Burrows Wheeler Transform. PLoS Genet. 16, e1009049. doi:10. 1371/journal.pgen.1009049 Copyright © 2022 Erven, Çakirlar, Bradley, Raemaekers and Madsen. 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. Rubinacci, S., Ribeiro, D. M., Hofmeister, R. J., and Delaneau, O. (2021). Efficient Phasing and Imputation of Low-Coverage Sequencing Data Using Large Reference Panels. Nat. Genet. 53, 120–126. doi:10.1038/s41588-020-00756-0 Sánchez-Quinto, F., Schroeder, H., Ramirez, O., Ávila-Arcos, M. C., Pybus, M., Olalde, I., et al. (2012). Genomic Affinities of Two 7,000-Year-Old Iberian Hunter-Gatherers. Curr. 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Asthma Mortality Among Persons Aged 15–64 Years, by Industry and Occupation — United States, 1999–2016
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Asthma Mortality Among Persons Aged 15–64 Years, by Industry and Occupation — United States, 1999–2016 Opal Patel, MPH1,2; Girija Syamlal, MBBS2; John Wood, MS2; Katelynn E. Dodd, MPH2; Jacek M. Mazurek, MD, PhD2 In 2015, an estimated 18.4 million U.S. adults had current asthma, and 3,396 adult asthma deaths were reported (1). An estimated 11%–21% of asthma deaths might be attributable to occupational exposures (2). To describe asthma mortality among persons aged 15–64 years,* CDC analyzed multiple cause-of-death data† for 1999–2016 and industry and occupation information collected from 26 states§ for the years 1999, 2003, 2004, and 2007– 2012. Proportionate mortality ratios (PMRs)¶ for asthma among persons aged 15–64 years were calculated. During 1999–2016, a total of 14,296 (42.9%) asthma deaths occurred among males and 19,011 (57.1%) occurred among females. Based on an estimate that 11%–21% of asthma deaths might be related to occupational exposures, during this 18-year period, 1,573–3,002 asthma deaths in males and 2,091–3,992 deaths in females might have resulted from occupational exposures. Some of these deaths might have been averted by instituting measures to prevent potential workplace exposures. The annual age-adjusted asthma death rate** per 1 mil- lion persons aged 15–64 years declined from 13.59 in 1999 to 9.34 in 2016 (p<0.001) among females, and from 9.14 (1999) to 7.78 (2016) (p<0.05) among males. The highest significantly elevated asthma PMRs for males were for those in the food, beverage, and tobacco products manufacturing industry (1.82) and for females were for those in the social assistance industry (1.35) and those in community and social services occupations (1.46). Elevated asthma mortality among workers in certain industries and occupations underscores the importance of optimal asthma management and identification and prevention of potential workplace exposures. persons aged 15–64 years. Asthma deaths were identified from death certificates using International Classification of Diseases, 10th Revision underlying cause-of-death codes J45 (asthma) and J46 (status asthmaticus). Death rates per 1 million persons aged 15–64 years by sex, race, ethnicity, and year were age-adjusted using the 2000 U.S. Census standard population. Time trends were assessed using a first-order autoregressive linear regression model to account for the serial correlation. Industry and occupa- tion information available from 26 states for the years 1999, 2003, 2004, and 2007–2012†† was coded§§ using the U.S. Census 2000 Industry and Occupation Classification System. PMRs, adjusted by 5-year age groups and race, were generated by industry and occupation for males and females. In addition, 95% confidence intervals (CIs) were calculated assuming Poisson distribution of the data. Morbidity and Mortality Weekly Report Morbidity and Mortality Weekly Report †† Information on industry and occupation was available only for decedents from 26 states for the years 1999, 2003, 2004, and 2007–2012. https://www.cdc. gov/niosh/topics/noms/default.html. §§ https://webappa.cdc.gov/ords/norms-glossary.html#ind-occ. ¶¶ For 98 residents of these 26 states, deaths occurred in states that did not provide the industry and occupation information to the National Institute for Occupational Safety and Health. Retired, unemployed, and non-paid (229 males and 687 females) and unknown or not reported (90 males and 78 females) industries, and retired, students, volunteers, homemakers and unemployed (233 males and 688 females) and unknown or not reported (78 males and 68 females) occupations were excluded from PMR analyses. * https://www.dol.gov/general/topic/youthlabor/agerequirements. † Decedents who had the International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of death (the disease or injury that initiated the chain of events that led directly and inevitably to death). https://wonder.cdc.gov/. § Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, North Carolina, North Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States represent the state where the death took place. ¶ PMR was defined as the observed number of deaths from asthma in a specified industry/occupation, divided by the expected number of deaths from asthma. The expected number of deaths was the total number of deaths in industry or occupation of interest multiplied by a proportion defined as the number of asthma deaths in all industries and/or occupations, divided by the total number of deaths in all industries/occupations. The asthma PMRs for each sex were internally adjusted by 5-year age groups and race. ** Age-adjusted death rates were calculated by applying age-specific death rates to the 2000 U.S. Census standard population age distribution. https://wonder. cdc.gov/wonder/help/mcd.html#Age-AdjustedRates. †† Information on industry and occupation was available only for decedents from 26 states for the years 1999, 2003, 2004, and 2007–2012. https://www.cdc. gov/niosh/topics/noms/default.html. §§ h b d d l h l d US Department of Health and Human Services/Centers for Disease Control and Prevention y y p g § Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, North Carolina, North Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States represent the state where the death took place. * https://www.dol.gov/general/topic/youthlabor/agerequirements. † Decedents who had the International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of death (the disease or injury that initiated the chain of events that led directly and inevitably to death). https://wonder.cdc.gov/. g §§ https://webappa.cdc.gov/ords/norms-glossary.html#ind-occ. ¶¶ d f h d h d , , , , d d, u , Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States represent the state where the death took place. ¶ PMR was defined as the observed number of deaths from asthma in a specified industry/occupation, divided by the expected number of deaths from asthma. The expected number of deaths was the total number of deaths in industry or occupation of interest multiplied by a proportion defined as the number of asthma deaths in all industries and/or occupations, divided by the total number of deaths in all industries/occupations. The asthma PMRs for each sex were internally adjusted by 5-year age groups and race. ** Age-adjusted death rates were calculated by applying age-specific death rates to the 2000 U.S. Census standard population age distribution. https://wonder. cdc.gov/wonder/help/mcd.html#Age-AdjustedRates. ¶¶ For 98 residents of these 26 states, deaths occurred in states that did not provide the industry and occupation information to the National Institute for Occupational Safety and Health. Retired, unemployed, and non-paid (229 males and 687 females) and unknown or not reported (90 males and 78 females) industries, and retired, students, volunteers, homemakers and unemployed (233 males and 688 females) and unknown or not reported (78 males and 68 females) occupations were excluded from PMR analyses. Asthma Mortality Among Persons Aged 15–64 Years, by Industry and Occupation — United States, 1999–2016 Retired, unemployed, and nonpaid workers and those with information that was unknown or not reported for industry or occupation were excluded from PMR analyses. During 1999–2016, a total of 33,307 U.S. decedents aged 15–64 years had asthma or status asthmaticus assigned as the underlying cause of death (Table 1) for an overall death rate of 8.89 per 1 million persons. The highest asthma death rates were among adults aged 55–64 years (16.32 per 1 million persons), females (9.95 per 1 million persons), persons who were not Hispanic or Latino (9.39 per 1 million), and blacks or African Americans (25.60 per 1 million persons). The age-adjusted asthma death rate per 1 million persons aged 15–64 years decreased 24.6% from 11.41 in 1999 to 8.60 in 2016 (p<0.01). The age-adjusted asthma death rates among females aged 15–64 years decreased from 13.59 per 1 million in 1999 to 9.34 in 2016 (p<0.001), and among males decreased from 9.14 (1999) to 7.78 (2016) (p<0.05). By state, annualized age-adjusted asthma death rates ranged from 4.59 per 1 million in Maine to 14.72 in the District of Columbia for males and from 6.70 per 1 million in North Dakota to 15.30 in Mississippi for females (Figure). National Vital Statistics System’s multiple cause-of-death data for 1999–2016 were analyzed to examine asthma mortality among Industry and occupation data were available for 3,393¶¶ (97.2%) of 3,491 asthma deaths, (1,398 of 1,435 [97.4%] MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention 60 Morbidity and Mortality Weekly Report TABLE 1. Number of asthma deaths* and age-adjusted asthma death rates† among persons aged 15–64 years, by sex and selected characteristics — United States, 1999–2016§ Characteristic Males Females Overall No. of deaths (% of asthma deaths) Death rate No. of deaths (% of asthma deaths) Death rate No. US Department of Health and Human Services/Centers for Disease Control and Prevention Asthma Mortality Among Persons Aged 15–64 Years, by Industry and Occupation — United States, 1999–2016 of deaths (% of asthma deaths) Death rate Overall (% of all asthma deaths) 14,296 (42.9) 7.78 19,011 (57.1) 9.95 33,307 (100.0) 8.89 Age group (yrs)¶ 15–24 1,731 (12.1) 4.42 1,035 (5.4) 2.78 2,766 (8.3) 3.62 25–34 2,272 (15.9) 6.12 1,818 (9.6) 4.97 4,090 (12.3) 5.55 35–44 2,874 (20.1) 7.55 3,692 (19.4) 9.60 6,566 (19.7) 8.58 45–54 3,853 (27.0) 10.28 6,284 (33.1) 16.22 10,137 (30.4) 13.30 55–64 3,566 (24.9) 12.39 6,182 (32.5) 19.98 9,748 (29.3) 16.32 Race** American Indian or Alaska Native 138 (1.0) 6.28 198 (1.0) 9.15 336 (1.0) 7.75 Asian or Pacific Islander 525 (3.7) 5.67 439 (2.3) 4.23 964 (2.9) 4.92 Black or African American 5,695 (39.8) 25.21 6,463 (34.0) 25.76 12,158 (36.5) 25.60 White 7,938 (55.5) 5.28 11,911 (62.7) 7.74 19,849 (59.6) 6.52 Ethnicity†† Hispanic or Latino 1,348 (9.4) 5.49 1,474 (7.8) 6.37 2,822 (8.5) 5.96 Not Hispanic or Latino 12,862 (90.0) 8.21 17,468 (91.9) 10.48 30,330 (91.1%) 9.39 Unknown 86 (0.6) N/A 69 (0.4) N/A 155 (0.5) N/A Year 1999 824 9.14 1,257 13.59 2,081 11.41 2000 878 9.60 1,150 12.24 2,028 10.95 2001 792 8.47 1,192 12.41 1,984 10.49 2002 872 9.14 1,148 11.71 2,020 10.49 2003 828 8.54 1,162 11.62 1,990 10.12 2004 770 7.82 1,044 10.21 1,814 9.06 2005 720 7.21 1,102 10.59 1,822 8.96 2006 721 7.12 1,039 9.81 1,760 8.52 2007 745 7.22 908 8.51 1,653 7.89 2008 667 6.47 931 8.54 1,598 7.52 2009 699 6.69 996 9.08 1,695 7.92 2010 747 7.04 982 8.86 1,729 7.97 2011 732 6.82 953 8.45 1,685 7.67 2012 850 7.91 988 8.71 1,838 8.31 2013 852 8.01 999 8.77 1,851 8.43 2014 875 8.19 1,089 9.63 1,964 8.94 2015 885 8.14 997 8.65 1,882 8.43 2016 839 7.78 1,074 9.34 1,913 8.60 p-value§§ 0.72 <0.05 0.004 <0.001 0.11 <0.001 Abbreviation: N/A = not available. * Decedents who had International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of death (i.e., the disease or injury that initiated the chain of morbid events leading directly to death, or the circumstances of the accident or violence that produced the fatal injury). † Age-adjusted asthma death rates per 1 million persons calculated using the 2000 U.S. Census standard population. § National Vital Statistics System. https://wonder.cdc.gov/. ¶ Age-specific asthma death rates per 1 million persons. Discussion The annual number of asthma deaths among persons aged 15–64 years has declined significantly from 1999 through 2016, most likely reflecting improvements in asthma manage- ment and effectiveness of prevention efforts (3,4). For example, replacing powdered latex gloves with powder-free natural rub- ber latex or nonlatex gloves reduced latex allergen exposure and substantially reduced work-related asthma*** among health care workers (4). Differences in asthma mortality by age, sex, and race/ethnicity have been previously reported (5). Based on an estimate that 11%–21% of asthma deaths might be attribut- able to occupational exposures (2), an estimated 3,664–6,994 asthma deaths during 1999–2016 (1,573–3,002 among males and 2,091–3,992 among females) might have been job-related, and therefore potentially preventable. population aged 15 64 years, by sex and state United States, 1999–2016¶ >10.06 8.89–10.06 8.07–8.88 6.99–8.06 5.90–6.98 <5.90 Males Females Males Females Males p y p Female workers in the health care industry and male work- ers in the construction industry accounted for the highest industry-related numbers of asthma deaths. The PMRs were significantly elevated among males in the food, beverage, and tobacco products manufacturing, other retail trade, and miscel- laneous manufacturing industries; and among females in the social assistance industry and in the community and social ser- vices occupations. A higher proportion of females with current asthma and a high frequency of exposures associated with work- related respiratory diseases have been observed in the health care and social assistance industries (6,7). National survey data indicate that approximately 9.1% (1.3 million) of 13.9 million female workers in the health care and social assistance indus- tries, and 4.2% (394,000) of 9.4 million male workers in the construction industry, have current asthma.††† Approximately 13.4% of health care and social assistance workers, 51.1% of construction workers, 31.8% of food manufacturing workers, 36.1% of beverage and tobacco product manufacturing work- ers, 40.0% of miscellaneous manufacturing workers, 21.5% of retail trade workers, and 3.7% of community and social services workers are frequently exposed to vapors, gas, dust, or fumes in the workplace (6). Workplace exposures to asthma-causing agents,§§§ such as cleaners, disinfectants, antibiotics, natural rubber latex among health care workers, and welding fumes and isocyanates (e.g., paints) among construction workers¶¶¶ have been associated with work-related asthma (8,9). Higher Females >10.06 8.89–10.06 8.07–8.88 6.99–8.06 5.90–6.98 <5.90 Females * Age-adjusted death rates were calculated by applying age-specific death rates to the 2000 U.S. Census standard population age distribution. https://wonder. cdc.gov/wonder/help/mcd.html#Age-Adjusted Rates. *** Work-related asthma includes occupational asthma (i.e., new-onset asthma caused by factors related to work) and work-exacerbated asthma (i.e., preexisting or concurrent asthma worsened by factors related to work). https://www.cdc.gov/niosh/topics/asthma/default.html. ††† https://wwwn.cdc.gov/eworld/Grouping/Asthma/97. §§§ Association of Occupational and Environmental Clinics list of occupational asthmagens. http://www.aoecdata.org/ExpCodeLookup.aspx. ¶¶¶ Occupational Safety and Health Administration. Health Hazards in Construction. https://www.osha.gov/dte/grant_materials/fy09/sh-19495-09/ health_hazards_workbook.pdf. Asthma Mortality Among Persons Aged 15–64 Years, by Industry and Occupation — United States, 1999–2016 of asthma deaths* and age-adjusted asthma death rates† among persons aged 15–64 years, by sex and selec 9 2016§ ge-adjusted asthma death rates† among persons aged 15–64 years, by sex and selected characteristics — Abbreviation: N/A not available. * Decedents who had International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of death (i.e., the disease or injury that initiated the chain of morbid events leading directly to death, or the circumstances of the accident or violence that produced the fatal injury). † Age-adjusted asthma death rates per 1 million persons calculated using the 2000 U.S. Census standard population. § N ti l Vit l St ti ti S t htt // d d / males and 1,995 of 2,056 [97.0%] females) among persons aged 15–64 years that occurred in residents of 26 states during 1999, 2003, 2004, and 2007–2012 (Table 2). By industry, the highest number of asthma deaths occurred among males in the construction industry (184; 13.2% of asthma deaths in males) and among females in the health care industry (279; 14.0% of asthma deaths in females). By occupation, the highest number of asthma deaths occurred among male construction trades workers (149; 10.7%) and among female office and adminis- trative support workers (186; 9.3%). By industry, PMRs were MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 rtment of Health and Human Services/Centers for Disease Control and Prevention 61 61 Morbidity and Mortality Weekly Report FIGURE. Annualized age-adjusted asthma death rate* per 1 million population aged 15–64 years,† by sex and state§ — United States, 1999–2016¶ FIGURE. Annualized age-adjusted asthma death rate* per 1 million population aged 15–64 years,† by sex and state§ — United States, 1999–2016¶ US Department of Health and Human Services/Centers for Disease Control and Prevention MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention **** https://www.cdc.gov/nchs/data/misc/hb_occup.pdf. Discussion Industries and occupations with ≥25 asthma* 2007–2012 Characteristic Industry Male (n = 1,079) Food, beverage, and tobacco products manufacturing Other retail trade Miscellaneous manufacturing Arts, entertainment and recreation Public administration Health care Repair and maintenance Professional, scientific, technical and management services Transportation and warehousing Accommodation and food services Educational services Construction Transportation equipment Administrative and support, and waste management services All other industries Female (n = 1,230) Social assistance Arts, entertainment and recreation Food and beverage stores Private households Health care Other retail trade Public administration Accommodation and food services Administrative and support, and waste management services Transportation and warehousing Finance and Insurance Personal and laundry services Educational services Miscellaneous manufacturing Professional, scientific, technical and management services All other industries Discussion significantly elevated among males working in food, beverage, and tobacco products manufacturing (1.82; CI = 1.22–2.61), other retail trade (1.65; CI = 1.29–2.10), and miscellaneous manufacturing (1.45; CI = 1.13–1.86); and among females working in social assistance (e.g., individual and family ser- vices and child day care services) (1.35; CI = 1.00–1.79). By occupation, the PMR was significantly elevated among female community and social services workers (1.46; CI = 1.02–2.01). MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention 62 Morbidity and Mortality Weekly Report TABLE 2. Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and 2007–2012 Characteristic No. of deaths PMR§,¶ (95% CI) TABLE 2. Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and 2007–2012 Characteristic No. of deaths PMR§,¶ (95% CI) Industry Male (n = 1,079) Food, beverage, and tobacco products manufacturing 29 1.82 (1.22–2.61)** Other retail trade 69 1.65 (1.29–2.10)** Miscellaneous manufacturing 66 1.45 (1.13–1.86)** Arts, entertainment and recreation 29 1.30 (0.88–1.87) Public administration 52 1.09 (0.83–1.45) Health care 40 1.04 (0.74–1.42) Repair and maintenance 46 1.01 (0.73–1.34) Professional, scientific, technical and management services 34 1.00 (0.69–1.39) Transportation and warehousing 89 0.98 (0.79–1.21) Accommodation and food services 66 0.96 (0.75–1.23) Educational services 29 0.95 (0.64–1.37) Construction 184 0.92 (0.79–1.07) Transportation equipment 28 0.78 (0.52–1.12) Administrative and support, and waste management services 36 0.66 (0.46–0.91) All other industries 282 — Female (n = 1,230) Social assistance 49 1.35 (1.00–1.79)** Arts, entertainment and recreation 26 1.29 (0.84–1.89) Food and beverage stores 27 1.19 (0.78–1.73) Private households 31 1.16 (0.79–1.64) Health care 279 1.12 (1.00–1.27) Other retail trade 96 1.10 (0.89–1.34) Public administration 69 1.06 (0.83–1.35) Accommodation and food services 116 1.01 (0.84–1.21) Administrative and support, and waste management services 42 0.97 (0.70–1.31) Transportation and warehousing 37 0.90 (0.63–1.24) Finance and Insurance 48 0.90 (0.66–1.19) Personal and laundry services 29 0.86 (0.58–1.24) Educational services 94 0.85 (0.69–1.04) Miscellaneous manufacturing 29 0.75 (0.50–1.07) Professional, scientific, technical and management services 35 0.66 (0.46–0.92) All other industries 223 — See table footnotes on page 64 TABLE 2. Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and 2007–2012 TABLE 2. See table footnotes on page 64. * Decedents who had the International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of death (i.e., the disease or injury that initiated the chain of morbid events leading directly to death, or the circumstances of the accident or violence that produced the fatal injury). j y) † Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, North Carolina, North Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States represent the state where the death took place. y † Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, North Carolina, North Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States represent the state where the death took place. § PMR is defined as the observed number of deaths from asthma in a specified industry/occupation, divided by the expected number of deaths from asthma. The expected number of deaths is the total number of deaths in industry or occupation of interest multiplied by a proportion defined as the number of asthma deaths in all industries or occupations, divided by the total number of deaths in all industries/occupations. The asthma PMRs were internally adjusted by 5-year age groups and race. CIs were calculated assuming Poisson distribution of the data. and race. CIs were calculated assuming Poisson distribution of the data. ¶ Retired, unemployed, and unpaid (229 males and 687 females) and unknown or not reported (90 males and 78 females) workers in industries, and retired, students, volunteers, homemakers and unemployed (233 males and 688 females) and unknown or not reported (78 males and 68 females) occupations were excluded from PMR analyses. and race. CIs were calculated assuming Poisson distribution of the data. See table footnotes on page 64. The findings in this report are subject to at least five limita- tions. First, asthma and status asthmaticus diagnoses could not be validated. It is possible that some decedents were misdiagnosed. However, given the potential impact of asthma diagnosis and status asthmaticus on patients’ lives, it seems likely that asthma would be accurately recorded on death certificates. Second, no information was available to assess whether workplace exposures triggered asthma attacks that led directly to death. Some attacks might have been triggered by exposures outside of the work environment. Third, to the extent that asthma attacks were triggered by workplace expo- sures, industry and occupation information reported on death certificates might not be the industry and occupation in which workplace exposures actually occurred because guidelines for reporting industry and occupation on death certificates**** instruct recorders to report decedent’s “usual” industry and occupation (i.e., “the type of job the individual was engaged in for most of his or her working life”). Fourth, no work history was available to assess changes in employment. Retired and PMRs in certain groups might also be explained in part by workers leaving employment in industries and occupations with workplace exposures that exacerbate their asthma and moving to jobs with fewer workplace exposures (10). Likewise, retired, unemployed, and nonpaid workers might have left the workforce because of workplace exposures. p p Differences in asthma mortality by industry and occupa- tion underscore the need for identifying workplace exposures, early diagnosis, and treatment and management of asthma cases, especially among industries and occupations with higher mortality. Pharmaceutical treatment of asthma related to occupational exposures is similar to that for asthma that is not work-related (3). Early identification and elimination of exposures is the preferred means of primary prevention to reduce asthma related to occupational exposures; however, reduction of exposure might be considered when elimination of exposures is not possible (4). Establishing an accurate diag- nosis and recommending appropriate management for workers with asthma related to occupational exposures is necessary to improve outcomes and could prevent asthma deaths (4). MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 63 Morbidity and Mortality Weekly Report TABLE 2. (Continued) Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and 2007–2012 Characteristic No. See table footnotes on page 64. ¶ Retired, unemployed, and unpaid (229 males and 687 females) and unknown or not reported (90 males and 78 females volunteers, homemakers and unemployed (233 males and 688 females) and unknown or not reported (78 males and 6 PMR analyses ales and 687 females) and unknown or not reported (90 males and 78 females) workers in industries, and retired, students, d (233 males and 688 females) and unknown or not reported (78 males and 68 females) occupations were excluded from y ** Statistically significant elevated PMR unemployed persons might have left the workforce because of severe asthma in relation to work. Finally, information on industry and occupation might not be nationally representative because only selected states provided information on industry and occupation, and only for certain years. asthma mortality among workers in certain industries and occupations underscores the importance of optimal asthma management, and identification and elimination or reduction of potential workplace exposures (3,4,9). 7  /  No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention See table footnotes on page 64. of deaths PMR§,¶ (95% CI) Occupation Male (n = 1,087) Office and administrative support occupations 62 1.25 (0.97–1.61) Other production occupations, including supervisors 51 1.21 (0.91–1.61) Sales and related occupations 89 1.17 (0.95–1.45) Laborers and material movers, hand 92 1.09 (0.88–1.34) Motor vehicle operators 74 1.07 (0.85–1.36) Metal workers and plastic workers 35 0.95 (0.66–1.33) Food preparation and serving related occupations 46 0.91 (0.66–1.21) Construction trades workers 149 0.89 (0.76–1.05) Management occupations, except agricultural 61 0.89 (0.69–1.15) Building and grounds cleaning and maintenance occupations 54 0.88 (0.67–1.16) Electrical equipment mechanics and other installation, maintenance, and repair workers 26 0.85 (0.56–1.25) Vehicle and mobile equipment mechanics, installers, and repairers 32 0.82 (0.56–1.15) All other occupations 316 — Female (n = 1,239) Community and social services occupations 36 1.46 (1.02–2.01)** Laborers and material movers, hand 47 1.19 (0.88–1.59) Healthcare support occupations 110 1.15 (0.95–1.39) Food preparation and serving related occupations 100 1.12 (0.92–1.37) Personal care and service occupations 75 1.09 (0.87–1.38) Sales and related occupations 134 1.09 (0.92–1.30) Health diagnosing and treating practitioners and technical occupations 59 1.00 (0.77–1.31) Building and grounds cleaning and maintenance occupations 62 1.00 (0.78–1.30) Management occupations, except agricultural 85 0.99 (0.80–1.24) Business operations specialists 25 0.96 (0.62–1.42) Education, training, and library occupations 70 0.93 (0.73–1.18) Health technologists and technicians 28 0.91 (0.61–1.32) Office and administrative support occupations 186 0.90 (0.77–1.04) All other occupations 222 — Abbreviations: CI = confidence interval; PMR = proportionate mortality ratio. * D d h h d h I i l Cl ifi i f Di 10 h R i i d J45 ( h ) J46 ( h i ) i d h d l i f TABLE 2. (Continued) Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and 2007–2012 TABLE 2. (Continued) Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and 2007–2012 Characteristic No. of deaths PMR§,¶ (95% CI) Building and grounds cleaning and maintenance occupation Management occupations, except agricultural Education, training, and library occupations Health technologists and technicians Office and administrative support occupations All other occupations Abbreviations: CI = confidence interval; PMR = proportionate mortality ratio. Summary What is already known about this topic? In 2015, a total of 3,396 asthma deaths were reported among adults aged ≥18 years in the United States. An estimated 11%–21% of asthma deaths might be attributable to occupa- tional exposures. Asthma deaths are preventable with proper asthma management and rapid response to asthma attacks. Corresponding author: Jacek M. Mazurek, jmazurek1@cdc.gov, 304-285-5983. Corresponding author: Jacek M. Mazurek, jmazurek1@cdc.gov, 304-285-5983. Acknowledgments Effective asthma management tools are available from CDC at https://www.cdc.gov/asthma/tools_for_control.htm, and information on the evaluation and treatment of asthma is available from the American Thoracic Society at https://www. thoracic.org/statements/allergy-asthma.php. Additional guid- ance for diagnosing work-related asthma is available from the Occupational Safety and Health Administration at https:// www.osha.gov/SLTC/occupationalasthma/. The elevated Eileen Storey, MD, Respiratory Health Division, National Institute for Occupational Safety and Health, CDC; Carol Johnson, MPH, Division of Environmental Hazards and Health Effects, National Center for Environmental Health, CDC; Vital Statistics Offices from the following 26 states providing industry and occupation information: Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention 64 Morbidity and Mortality Weekly Report 1Association of Schools and Programs of Public Health/CDC Public Health Fellowship Program; 2Respiratory Health Division, National Institute for Occupational Safety and Health, CDC. References 1. CDC. Asthma: most recent asthma data. 2017. Atlanta, GA: US Department of Health and Human Services, CDC; 2017. https://www. cdc.gov/asthma/most_recent_data.htm MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 No conflicts of interest were reported. US Department of Health and Human Services/Centers for Disease Control and Prevention What is added by this report? 2. Steenland K, Burnett C, Lalich N, Ward E, Hurrell J. Dying for work: the magnitude of US mortality from selected causes of death associated with occupation. Am J Ind Med 2003;43:461–82. https://doi.org/10.1002/ ajim.10216 Among U.S. adults aged 15–64 years, 33,307 deaths from asthma occurred during 1999–2016, including an estimated 3,664–6,994 (approximately 204–389 annually) that could be attributable to occupational exposures and were therefore potentially preventable. The highest asthma death rates were among adults aged 55–64 years, females, persons who were not Hispanic or Latino, and blacks or African Americans. By industry, asthma mortality was significantly elevated among males in food, beverage, and tobacco products manufacturing, other retail trade, and miscellaneous manufacturing, and among females in social assistance. By occupation, asthma mortality was significantly elevated among females in community and social services. 3. National Asthma Education and Prevention Program. Expert panel report 3 (EPR-3): guidelines for the diagnosis and management of asthma—summary report 2007. J Allergy Clin Immunol 2007;120(Suppl):S94–138. https://doi.org/10.1016/j.jaci.2007.09.029 4. Heederik D, Henneberger PK, Redlich CA; ERS Task Force on the Management of Work-related Asthma. Primary prevention: exposure reduction, skin exposure and respiratory protection. Eur Respir Rev 2012;21:112–24. https://doi.org/10.1183/09059180.00005111 g 5. Moorman JE, Akinbami LJ, Bailey CM, et al. National surveillance of asthma: United States, 2001–2010. Vital Health Stat 3 2012;35:1–58. 6. Calvert GM, Luckhaupt SE, Sussell A, Dahlhamer JM, Ward BW. The prevalence of selected potentially hazardous workplace exposures in the US: findings from the 2010 National Health Interview Survey. Am J Ind Med 2013;56:635–46. https://doi.org/10.1002/ajim.22089 What are the implications for public health practice? Elevated asthma mortality among male and female workers in certain industries and occupations highlights the importance of optimal asthma management, and identification and preven- tion of workplace exposures. 7. White GE, Seaman C, Filios MS, et al. Gender differences in work-related asthma: surveillance data from California, Massachusetts, Michigan, and New Jersey, 1993–2008. J Asthma 2014;51:691–702. https://doi.org/1 0.3109/02770903.2014.903968 8. Baur X, Bakehe P, Vellguth H. Bronchial asthma and COPD due to irritants in the workplace - an evidence-based approach. J Occup Med Toxicol 2012;7:19. https://doi.org/10.1186/1745-6673-7-19 New Hampshire, New Jersey, New Mexico, North Carolina, North Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. 9. Tarlo SM, Balmes J, Balkissoon R, et al. Diagnosis and management of work-related asthma: American College Of Chest Physicians consensus statement. Chest 2008;134(Suppl):1S–41S. https://doi.org/10.1378/ chest.08-0201 Conflict of Interest Conflict of Interest No conflicts of interest were reported. 10. Le Moual N, Kauffmann F, Eisen EA, Kennedy SM. The healthy worker effect in asthma: work may cause asthma, but asthma may also influence work. Am J Respir Crit Care Med 2008;177:4–10. https:// doi.org/10.1164/rccm.200703-415PP MMWR  /  January 19, 2018  /  Vol. 67  /  No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention 65
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https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0007560&type=printable
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Potent and selective inhibitors for M32 metallocarboxypeptidases identified from high-throughput screening of anti-kinetoplastid chemical boxes
PLoS neglected tropical diseases
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10,896
Editor: Laura-Isobel McCall, University of Oklahoma, UNITED STATES Editor: Laura-Isobel McCall, University of Oklahoma, UNITED STATES Received: March 6, 2019 Accepted: June 18, 2019 Published: July 22, 2019 Editor: Laura-Isobel McCall, University of Oklahoma, UNITED STATES Received: March 6, 2019 Accepted: June 18, 2019 Published: July 22, 2019 Copyright: © 2019 Salas-Sarduy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was supported by PICT 2014- 3510 from the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (ANPCyT) to GTN (https:// www.argentina.gob.ar/ciencia/agencia), PIP 11220130100303CO from Argentinian National Research Council to GTN (https://www.conicet. gov.ar/), and PICTO-2013-0067 from ANPCyT to RESEARCH ARTICLE Emir Salas-Sarduy1, Lionel Ura´n LandaburuID1, Adriana K. CarmonaID2, Juan Jose´ Cazzulo1, Ferna´n Agu¨ero1, Vanina E. Alvarez1, Gabriela T. NiemirowiczID1* Emir Salas-Sarduy1, Lionel Ura´n LandaburuID1, Adriana K. CarmonaID2, Juan Jose´ Cazzulo1, Ferna´n Agu¨ero1, Vanina E. Alvarez1, Gabriela T. NiemirowiczID1* 1 Instituto de Investigaciones Biotecnolo´gicas “Dr. Rodolfo Ugalde”–Universidad Nacional de San Martı´n– CONICET, San Martı´n, B1650HMP, Buenos Aires, Argentina, 2 Departamento de Biofı´sica, Universidade Federal de São Paulo, São Paulo, Brazil a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * gniemiro@iibintech.com.ar * gniemiro@iibintech.com.ar Abstract Enzymes of the M32 family are Zn-dependent metallocarboxypeptidases (MCPs) widely distributed among prokaryotic organisms and just a few eukaryotes including Trypanosoma brucei and Trypanosoma cruzi, the causative agents of sleeping sickness and Chagas dis- ease, respectively. These enzymes are absent in humans and several functions have been proposed for trypanosomatid M32 MCPs. However, no synthetic inhibitors have been reported so far for these enzymes. Here, we present the identification of a set of inhibitors for TcMCP-1 and TbMCP-1 (two trypanosomatid M32 enzymes sharing 71% protein sequence identity) from the GlaxoSmithKline HAT and CHAGAS chemical boxes; two col- lections grouping 404 compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. For this purpose, we adapted continuous fluorescent enzy- matic assays to a medium-throughput format and carried out the screening of both collec- tions, followed by the construction of dose-response curves for the most promising hits. As a result, 30 micromolar-range inhibitors were discovered for one or both enzymes. The best hit, TCMDC-143620, showed sub-micromolar affinity for TcMCP-1, inhibited TbMCP-1 in the low micromolar range and was inactive against angiotensin I-converting enzyme (ACE), a potential mammalian off-target structurally related to M32 MCPs. This is the first inhibitor reported for this family of MCPs and considering its potency and specificity, TCMDC- 143620 seems to be a promissory starting point to develop more specific and potent chemi- cal tools targeting M32 MCPs from trypanosomatid parasites. OPEN ACCESS Citation: Salas-Sarduy E, Landaburu LU, Carmona AK, Cazzulo JJ, Agu¨ero F, Alvarez VE, et al. (2019) Potent and selective inhibitors for M32 metallocarboxypeptidases identified from high- throughput screening of anti-kinetoplastid chemical boxes. PLoS Negl Trop Dis 13(7): e0007560. https://doi.org/10.1371/journal.pntd.0007560 Potent and selective inhibitors for M32 metallocarboxypeptidases identified from high-throughput screening of anti- kinetoplastid chemical boxes Emir Salas-Sarduy1, Lionel Ura´n LandaburuID1, Adriana K. CarmonaID2, Juan Jose´ Cazzulo1, Ferna´n Agu¨ero1, Vanina E. Alvarez1, Gabriela T. NiemirowiczID1* Introduction Members of the Trypanosomatidae family comprise parasitic organisms that cause highly dis- abling and often fatal diseases in humans and animals. The species that are responsible for human infections are Trypanosoma brucei, which cause Human African trypanosomiasis (HAT), Trypanosoma cruzi, the etiological agent of Chagas disease (American trypanosomia- sis), and Leishmania spp., which cause different forms of leishmaniasis. Together, these vector- borne diseases constitute a substantial public health problem for which there is not a satisfac- tory treatment [1]. Major side-effects, and in some cases low effectiveness, are common prob- lems associated with existing therapy. This situation makes imperative the development of new chemotherapeutic options. In this context, new drugs based on unique aspects of parasite biology and biochemistry are of great interest, particularly in the case of emerging resistance to traditional treatments [2–4]. In this scenario, proteases have become popular targets as these enzymes play key functions in parasite biology; namely nutrition, cell cycle progression, invasion and pathogenesis, among others. The M32 family of metallocarboxypeptidases (MCPs) contains a group of hydrolases, which although being broadly distributed among prokaryotic organisms, are only present in a few eukaryotes including some green algae and trypanosomatids [5]. This unique phylogenetic distribution, in particular the absence of M32 enzymes in metazoans, has been considered an attractive trait due to the high specificity/selectivity potential of this family for drug target development. Within the Trypanosomatidae family several conserved M32 MCPs have been characterized [5–10]. Nonetheless, the cellular or biological functions of these proteins are cur- rently unknown, as well as their essentiality status. In T. brucei, the genome-wide study by Als- ford et al. (2011) reported no significant lost-of-fitness after induction of T. brucei MCP-1 (TbMCP-1) RNAi in bloodstream and procyclic stages, as well as in the differentiation from procyclic to bloodstream forms [11]. More recently, however, it has been shown that TbMCP- 1 null mutant strains display extended doubling times in culture, suggesting that this enzyme might contribute to the adaptive fitness of the bloodstream form [12]. On the basis of their bio- chemical properties and stage-specific expression, the L. major M32 carboxypeptidase has been implicated in the catabolism of peptides and proteins to single amino acids required for protein synthesis [7]. The restricted substrate preference of T. Author summary In recent years, the pharmaceutical company GlaxoSmithKline announced the disclosure of small collections of antiparasitic compounds to facilitate research and drug develop- ment for three of the main Tropical Neglected Diseases- i.e. Human African 1 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases FA and VEA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Trypanosomiasis, Leishmaniasis and Chagas Disease. These collections include new chemical entities with potential novel mechanisms of action that are likely to be active against a wide variety of targets. Taking advantage of these open access molecules, we suc- cessfully set up medium-throughput screening assays to find the first inhibitors of two metallocarboxypeptidases of the M32 family, a group of proteolytic enzymes proposed to play several roles in the biology of trypanosomatids including peptide catabolism, mainte- nance of parasite adaptive fitness and hydrolysis of bioactive peptides from the human host. Competing interests: The authors have declared that no competing interests exist. Development of continuous metallocarboxypeptidase assays To evaluate compounds in the HAT and CHAGAS chemical boxes, we devised a continuous assay for each MCP, based on FRET (fluorescence resonance energy transfer) peptides. We carried out the optimization process in 384 well plates, the same format used for the screening of the compound collections. For the selection of the most suitable substrate for the HTS assay, we initially assayed six FRET peptides against both enzymes. These were recently designed considering subsite preferences (P1´-P4) of TcMCP-1 and TbMCP-1 [12]. However, because no peptide was completely satisfactory for both enzymes, we selected independent substrates, Abz-LKFK(Dnp)-OH and Abz-RFFK(Dnp)-OH, for TcMCP-1 and TbMCP-1 assays, respec- tively. After substrate selection, a convenient enzyme concentration in the assay was deter- mined through the activity of 2-fold dilutions of TcMCP-1 and TbMCP-1 at a fixed substrate concentration (Fig 1A and 1B). Moreover, the Selwyn test [17] revealed no enzyme inactiva- tion under the conditions tested (Fig 1C and 1D). Thus, for a wide range of enzyme concentra- tions (for both MCPs), the V0 vs. [E]0 curves showed a linear behavior (Fig 1E and 1F). In particular, for [TcMCP-1]0 < 0,34 nM and [TbMCP-1]0 < 1,53 nM, the rate of the substrate hydrolysis remained constant for at least 40 minutes, a suitable time to perform the screening (Fig 1A and 1B). The best balance between TcMCP-1 activity on Abz-LKFK(Dnp)-OH substrate (estimated as dF/dt) and the time over which the reaction displayed linear kinetics was achieved at [TcMCP-1]0 = 0,17 nM. Under these conditions, the enzyme showed the typical hyperbolic behavior predicted by the Michaelis-Menten equation (Hill coefficient = 1,06) and an esti- mated KM value of 2,23 ± 0,28 μM (Fig A in S1 Text). Similarly, when the TbMCP-1 concentra- tion was fixed at 1,25 nM we obtained a KM value on Abz-RFFK(Dnp)-OH substrate of 0,37 ± 0,06 μM (Hill coefficient = 1,03) (Fig A in S1 Text). To afford the best opportunity to find compounds with different inhibition modalities, we decided to employ balanced assay conditions (i.e. KM/[S] = 1)[18]. Using these conditions, preliminary characterization experi- ments of both optimized assays showed good general performance, with a dynamic range (μC +—μC-) higher than 15 RFU/sec, a μC+/μC- ratio  50, good reproducibility (VC < 5%) and a Z ´ factor value in the range 0,6–0,8. Synthetic inhibitors for M32 metallocarboxypeptidases activity would be of great value to ask mechanistic and phenotypic questions in both biochemi- cal and cell-based studies. However, no inhibitors have been reported to date for these enzymes or other members of this family. Recently, a diverse collection of ~ 1.8 million compounds from the proprietary library of GlaxoSmithKline (GSK) has been run through whole-cell phenotypic screens against L. dono- vani, T. cruzi and T. brucei. As a result, three anti-kinetoplastid chemical boxes of ~200 com- pounds each were assembled and open sourced [16]. The guiding design criteria for these molecule sets were chosen to include structures from different chemical families that are likely to be active against a wide variety of targets. By taking advantage of this diversity, we identified the first inhibitors of the M32 family of MCPs within the GSK HAT and CHAGAS chemical boxes. As model enzymes of the M32 family we employed TcMCP-1 and TbMCP-1, which have similar basic amino acid preference at the P1´ position and share 71% of protein sequence identity [5, 6]. Introduction cruzi MCP-1 (TcMCP-1), plus its strong structural similarity to angiotensin I-converting enzyme (ACE), neurolysin and thi- met oligopeptidase [8], have also pointed out a possible regulatory role of this family in the metabolism of small peptides. In fact, it has been shown that TcMCP-1 can produce des- Arg9-bradykinin [6], a peptide that promotes the process of cell invasion through B1 receptors by the T. cruzi trypomastigotes [13]. In this sense, two reports have suggested that M32 pepti- dases are secreted by trypanosomatids [14, 15], a fact that is in agreement with this hypothesis. In the current scenario, the availability of selective small-molecule modulators of M32 MCPs PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 2 / 20 Primary screening of HAT and CHAGAS chemical boxes Using the same lot of substrate and enzyme, the 404 compounds present in the HAT and CHAGAS chemical boxes were screened at a single fixed dose (25 μM). Each plate included 24 3 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Fig 1. Continuous fluorogenic assays for recombinant MCPs. (A) Kinetic progression curves for different TcMCP-1 concentrations at a fixed Abz-LKFK(Dnp)-OH dose (1,25 μM). (B) The activity of 2-fold dilutions of TbMCP-1 was analyzed with Abz-RFFK(Dnp)-OH as substrate (4,8 μM). For both enzymes, working dilutions were selected from those that showed linear kinetics for more than 40 minutes. (C, D) Selwyn test for different TcMCP-1 and TbMCP-1 concentrations. In both cases, the global fitting of experimental data from different enzyme dilutions to a unique curve was good, indicating that enzymes remained stable during the whole assay. To facilitate observation, curves were slightly displaced from each other in the Y axis. (E, F) Curve of V0 vs. [E]0 for TcMCP-1 and TbMCP-1 respectively. In both cases, the expected linear behavior was observed. https://doi.org/10.1371/journal.pntd.0007560.g001 Synthetic inhibitors for M32 metallocarboxypeptidases Fig 1. Continuous fluorogenic assays for recombinant MCPs. (A) Kinetic progression curves for different TcMCP-1 concentrations at a fixed Abz-LKFK(Dnp)-OH dose (1,25 μM). (B) The activity of 2-fold dilutions of TbMCP-1 was analyzed with Abz-RFFK(Dnp)-OH as substrate (4,8 μM). For both enzymes, working dilutions were selected from those that showed linear kinetics for more than 40 minutes. (C, D) Selwyn test for different TcMCP-1 and TbMCP-1 concentrations. In both cases, the global fitting of experimental data from different enzyme dilutions to a unique curve was good, indicating that enzymes remained stable during the whole assay. To facilitate observation, curves were slightly displaced from each other in the Y axis. (E, F) Curve of V0 vs. [E]0 for TcMCP-1 and TbMCP-1 respectively. In both cases, the expected linear behavior was observed. https://doi.org/10.1371/journal.pntd.0007560.g001 positive and negative controls, plus 16 wells containing 31,25 mM EDTA (inhibition control) alternately located in columns 11, 12, 23 and 24. In general, for each MCP, both plates pre- sented highly similar Z´ scores although best values were obtained for the TbMCP-1 assay pre- sumably due to the lower background signal of the Abz-RFFK(Dnp)-OH substrate. To avoid positive and negative controls, plus 16 wells containing 31,25 mM EDTA (inhibition control) alternately located in columns 11, 12, 23 and 24. Synthetic inhibitors for M32 metallocarboxypeptidases Table 1. Statistics for the plates during primary screening. TcMCP-1 TbMCP-1 Plate 1 Plate 2 Plate 1 Plate 2 Compounds (n) 320 84 320 84 Mean SD Mean SD Mean SD Mean SD Enzyme control (C+) (RFU/sec) 19,12 1,60 20,19 2,14 16,80 0,57 21,23 0,57 Substrate control (C-) (RFU/sec) 2,98 0,19 3,63 0,24 0,01 0,20 -0,12 0,38 EDTA control (RFU/sec) 1,28 0,24 2,32 0,34 0,13 0,20 0,39 0,11 Z´ factor 0,67 0,57 0,86 0,87 TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2 containing 0,01% Triton X-100 (C+). Final substrate concentration was set to a value KM /[S] ~ 1. Additionally, 24 negative or substrate controls (no enzyme added, C-) plus 16 inhibition controls (EDTA final concentration 31,25 mM) were included in each plate. Z factor was calculated as in [19]. Table 1. Statistics for the plates during primary screening. TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2 containing 0,01% Triton X-100 (C+). Final substrate concentration was set to a value KM /[S] ~ 1. Additionally, 24 negative or substrate controls (no enzyme added, C-) plus 16 inhibition controls (EDTA final concentration 31,25 mM) were included in each plate. Z factor was calculated as in [19]. TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2 containing 0,01% Triton X-100 (C+). Final substrate concentration was set to a value KM /[S] ~ 1. Additionally, 24 negative or substrate controls (no enzyme added, C-) plus 16 inhibition controls (EDTA final concentration 31,25 mM) were included in each plate. Z factor was calculated as in [19]. https://doi.org/10.1371/journal.pntd.0007560.t001 the interference of highly fluorescent compounds, an auto-fluorescence cut-off value equal to 2x105 RFU was used to accept or discard a molecule from the subsequent analysis. Using this limit, ~19% of the compounds were eliminated for TcMCP-1 and TbMCP-1 assays. Statistics are summarized in Table 1. As shown in Table 2, if we consider a cut-off value  3 standard deviations from the control mean (μc+ - 3σc+), 70 and 132 inhibitory molecules were retrieved for TcMCP-1 and TbMCP- 1, respectively. To reduce the number of resultant hits, we explored other two thresholds focus- ing only in outliers: i) those compounds showing slopes >3σ standard deviations above the average of all slopes in the plate (control independent) and ii) those compounds showing an inhibition percentage >3σ standard deviations above the average for the plate (control depen- dent). Interestingly, both criteria retrieved exactly the same list of compounds for TcMCP-1 (n = 5) while for TbMCP-1 the intersection between this two groups was lower (2 out of 4 compounds). Primary screening of HAT and CHAGAS chemical boxes In general, for each MCP, both plates pre- sented highly similar Z´ scores although best values were obtained for the TbMCP-1 assay pre- sumably due to the lower background signal of the Abz-RFFK(Dnp)-OH substrate. To avoid positive and negative controls, plus 16 wells containing 31,25 mM EDTA (inhibition control) alternately located in columns 11, 12, 23 and 24. In general, for each MCP, both plates pre- sented highly similar Z´ scores although best values were obtained for the TbMCP-1 assay pre- sumably due to the lower background signal of the Abz-RFFK(Dnp)-OH substrate. To avoid 4 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 https://doi.org/10.1371/journal.pntd.0007560.t002 () Different hit selection criteria were applied to both HAT and CHAGAS boxes. 1) Compounds showing an inhibition  three standard deviation from control mean. 2) Compounds showing slopes >3σ standard deviations above the average of all slopes in the plate and 3) those compounds showing a percent inhibition >3σ standard deviations above the average for the plate. https://doi.org/10.1371/journal.pntd.0007560.t001 () Highly fluorescent compounds were discarded from the analysis. Finally, another 7 molecules performed better in the secondary screening than in the first round. Despite the observed round to round discrepancies (Table A in S1 Text), we decided to continue curve analysis for all the compounds, with the exception of the three that showed no inhibition at 31,5 μM dur- ing secondary screening. recombinant MCPs using identical assay conditions as in the primary screening. Prior to the analysis of the complete dataset, we examined whether there was a correlation between the inhibition percentages in the primary (compound concentration 25 μM) and secondary screening, using only the data corresponding to a compound concentration of 31,5 μM. This was important to assess consistency of data, as both screening rounds were performed without technical replicates due to limitation of compound stocks. For TcMCP-1, 9 compounds pre- sented similar behavior in both screenings (correlation coefficient r2 = 0,9868; slope = 1,146) (Fig 2A) whereas 7 molecules failed to reach  40% of inhibition threshold (n = 6) or displayed no inhibition (n = 1) (correlation coefficient r2 = -0,518; slope = 0,2595). Additionally, 7 com- pounds performed better in the secondary screening (correlation coefficient r2 = 0,5156; slope = 1,2749). For the T. brucei enzyme, consistent results in both assays were achieved only by 8 compounds (correlation coefficient r2 = 0,9349; slope = 1,080) (Fig 2B). About 45% of the samples did not repeat the  40% of inhibition criterium (n = 10) or did not inhibit (n = 2) TbMCP-1 (correlation coefficient r2 = 0,1163; slope = 0,3173). Finally, another 7 molecules performed better in the secondary screening than in the first round. Despite the observed round to round discrepancies (Table A in S1 Text), we decided to continue curve analysis for all the compounds, with the exception of the three that showed no inhibition at 31,5 μM dur- ing secondary screening. For TcMCP-1, five compounds (TCMDC-143620, TCMDC-143422, TCMDC-143456, TCMDC-143209 and TCMDC-143385) showed an IC50 value  10 μM (Fig 3A and Table 3). In good agreement, the four more potent molecules (TCMDC-143620, TCMDC-143422, TCMDC-143456 and TCMDC-143209) also inhibited the T. brucei enzyme (Table 3). Com- pounds TCMDC-143385 and TCMDC-143172 (which display an IC50 ~10 μM for TcMCP-1) did not reach the 40% inhibition threshold in the TbMCP-1 primary screening and were left out from the secondary analysis. Other potent molecules, namely TCMDC-143409 and TCMDC-143323 were specific inhibitors of T. Synthetic inhibitors for M32 metallocarboxypeptidases Fig 2. Correlation between the inhibition percentages in the primary and secondary screenings for the most reproducible compounds. As both screening rounds were performed without technical replicates (see Material and Methods), we introduced this analysis to assess data consistency. The analysis was performed using the 25μM and 31,5μM data points from the primary and secondary screenings, respectively. The main panel shows correlation of the most reproducible hits, whereas the insets show the correlation analysis for all the hit compounds at indicated concentrations. (A) TcMCP-1 (B) TbMCP-1. https://doi org/10 1371/journal pntd 0007560 g002 Fig 2. Correlation between the inhibition percentages in the primary and secondary screenings for the most reproducible compounds. As both screening rounds were performed without technical replicates (see Material and Methods), we introduced this analysis to assess data consistency. The analysis was performed using the 25μM and 31,5μM data points from the primary and secondary screenings, respectively. The main panel shows correlation of the most reproducible hits, whereas the insets show the correlation analysis for all the hit compounds at indicated concentrations. (A) TcMCP-1 (B) TbMCP-1. h //d i /10 13 1/j l d 000 60 002 https://doi.org/10.1371/journal.pntd.0007560.g002 recombinant MCPs using identical assay conditions as in the primary screening. Prior to the analysis of the complete dataset, we examined whether there was a correlation between the inhibition percentages in the primary (compound concentration 25 μM) and secondary screening, using only the data corresponding to a compound concentration of 31,5 μM. This was important to assess consistency of data, as both screening rounds were performed without technical replicates due to limitation of compound stocks. For TcMCP-1, 9 compounds pre- sented similar behavior in both screenings (correlation coefficient r2 = 0,9868; slope = 1,146) (Fig 2A) whereas 7 molecules failed to reach  40% of inhibition threshold (n = 6) or displayed no inhibition (n = 1) (correlation coefficient r2 = -0,518; slope = 0,2595). Additionally, 7 com- pounds performed better in the secondary screening (correlation coefficient r2 = 0,5156; slope = 1,2749). For the T. brucei enzyme, consistent results in both assays were achieved only by 8 compounds (correlation coefficient r2 = 0,9349; slope = 1,080) (Fig 2B). About 45% of the samples did not repeat the  40% of inhibition criterium (n = 10) or did not inhibit (n = 2) TbMCP-1 (correlation coefficient r2 = 0,1163; slope = 0,3173). Secondary screening In the secondary screening we decided to include all compounds that showed  40% of inhibi- tion (TcMCP-1: 23 compounds; TbMCP-1: 27 compounds). To estimate IC50 for the resulting hits, two-fold serial dilutions, ranging from 7,5 pM to 62,5 μM, were analyzed against both Table 2. Primary screening results. TcMCP-1 TbMCP-1 Plate 1 Plate 2 Plate 1 Plate 2 Compounds (n) 320 84 320 84 Analyzed compounds (n) 256 72 254 71 1 51 19 92 40 2 4 1 2 2 3 4 1 0 2 40% inhibition 19 4 22 5 () Highly fluorescent compounds were discarded from the analysis. () Different hit selection criteria were applied to both HAT and CHAGAS boxes. 1) Compounds showing an inhibition  three standard deviation from control mean. 2) Compounds showing slopes >3σ standard deviations above the average of all slopes in the plate and 3) those compounds showing a percent inhibition >3σ standard deviations above the average for the plate. https://doi.org/10.1371/journal.pntd.0007560.t002 Table 2. Primary screening results. TcMCP-1 TbMCP-1 Plate 1 Plate 2 Plate 1 Plate 2 Compounds (n) 320 84 320 84 Analyzed compounds (n) 256 72 254 71 1 51 19 92 40 2 4 1 2 2 3 4 1 0 2 40% inhibition 19 4 22 5 () Highly fluorescent compounds were discarded from the analysis. () Highly fluorescent compounds were discarded from the analysis. () Highly fluorescent compounds were discarded from the analysis. () Different hit selection criteria were applied to both HAT and CHAGAS boxes. 1) Compounds showing an inhibition  three standard deviation from control mean. 2) Compounds showing slopes >3σ standard deviations above the average of all slopes in the plate and 3) those compounds showing a percent inhibition >3σ standard deviations above the average for the plate. 5 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases indicated in Material and Methods. For each compound, solid line represents the best fit of four-parameter Hill equation to experimental data (closed circles). (A) Dose-response curves corresponding to the most potent TcMCP-1 inhibitors. (B) Equivalent analysis for the top-five TbMCP-1 inhibitors. (C) Structure and identifiers corresponding to the most potent hit compounds identified for both enzymes. Subgroup (a) contains those molecules that inhibited both MCPs. Subgroup (b) is formed by TCMDC-133485, which selectively acts on TcMCP-1 whereas subgroup (c) includes TbMCP-1 specific inhibitors. https://doi.org/10.1371/journal.pntd.0007560.g003 https://doi.org/10.1371/journal.pntd.0007560.g003 TcMCP-1 (< 30%) (Fig 3B and Table 3). The structure of the top-five inhibitors for each enzyme is shown in Fig 3C. brucei enzyme or produced little inhibition on 6 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the inhibitory activity, enzymes were incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition percentages determined for each condition as Synthetic inhibitors for M32 metallocarboxypeptidases Fig 3. Dose-response curves and structures of top-five inhibitors identified for eac incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selec Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the inhibitory activity, enzymes were incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition percentages determined for each condition as Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the inhibitory activity, enzymes were incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition percentages determined for each condition as Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the ncubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition perce PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 7 / 20 Compounds that presented similar behavior in both primary and secondary screening (<15% variation between both assays https://doi.org/10.1371/journal.pntd.0007560.t003 https://doi.org/10.1371/journal.pntd.0007560.t003 ted similar behavior in both primary and secondary screening (<15% variation between both assays) are highlighted in grey. Lead compounds have low structural redundancy Red squares for TbMCP-1, blue squares for TcMCP-1, grey squares for non-active in TbMCP-1/TcMCP-1, accordingly. https://doi.org/10.1371/journal.pntd.0007560.g004 physicochemical properties (Fig 4). Whereas the Tanimoto clustering was expected to be inconclusive based on the premises used to assemble the chemical boxes (one or two putative chemotypes per box [16]); the clustering based on physicochemical properties also showed no significant correlation between these properties and the observed IC50s. Similarly, MCS clus- tering provided no insights into candidate substructures guiding the activity or specificity of the compounds against each enzyme. In all three strategies, the clusters not only group up dis- similar potencies, but also mix compounds with different enzyme specificity. Lead compounds have low structural redundancy To first assess the possibility that these lead compounds have shared structural features that help explain their bioactivity profile, we performed three different clustering strategies: one using Tanimoto similarity (Fig B in S1 Text), one based on shared substructures (overlap of Maximum Common Subgraphs, MCS) (Fig C in S1 Text), and the third one based on shared Table 3. IC50 values and Hill slopes for identified hits. Table 3. IC50 values and Hill slopes for identified hits. Compound Chemical Box TcMCP-1 TbMCP-1 IC50 (μM) Hill Slope R square IC50 (μM) Hill Slope R square TCMDC-143620 CHAGAS 0,6939 -1,06 0,9821 4,989 -1,461 0,9674 TCMDC-143422 CHAGAS 1,52 -1,038 0,8644 10,46 -1,206 0,8407 TCMDC-143456 HAT 2,206 -0,9597 0,9609 14,74 -1,247 0,9121 TCMDC-143209 CHAGAS 4,182 -1,019 0,8086 28,34 -1,2405 0,9851 TCMDC-143385 CHAGAS 9,473 -0,9614 0,838 - TCMDC-143172 HAT 11,21 -2,825 0,9751 - TCMDC-143513 HAT 13,52 -0,9734 0,8486 28,33 -0,8371 0,9523 TCMDC-143551 HAT 13,84 -1,834 0,4295 34,51 -1,562 0,5659 TCMDC-143462 HAT 15,48 -0,9436 0,8296 - TCMDC-143382 HAT 20,59 -0,7277 0,861 22,55 -0,7258 0,9649 TCMDC-143515 HAT 21,83 -1,149 0,7376 31,71 -0,8447 0,9624 TCMDC-143432 CHAGAS 26,26 -0,6129 0,9382 - TCMDC-143242 HAT 26,5 -1,237 0,851 - TCMDC-143592 CHAGAS 27,91 -0,8919 0,8208 - TCMDC-143408 CHAGAS 32,86 -1,153 0,9473 - TCMDC-143496 HAT 34,9 -0,822 0,6521 - TCMDC-143071 CHAGAS 38,91 -1,235 0,8373 - TCMDC-143263 HAT 40,74 -0,9946 0,8174 - TCMDC-143543 HAT >60 TCMDC-143323 HAT - 3,938 -0,894 0,9353 TCMDC-143409 CHAGAS - 10,42 -1,2409 0,9815 TCMDC-143191 CHAGAS - 16,11 -1,267 0,977 TCMDC-143645 HAT - 20,79 -1,127 0,9238 TCMDC-143143 CHAGAS - 21,78 -1,466 0,9195 TCMDC-143332 CHAGAS - 23,27 -1,063 0,9614 TCMDC-143158 HAT - 27,64 -1,014 0,8757 TCMDC-143254 HAT - 43,43 -0,8163 0,768 TCMDC-143265 HAT - 44,2 -2,036 0,7989 TCMDC-143454 HAT - >60 TCMDC-143187 CHAGAS - >60 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 8 / 20 Synthetic inhibitors for M32 metallocarboxypeptidases Fig 4. A dendrogram representing compound clustering using molecular features, and summarizing the activity distribution among tested MCPs. Squares next to the names give an idea of IC50 observed: the brighter the color, the lower the IC50. Red squares for TbMCP-1, blue squares for TcMCP-1, grey squares for non-active in TbMCP-1/TcMCP-1, accordingly. https://doi org/10 1371/journal pntd 0007560 g004 Fig 4. A dendrogram representing compound clustering using molecular features, and summarizing the activity distribution among tested MCPs. Squares next to the names give an idea of IC50 observed: the brighter the color, the lower the IC50. Synthetic inhibitors for M32 metallocarboxypeptidases compounds), pyrazole (3 compounds), diol (1 compound) and hydrazide (1 compound). The majority of compounds (24 out of 30) presented at least one ZBG in the structure. More specif- ically, 15 with a single group and 9 with two groups were found. All compounds and their cor- responding ZBGs have been summarized in Fig D in S1 Text. MCP inhibitors are specific Considering the abundance of ZBGs and heteroatom-containing moieties in the hits, we evalu- ated the possibility of a nonspecific mechanism of inhibition (involving metal chelation) for the top-five inhibitors identified in the screening for each enzyme. Because M32 MCPs show a strong topological similarity with ACE [22], we chose this enzyme to estimate the IC50 value for each molecule. As done for the MCPs essays, ACE activity was analyzed employing a FRET substrate, Abz-FRK(Dnp)P-OH, at a concentration equal to the apparent KM of the enzyme ~3 μM [23]. Experiment set up is summarized in Figs E and F in S1 Text. For comparative pur- poses, captopril, a potent competitive ACE inhibitor, was included in the analysis (IC50 ~1 nM) (Fig 5A). Under these conditions, no inhibition could be detected for any of the com- pounds evaluated, thus suggesting that these molecules are not promiscuous metallocarboxy- peptidase inhibitors (Fig 5B, 5C and 5D) but are instead specific inhibitors of M32 MCPs. Most compounds have at least one Zinc-biding group To determine the number and type of Zinc-binding groups (ZBGs) among the compound leads, an MCS analysis was performed using an ad hoc curated [20, 21] database of ZBGs. From a total of 48 groups available in the database, only six of them were found among 24 of the 30 lead compounds: pyridine (14 compounds), sulfonamide (7 compounds), imidazole (4 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 9 / 20 Discussion M32 MCPs have an unusual phylogenetic distribution (with trypanosomatids being among the few eukaryotic genomes encoding these enzymes). Hence M32 MCPs from parasites arose naturally as interesting candidates for drug target development. Furthermore, the current lack of knowledge about the cellular and/or physiological role(s) of these enzymes makes the identi- fication of potent inhibitors a task of great significance, as these compounds may be used as molecular probes to potentially identify natural substrates, to recognize the specific pathways in which they are involved or, hopefully, to perform their chemical validation as drug targets. In this work, we describe the first drug-like inhibitors of TcMCP-1 and TbMCP-1, two closely related MCPs from the human pathogens T. cruzi and T. brucei, respectively. Our starting point were the GSK HAT and CHAGAS boxes, two small collections containing non-redun- dant, chemically diverse and highly bioactive compounds [16], which could facilitate future optimization efforts. Although we initially aimed for a common assay for both MCPs, we soon realized that the use of different FRET substrates for each enzyme resulted in better general performance of the individual assays (considering signal robustness, temporal duration of linear kinetics, dynamic range, μC+/μC- ratio and Z´ factor). Surprisingly, the substrates that resulted most suitable for the developed HTS assays were not, in any case, those that showed the best values of kcat, KM and kcat/KM in their previous kinetic characterization [12]. Although different assays were used to screen these collections, we were able to find specific inhibitors for both enzymes, and perhaps more important, mutual inhibitors; suggesting the consistency of inter-assay results. Of note, specific inhibitors for each enzyme were distributed evenly among HAT and CHA- GAS boxes with no apparent bias. This fact confirms the importance of not circumscribing the search to just the pathogen-specific box, but instead to widen the search to all the boxes avail- able, as previously observed for T. cruzi cysteine peptidase cruzipain [24]. Due to the limited amount of compound stocks, we decided to implement the screening of chemical boxes in singlet, with primary evaluation of all compounds at a fixed dose and further dose-response analysis of unconfirmed hits in a secondary screening. Synthetic inhibitors for M32 metallocarboxypeptidases Fig 5. Dose-response curves for ACE. (A) Purified rabbit lung ACE was assayed at 37˚C in 0,1 M Tris-HCl pH 7,0 buffer containing 50 mM NaCl, 10 mM ZnCl2 and 0,01% Triton X-100. Captopril, a potent competitive ACE inhibitor, was included as a positive inhibition control (IC50~ 1 nM). NI, no inhibitor added. (B, C) Data corresponding to TcMCP-1 and TbMCP-1 specific inhibitors, respectively. (D) Dose-response curves corresponding to those molecules that inhibited both MCPs. In all cases, solid lines represent the best fit of four-parameter Hill equation to experimental data (closed circles). Yellow, dark-green and orange colors were used for ACE, TcMCP-1 and TbMCP-1, respectively. https://doi.org/10.1371/journal.pntd.0007560.g005 comparison to secondary dose-response evaluation. These discrepancies are common and may be due to a variety of factors [25]. Besides intrinsic compound-specific and experimental data variability [26], these factors may include solubility issues (given that in primary and sec- ondary screenings both the final concentration and serial-dilution protocol were different), differential stability of compounds in stock (10 mM) and working (2 mM) solutions [27], unintended absorption of the compounds to different containing materials during storage, moderate dose-dependent quenching effects of compounds on fluorescence readouts, among others [28]. In addition, although we included 0,01% Triton X-100 in assay buffer, compound- specific aggregate formation was not tested and thus, cannot be dismissed. As mentioned, we identified in this work eight molecules able to inhibit both MCPs. These mutual inhibitors came from both boxes in similar numbers, as previously noted for enzyme- specific compounds. Interestingly, in all cases they were more potent inhibitors of TcMCP-1, for reasons that are as yet unclear. Importantly, four of these compounds proved to be inactive on ACE, a Zinc-dipeptidyl carboxypeptidase involved in various physiological and physiopath- ological conditions in mammals [29] which shows significant structural similarity to M32 enzymes [22, 30]. This fact strongly suggests that despite the structural resemblance and the small number of compounds tested here, the identification of inhibitors with high selectivity for trypanosomatid M32 MCPs over ACE can be achieved, a point in favor to the specific druggability of these enzymes. The identified inhibitors display high structural diversity, with many showing only mar- ginal similarity to the other hits, hence representing different structural clusters and presum- ably, different inhibitory scaffolds. Discussion As expected, given the error-prone nature of the single-well (single dose, single replicate) measurements used in pri- mary screening, significant discrepancies in inhibition were observed for some compounds in 10 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Synthetic inhibitors for M32 metallocarboxypeptidases glected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 11 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 11 / 20 https://doi.org/10.1371/journal.pntd.0007560.g005 Synthetic inhibitors for M32 metallocarboxypeptidases presumptive explanation can be put forward. As in the case of many other metallopeptidase inhibitors, it is likely that inhibition of trypanosomatid M32 MCPs occurs throughout the per- turbation of the coordination sphere of the catalytic metal ion (presumably Zn2+ in the case of TcMCP-1 and TbMCP-1, by extension from other M32 enzymes [31]). Typically, synthetic metallopeptidase inhibitors achieve preliminary affinity and target selectivity through the for- mation of stabilizing interactions with specific residues within the active site; while a ZBG is responsible for metal chelation, enhancing binding affinity, modulating selectivity and dis- rupting catalytic activity [32]. For the majority of the inhibitors presented here, it was possible to identify typical ZBG or at least, heteroatom-containing groups able to establish a coordi- native bond with a Zn2+ ion (Fig D in S1 Text). For those compounds, an inhibition mecha- nism like the one described above is possible. For other molecules not having a Zn- coordinating group, the most plausible explanation is that inhibition occurs as a result of the prevention of substrate binding by the partial occupancy or the deformation of the catalytic cleft by the inhibitor molecule, as previously observed for Non-Zinc-Binding inhibitors of other metallopeptidases [33]. The vast majority of the hits identified here inhibit one or both MCPs in the micromolar range, with only a few of them showing potencies <10 μM. Outstandingly, TCMDC-143620 inhibits TcMCP-1 in the sub-micromolar range (it also inhibits TbMCP-1, but with potency ~7-fold lower). This is the most potent inhibitor described so far for an enzyme of the M32 family and seems a promising candidate for further structure-based optimization. The unusu- ally high flexibility of the M32 MCPs around the active site [31, 34] prevented us to use a dock- ing approach to get insights of the binding mode of this compound within TcMCP-1 and TbMCP-1 catalytic clefts. However, the TCMDC-143620 molecule seems able to form a variety of stabilizing interactions. These may include hydrophobic and electrostatic interactions, hydrogen bonding and the coordination to the metal ion through the pyridine ring. In addi- tion, the presence of a central sulfonamide group and a distal nitrile group add further interac- tion possibilities to this molecule. For example, the sulfonamide group has been extensively incorporated into metallopeptidase inhibitors due to its ability to improve the enzyme-inhibi- tor binding by different mechanisms. In this regard, the presence of “unpaired” hits is not surprising, considering that no more than two members of the same structural cluster were included per box during collection assembly [16] and that “twin” compounds might well not pass the activity or auto-fluorescence filters included in this work. Among the identified inhib- itors, only TCMDC-143265 and TCMDC-143551 share similar core structures, thus probably populating the same cluster and sharing a common active scaffold. A significant part of both molecules is identical and adopts the same spatial conformation (Fig G in S1 Text), with the largest differences located around the benzamide ring. Besides the obvious differences in the length and position of sulfonamide substituents, the chlorine substitution in position 2 imposes a ~90˚ rotation of the benzamide ring in TCMDC-143265 compared to TCMDC- 143551, where all ring systems are almost coplanar. Interestingly, these structural differences seem to dictate the selectivity toward TcMCP-1, as TCMDC-143551 inhibits both enzymes whereas TCMDC-143265 is specific for TbMCP-1. Even for this pair of compounds, there is no evident substructure responsible for M32 MCPs bioactivity; though this is probably a biased observation due to the lack of well-defined structural features for M32 MCPs inhibitors. Although the crystallographic structure of TcMCP-1 has been determined [8] and subsite specificity have been explored for both enzymes using FRET substrate libraries [12] and muta- genesis [6, 8], little is yet known about how substrates are accommodated into the catalytic groove, which residues are key determinants of subsite specificity and the significance of the hinge-type movement between L and R domains in the stabilization of enzyme-substrate or enzyme-inhibitor complexes. With all these gaps to fill, it seems risky to speculate about the modes of interaction of these new inhibitors with TcMCP-1 and TbMCP-1. However, a 12 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Enzymes TcMCP-1 (MEROPS ID: M32.003) and TbMCP-1 were expressed as GST fusion proteins in E. coli BL21 (DE3) Codon Plus and purified as previously described [6, 8]. Reagents Triton X-100, MOPS (3-(N-morpholino)propanesulfonic acid), DMSO, EDTA and captopril were purchased from Sigma-Aldrich. Substrates Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)- OH were from GenScript (Piscataway, NJ, USA). Black solid bottom polystyrene Corning NBS 384-well plates were from Sigma-Aldrich (CLS3654-100EA). Anti-kinetoplastid chemical boxes The HAT and CHAGAS chemical boxes [16] were provided by GlaxoSmithKline. The collec- tion comprised 404 compounds, prepared as 10 mM stock solutions in DMSO (10 μL each) and dispensed in 96 well plates. For primary screening, a working solution (final concentration of 2 mM) for each compound was prepared by 1/5 dilution in DMSO while 1 μL of the 10 mM stock solution was used for secondary screening of selected compounds, as previously described [24]. The final concentration of compounds tested in primary screening was 25 μM, while the com- pound concentrations assayed in secondary screening ranged from 7,5 pM to 62,5 μM. Synthetic inhibitors for M32 metallocarboxypeptidases its reported anti-T. cruzi activity. This might also explain, at least partially, the moderate cyto- toxic and anti-T. brucei and L. donovani activities reported for this compound, considering the global similarities of enzymes within CYP51 family [40, 41]. Although involved in other stud- ies as part of the GSK CHAGAS Box [42], no further information is currently available from the evaluation of TCMDC-143620 against other molecular targets, except for our previous cru- zipain study [24] where it was found to be inactive (~7,5% of cruzipain inhibition at 25 μM). A complete profile of the off-target activity of TCMDC-143620 would be critical for future opti- mization efforts in order to achieve a suitable M32 MCPs probe from this compound. In summary, 30 micromolar-range inhibitors, presenting both high structural diversity and novelty, have been discovered for TcMCP-1 and/or TbMCP-1 by using continuous, fluores- cent-based and HTS-capable enzymatic assays. The best hit shows sub-micromolar affinity for TcMCP-1, inhibits TbMCP-1 in the low micromolar range and, like other potent hits, is inac- tive on ACE. Considering its potency and specificity, this molecule seems to be a promissory starting point to develop more specific and potent tools to expand our understanding of the biochemistry and biological role(s) of M32 MCPs from trypanosomatid parasites and, hope- fully, to assess in a near future their value as drug targets. These mechanisms include: i) direct formation of hydro- gen bonds to the enzyme backbone, ii) properly redirection of bulky groups into enzyme pockets by inducing a twist in the structure of the inhibitor molecule and iii) even cooperate with other chelating groups in the coordination of the catalytic metal ion [35]. Similarly, the nitrile group in TCMDC-143620 can establish polar interactions, hydrogen bonds or react with serine or cysteine side chains to form covalent adducts which would greatly stabilize inhibitor binding [36]. Interestingly, the nitrile group is also able to form coordinative bonds with a variety of metal ions including Co2+, Mn2+, Fe3+, Cu2+ and Zn2+ [37]. Thus, a possible role of this group in the direct coordination of the catalytic metal ion cannot be discarded at present. The determination of the crystallographic structure of TcMCP-1 or TbMCP-1 in com- plex with TCMDC-143620 would provide a definitive answer to these questions as well as important clues to undertake the future lead-optimization of this hit. A preliminary analysis of the bioactivity profile of TCMDC-143620 (https://pubchem.ncbi. nlm.nih.gov/compound/91800813) indicates that it shows potent activity against T. cruzi in culture and only moderate but measurable activity on T. brucei and L. donovani. Also, this compound exhibits moderate cytotoxicity on mammalian cell NIH 3T3 (IC50 = 13 μM) but resulted inactive on HepG2 (IC50 > 100 μM). Considering target-specific assays; this com- pound has a single bioactivity report. TCMDC-143620 was found to be a potent inhibitor (IC50 = 79 nM) of T. cruzi sterol 14-α demethylase (CYP51) enzyme, which is involved in the ergosterol biosynthesis pathway and was considered until recent years as a promissory thera- peutic target for Chagas disease [38, 39]. The inhibition of this target is probably the cause of 13 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases (iii) display linear kinetics for enough time to perform several reading cycles (at least 8 cycles, minimum time between cycles: 264 sec) through the 384-wells. In all cases, EDTA (final con- centration 31,25 mM) was used as positive inhibition control. Primary screening To perform the primary screening, 1 μL of each compound (2 mM in DMSO, final concentra- tion in the assay: 25 μM), EDTA (500 mM, final concentration in the assay: 31,25 mM) were dispensed into 384-well Corning black solid-bottom assay plates. Then, 40 μL of 100 mM MOPS, 0,01% Triton X-100 pH 7,2 containing TbMCP-1 (2,50 nM) or TcMCP-1 (0,34 nM) were added to each well, plates were homogenized (30 seg, orbital, medium intensity) and each well subjected to a single autofluorescence read (ex/em = 320/420 nm). Plates were incu- bated in darkness for 15 min at 30 ˚C and then 40 μL of Abz-RFFK(Dnp)-OH (4 μM) or Abz- LKFK(Dnp)-OH (0,8 μM) in assay buffer were added to each well to start the reaction. After homogenization (30 seg, orbital, medium intensity), the fluorescence of the Abz group (ortho- aminobenzoic acid) (ex/em = 320/420 nm) was acquired kinetically for each well (8 read cycles, one cycle every 300 seconds). Considering our previous experiences, the auto-fluores- cent cut-off was arbitrarily set at 2x105 RFU to discard highly interfering compounds. All com- pounds were assayed in singlet (without replicates) due to the limited availability of stocks. Raw screening measurements were used to determine the slope (dF/dt) of progression curves by linear regression for control and non-interfering compound wells. In the case of control-dependent hit selection criteria, percent inhibition percentage (%Inh) was calculated for each compound according to the following equation: Inh ¼ 100  1 dF dt WELL mC  ðmCþ mCÞ 2 4 3 5 ð1Þ ð1Þ where dF/dtWELL represents the slope of each compound well and μC+ and μC−the average of MCP (no-inhibition) and substrate (no-enzyme) controls, respectively. where dF/dtWELL represents the slope of each compound well and μC+ and μC−the average of MCP (no-inhibition) and substrate (no-enzyme) controls, respectively. MCPs assays TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2 containing 0,01% Triton X-100. Assays were performed in solid black 384-well plates (final reaction volume ~80 μL) and the hydrolysis of the K(Dnp)-OH group was monitored continuously at 30 ˚C with a Beckman Coulter DTX 880 Multimode Reader (Radnor, Pennsylvania, USA) using standard 320 nm excitation and 420 nm emission filter set. For each MCP, final substrate concentration was set to a value KM /[S] ~ 1. Optimal enzyme concentration was selected from 2-fold serial dilutions to match three criteria: (i) being linearly proportional to V0, (ii) display robust signal evolution at substrate concentration chosen and PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 14 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Table 4. Molecular features used to build compound clustering dendrogram. NAME MW HBD HBA RB RINGS XLOGP TCMDC-143071 416,3 1 3 6 4 3,18 TCMDC-143143 242,2 1 3 2 2 4,36 TCMDC-143158 443,3 1 3 6 6 1,46 TCMDC-143172 299,1 1 2 3 3 4,6 TCMDC-143187 463,8 3 4 7 5 3,67 TCMDC-143191 354,3 0 3 3 5 3,04 TCMDC-143209 378,2 2 4 8 3 3,44 TCMDC-143242 374,3 1 2 6 5 2,78 TCMDC-143254 415,3 3 3 7 4 3,99 TCMDC-143263 302,2 2 3 6 3 2,71 TCMDC-143265 407,8 1 5 6 3 2,53 TCMDC-143323 386,3 3 5 8 4 -0,04 TCMDC-143332 430,3 1 4 6 4 2,37 TCMDC-143382 378,3 3 4 5 4 2,08 TCMDC-143385 443,3 1 4 7 4 2,68 TCMDC-143408 340,3 1 4 5 4 1,81 TCMDC-143409 375,1 0 3 3 4 2,5 TCMDC-143422 279,2 0 1 2 4 3,31 TCMDC-143432 350,3 0 1 6 3 3,74 TCMDC-143454 259,6 2 2 3 3 3,38 TCMDC-143456 318,2 1 4 6 3 1,58 TCMDC-143462 434,3 2 5 7 4 3,11 TCMDC-143496 464,4 1 4 8 4 2,9 TCMDC-143513 461,8 2 4 8 5 2,4 TCMDC-143515 459,8 2 2 7 5 4,55 TCMDC-143543 460,4 0 2 6 6 4,23 TCMDC-143551 436,4 1 5 8 4 2,3 TCMDC-143592 351,7 1 1 6 3 3,9 TCMDC-143620 396,3 0 4 5 4 2,65 TCMDC-143645 363,3 3 1 7 3 4,32 MW: Molecular Weight; HBD: Hydrogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Number of Rings; XLOGP: XlogP (Partition Table 4. Molecular features used to build compound clustering dendrogram. MW: Molecular Weight; HBD: Hydrogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Number of Rings; XLOGP: XlogP (Partition coefficient score) ogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Number of Rings; XLOGP: XlogP (Partition MW: Molecular Weight; HBD: Hydrogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Numb coefficient score). where dF/dtWELL represents the slope of each compound well and μC+ and μC−the average of MCP (no-inhibition) and substrate (no-enzyme) controls, respectively. The IC50 and Hill slope parameters for each compound were estimated by fitting the four-parameter Hill equa- tion to experimental data from dose-response curves using the GraphPad Prism program (ver- sion 5.03). Secondary assay Compounds selected from primary screening were re-tested in a dose-response manner (final concentration ranging from 7,5 to 62,5 μM) using identical assay conditions. To avoid any positional and/or association bias, we randomly defined the row position for each compound. One μL of compounds stock (10 mM in DMSO) and EDTA (31,25 mM) were added to the first well of column 1, followed by addition of 40 μL of 100 mM MOPS, 0,01% Triton X-100 pH 7,2 buffer. After addition of 20 μL of the same buffer to subsequent wells of the plate, 22 serial 2-fold dilutions were made horizontally. The last two positions of every row were used, alternatively, for C+ and C−controls to reduce any positional and/or association bias. Then, 20 μL of activity buffer containing TbMCP-1 or TcMCP-1 were added to each well, except for those corresponding to C−; completed with 20 μL of activity buffer. After homogenization, 15 minutes of incubation at 30˚C and autofluorescence measurement, the substrate (in activity buffer) was added to the previous mix. Data collection and processing were performed exactly as described above. Percentage of M32 MCPs residual activity was calculated for each condi- tion according to the following equation: %Res:ActMCP ¼ 100  dF dt WELL mC  ðmCþ mCÞ 2 4 3 5 ð2Þ ð2Þ 15 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 https://doi.org/10.1371/journal.pntd.0007560.t004 Supporting information S1 Text. Supplementary information. (PDF) Compound clustering Three separate compound clustering routines were used. One of them derived from calculated or predicted molecular features, and the other two directly inferred from different distance metrics between compounds: one using Tanimoto similarity and another one using the over- lap score calculated in a MCS (Maximum Common Subgraph) pipeline. The Tanimoto dis- tance compound clustering was performed to rapidly find compound pairs, if available, within the leads. OpenBabel 2.4.1 [43] was used to export molecule MDLs from SMILES format, available from GSK chembox summary. For Tanimoto clustering, the indexes were calculated using ChemFP 1.3 [44] with ob2fps bindings and simsearch -NxN as parameter. ChemFP results were parsed and analyzed using an ad hoc perl script, setting the distance (D) between compounds as D = 1—Tindex. The dis- tance matrix was built using melt and acast from R Data table package [45]. To assess the MCS clustering, all compounds were imported into a R script using Chemmi- ner [46] and further analyzed using fmcsR [47] for batch MCS calculations. For the molecular feature clustering, a perl script was built to run XlogP3 v3.2.2 [48] through all lead compounds. Features used to build distance matrix, along with their corre- sponding values, can be found in Table 4. All clustering plots were achieved using the R base hierarchical clustering tool, hclust. Synthetic inhibitors for M32 metallocarboxypeptidases Acknowledgments ESS, GTN, JJC, VEA and FA are members of the research career of the National Research Council (CONICET, Argentina), and LUL has a doctoral fellowship from CONICET. Zinc-binding group assessment among lead compounds To find ZBGs among lead compounds, a curated database of such chemotypes was first created (Table B in S1 Text). Structures were drawn using Marvin Sketcher (Chemaxon) and exported to SMILES format. This database was then imported to R and processed similarly to the MCS clustering, though instead of calculating overlapping scores between compounds, the overlap- ping score was determined for each compound against all ZBGs in the database. Only those compound-ZBG pairs where overlap was complete (score = 1 and, hence, ZBG completely contained in the lead compound) were counted as a match. ACE assay Purified rabbit lung ACE (EC 3.4.15.1) was purchased from Sigma-Aldrich. Enzyme activity was assayed fluorimetrically with Abz-FRK(Dnp)P-OH (ex/em = 320/420 nm) as substrate in buffer 0,1 M Tris-HCl, 50 mM NaCl, 10 mM ZnCl2, pH 7.0 containing 0,01% Triton X-100 as indicated in [23]. Selected compounds were tested in a dose-response manner (final concen- tration ranging from 7,5 pM to 62,5 μM) using identical assay conditions employed with both MCPs. Captopril (15 pM—125 μM) was used as inhibition control. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560 July 22, 2019 16 / 20 References 1. Field MC, Horn D, Fairlamb AH, Ferguson MA, Gray DW, Read KD, et al. Anti-trypanosomatid drug dis- covery: an ongoing challenge and a continuing need. Nat Rev Microbiol. 2017; 15(4):217–31. Epub 2017/02/28. https://doi.org/10.1038/nrmicro.2016.193 [pii]. PMID: 28239154; PubMed Central PMCID: PMC5582623. 2. Fairlamb AH, Gow NA, Matthews KR, Waters AP. Drug resistance in eukaryotic microorganisms. Nature microbiology. 2016; 1(7):16092. Epub 2016/08/31. https://doi.org/10.1038/nmicrobiol.2016.92 PMID: 27572976; PubMed Central PMCID: PMC5215055. 3. Ponte-Sucre A, Gamarro F, Dujardin JC, Barrett MP, Lopez-Velez R, Garcia-Hernandez R, et al. Drug resistance and treatment failure in leishmaniasis: A 21st century challenge. PLoS neglected tropical dis- eases. 2017; 11(12):e0006052. Epub 2017/12/15. https://doi.org/10.1371/journal.pntd.0006052 PMID: 29240765; PubMed Central PMCID: PMC5730103. 4. Fairlamb AH, Horn D. Melarsoprol Resistance in African Trypanosomiasis. Trends in parasitology. 2018; 34(6):481–92. Epub 2018/05/01. https://doi.org/10.1016/j.pt.2018.04.002 PMID: 29705579. 5. Niemirowicz G, Parussini F, Aguero F, Cazzulo JJ. Two metallocarboxypeptidases from the protozoan Trypanosoma cruzi belong to the M32 family, found so far only in prokaryotes. Biochem J. 2007; 401 (2):399–410. Epub 2006/09/30. https://doi.org/10.1042/BJ20060973 [pii] PMID: 17007610; PubMed Central PMCID: PMC1820797. 6. Frasch AP, Carmona AK, Juliano L, Cazzulo JJ, Niemirowicz GT. Characterization of the M32 metallo- carboxypeptidase of Trypanosoma brucei: differences and similarities with its orthologue in Trypano- soma cruzi. Mol Biochem Parasitol. 2012; 184(2):63–70. Epub 2012/05/12. https://doi.org/10.1016/j. molbiopara.2012.04.008 [pii]. PMID: 22575602; PubMed Central PMCID: PMC3383389. 7. Isaza CE, Zhong X, Rosas LE, White JD, Chen RP, Liang GF, et al. A proposed role for Leishmania major carboxypeptidase in peptide catabolism. Biochem Biophys Res Commun. 2008; 373(1):25–9. Epub 2008/06/10. https://doi.org/10.1016/j.bbrc.2008.05.162 [pii]. PMID: 18539138; PubMed Central PMCID: PMC2561202. 8. Niemirowicz G, Fernandez D, Sola M, Cazzulo JJ, Aviles FX, Gomis-Ruth FX. The molecular analysis of Trypanosoma cruzi metallocarboxypeptidase 1 provides insight into fold and substrate specificity. Mol Microbiol. 2008; 70(4):853–66. Epub 2008/09/17. https://doi.org/10.1111/j.1365-2958.2008.06444. x [pii]. PMID: 18793339. 9. Isaza CE. Carboxypeptidase Taq A2. 2013. In: Handbook of Proteolytic Enzymes [Internet]. Academic Press.; [1249–53.]. 10. Niemirowicz GT, Frasch AP, Cazzulo JJ. Carboxypeptidase Taq-Like Peptidases from Trypanosoma- tids A2. In: Rawlings ND, Salvesen G., editor. Handbook of Proteolytic Enzymes: Academic Press.; 2013. p. 1253–7. 11. Alsford S, Turner DJ, Obado SO, Sanchez-Flores A, Glover L, Berriman M, et al. High-throughput phe- notyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome research. 2011; 21(6):915–24. Epub 2011/03/03. https://doi.org/10.1101/gr.115089.110 PMID: 21363968; PubMed Central PMCID: PMC3106324. 12. Frasch AP, Bouvier LA, Oppenheimer FM, Juliano MA, Juliano L, Carmona AK, et al. Synthetic inhibitors for M32 metallocarboxypeptidases Resources: Adriana K. Carmona, Juan Jose´ Cazzulo. Software: Lionel Ura´n Landaburu. Supervision: Emir Salas-Sarduy, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez. Validation: Emir Salas-Sarduy, Gabriela T. Niemirowicz. Visualization: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. Writing – original draft: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Writing – review & editing: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Adriana K. Car- mona, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Supervision: Emir Salas-Sarduy, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez. Validation: Emir Salas-Sarduy, Gabriela T. Niemirowicz. Visualization: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. Writing – original draft: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Writing – original draft: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Writing – review & editing: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Adriana K. Car- mona, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Author Contributions Conceptualization: Emir Salas-Sarduy, Lionel Ura´n Landaburu. Data curation: Emir Salas-Sarduy, Lionel Ura´n Landaburu. Formal analysis: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. Formal analysis: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. unding acquisition: Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Funding acquisition: Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Investigation: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Investigation: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina E. 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PyRhO: a virtual optogenetics laboratory
BMC neuroscience
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Acknowledgements Thi k g This work was supported by the UK BBSRC grant BB/L018268/1. From a minimal set of photocurrent data, PyRhO will fit and parameterize the Three [1], Four [1] and Six-state [2] rhodopsin models to capture the underlying biophysi- cal photocycle which defines their kinetics. These models are then used to accurately compute the photocurrents across a range of flux, voltage and other experimental conditions for the given rhodopsin. After selecting a sui- table model based on the desired balance between simu- lation accuracy and speed, the artificial rhodopsin can be seamlessly inserted into software such as NEURON and Brian for use in simulations from the cellular to the net- work level. We demonstrate the use of PyRhO in fitting models to channelrhodopsin-2 (ChR2) [3] data and pre- sent results for typical illumination strategies and experi- mental protocols designed to tease apart the effects of key model parameters. Published: 18 December 2015 Published: 18 December 2015 Evans et al. BMC Neuroscience 2015, 16(Suppl 1):P178 http://www.biomedcentral.com/1471-2202/16/S1/P178 Open Access PyRhO: a virtual optogenetics laboratory Benjamin D Evans1*, Sarah Jarvis2, Simon R Schultz2, Konstantin Nikolic1 From 24th Annual Computational Neuroscience Meeting: CNS*2015 Prague, Czech Republic. 18-23 July 2015 PyRhO: a virtual optogenetics laboratory Benjamin D Evans1*, Sarah Jarvis2, Simon R Schultz2, Konstantin Nikolic1 From 24th Annual Computational Neuroscience Meeting: CNS*2015 Prague, Czech Republic. 18-23 July 2015 open-source modules and software. An accompanying GUI running in IPython [4] has also been developed to facilitate more interactive exploration of the models for both experimental and didactic purposes. Furthermore, IPython has been identified as a particularly promising medium for sharing models and reproducing results in computational neuroscience [5]. Simulations based on these virtual opsins will enable neuroscientists to gain insight into their behaviour and rapidly identify the most suitable variant for application in a particular biological system, not only guiding choice, but potentially also rho- dopsin development. In this way, we expect PyRhO will help to significantly improve the effectiveness of optoge- netics as a tool for transforming biological sciences. Optogenetics has become a key tool for understanding the function of neural circuits and controlling their beha- viour. An array of opsins have been genetically isolated from several families of organism, including algae and bacteria, with a wide range of temporal and spectral properties. In an effort to develop more effective and tai- lored opsins, hybrids and genetic mutants are continually being created. Experimentally characterizing these new variants is a lengthy process requiring substantial effort before they can be harnessed to address questions in neu- roscience. Experimentally testing each combination of opsin and target cell type of interest is practically impos- sible, effectively limiting the use of optogenetics as a tool. To aid in this effort we propose PyRhO; an integrated suite of open-source, multi-scale computational tools to characterize rhodopsins, then rapidly develop and con- duct virtual experiments with them in silico. © 2015 Evans et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: benjamin.evans@imperial.ac.uk 1Institute of Biomedical Engineering, Department of Electrical & Electronic Engineering, Imperial College London, London, UK Full list of author information is available at the end of the article Authors’ details 1I i f Bi 1Institute of Biomedical Engineering, Department of Electrical & Electronic Engineering, Imperial College London, London, UK. 2Department of Bioengineering, Electrical Engineering, Imperial College London, London, UK. References 1. Nikolic K, Grossman N, Grubb MS, Burrone J, Toumazou C, Degenaar P: Photocycles of Channelrhodopsin-2. Photochemistry and Photobiology 2009, 85:400-411. 2. Grossman N, Simiaki V, Martinet C, Toumazou C, Schultz SR, Nikolic K: The spatial pattern of light determines the kinetics and modulates backpropagation of optogenetic action potentials. Journal of Computational Neuroscience 2013, 34(3):477-488. 3. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K: Millisecond- timescale, genetically targeted optical control of neural activity. Nature Neuroscience 2005, 8(9):1263-1268. 4. Perez F, Granger BE: IPython: a System for Interactive Scientific Computing. Computing in Science and Engineering 2007, 9(3):21-29. The tools are written in Python for easy scripting of experiments and compatibility with a large array of p g p g g g 5. Topalidou M, Leblois A, Boraud T, Rougier NP: A Long Journey into Reproducible Computational Neuroscience. Frontiers in Computational Neuroscience 2015, 9(30):1-2. doi:10.1186/1471-2202-16-S1-P178 Cite this article as: Evans et al.: PyRhO: a virtual optogenetics laboratory. BMC Neuroscience 2015 16(Suppl 1):P178. doi:10.1186/1471-2202-16-S1-P178 Cite this article as: Evans et al.: PyRhO: a virtual optogenetics laboratory. BMC Neuroscience 2015 16(Suppl 1):P178. © 2015 Evans et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The 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.
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Author Correction: The lengths of trachea and main bronchus in Chinese Shanghai population
Scientific reports
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www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Author Correction: The lengths of trachea and main bronchus in Chinese Shanghai population OPEN Xiahui Ge, Haidong Huang, Chong Bai, Xuejun Guo, Christoforos Kosmidis, Konstantinos Sapalidis, Sofia Baka, Kosmas Tsakiridis, Wolfgang Hohenforst‑Schmidt, Lutz Freitag, Anastasios Vagionas, Konstantinos Drevelegas & Paul Zarogoulidis Xiahui Ge, Haidong Huang, Chong Bai, Xuejun Guo, Christoforos Kosmidis, Konstantinos Sapalidis, Sofia Baka, Kosmas Tsakiridis, Wolfgang Hohenforst‑Schmidt, Lutz Freitag, Anastasios Vagionas, Konstantinos Drevelegas & Paul Zarogoulidis Correction to: Scientific Reports https://​doi.​org/​10.​1038/​s41598-​021-​81744-0, published online 26 January 2021 The original version of this Article contained errors in the Material and Methods section under subheading ‘Study population’, “Therefore, 153 adult patients were eligible for evaluation in the study, including 71 patients with unilateral mild pneumonia, 60 patients with pulmonary small nodule (major diameter ≤ 1 cm) and 21 patients with no lesion in chest CT scan but chronic cough or occasional bloody sputum.” now reads: “Therefore, 153 adult patients were eligible for evaluation in the study, including 72 patients with unilateral mild pneumonia, 60 patients with pulmonary small nodule (major diameter ≤ 1 cm) and 21 patients with no lesion in chest CT scan but chronic cough or occasional bloody sputum.” “Therefore, 153 adult patients were eligible for evaluation in the study, including 72 patients with unilateral mild pneumonia, 60 patients with pulmonary small nodule (major diameter ≤ 1 cm) and 21 patients with no lesion in chest CT scan but chronic cough or occasional bloody sputum.” Furthermore, in the Material and Methods section under subheading ‘Measurements.’, urthermore, in the Material and Methods section under subheading ‘Measurements.’, “Similarly, the length of right main bronchi was to calculate the distance from left carina 2 to the carina and the length of left main bronchi was to measure the distance from right carina 1 to the carina.” “Similarly, the length of right main bronchi was to calculate the distance from left carina 2 to the carina and the length of left main bronchi was to measure the distance from right carina 1 to the carina.” “Similarly, the length of right main bronchi was to calculate the distance from right carina 1 to the carina and the length of left main bronchi was to measure the distance from left carina 2 to the carina.” “Similarly, the length of right main bronchi was to calculate the distance from right carina 1 to the carina and the length of left main bronchi was to measure the distance from left carina 2 to the carina.” “However, height between men and women was significantly different either.” now reads: “However, height between men and women was significantly different.” The original Article has been corrected. | https://doi.org/10.1038/s41598-021-98467-x Scientific Reports | (2021) 11:19917 re.com/scientificreports/ 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. now reads: 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © Th A h ( ) 2021 www.nature.com/scientificreports/ 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-98467-x Scientific Reports | (2021) 11:19917 |
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Implementing the World Health Organization Pandemic Influenza Severity Assessment framework—Singapore's experience
Influenza and other respiratory viruses
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O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Received: 1 April 2019  |  Revised: 12 August 2019  |  Accepted: 4 September 2019 Received: 1 April 2019  |  Revised: 12 August 2019  |  Accepted: 4 September 2019 Received: 1 April 2019  |  Revised: 12 August 2019  |  Accepted: 4 September 2019 DOI: 10.1111/irv.12680 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Correspondence Methods: We assessed the severity of influenza by monitoring indicators of influenza transmissibility, seriousness of disease and impact on healthcare resource utilisation. Indicators were described by various parameters collected weekly from eight gov- ernment hospitals, 20 government and 30 private primary care clinics, and the na- tional public health laboratory. Transmissibility and seriousness of disease indicators were each represented by multiple parameters, and alert thresholds were set at the 70th and 90th percentile of a parameter's past 2‐year surveillance data. We derived a collective measure for each indicator using the average percentile rank of the related parameters. Alert thresholds for the single impact parameter were set at predefined values and evaluated for its sensitivity, specificity and positive predictive value. Results: For the transmissibility and seriousness of disease parameters, calculation of the percentile rank was simple and independent of a parameter's underlying distribu- tion. For the impact parameter, predefined alert thresholds had high sensitivity and specificity (>80%) but low positive predictive value (15%‐30%). Assessment scales were used to qualitatively classify the activity of an indicator as low, moderate or high together with a confidence level. Conclusion: We applied different methods for threshold setting depending on the attributes of each parameter and indicator. For indicators represented by multiple parameters, an aggregated assessment of the indicator's level of activity and confi- dence level of the assessment was needed for effective reporting. |  3 wileyonlinelibrary.com/journal/irv 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. © 2019 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd. Implementing the World Health Organization Pandemic Influenza Severity Assessment framework—Singapore's experience Rachael Pung  | Vernon Jian Ming Lee Communicable Diseases Division, Ministry of Health, Singapore, Singapore Communicable Diseases Division, Ministry of Health, Singapore, Singapore Abstract Background: We report our experience in evaluating the severity of local influ- enza epidemics using the World Health Organization Pandemic Influenza Severity Assessment framework. Correspondence Vernon Jian Ming Lee, Communicable Diseases Division, Ministry of Health, College of Medicine Building, 16 College Road, Singapore 169854, Singapore. Email: Vernon_Lee@moh.gov.sg K E Y W O R D S 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. © 2019 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd. |  3 wileyonlinelibrary.com/journal/irv |  3 wileyonlinelibrary.com/journal/irv Influenza Other Respi Viruses. 2020;14:3–10. 4  |     1 | BACKGROUND PUNG and LEE recommended indicators for monitoring severity were the transmis- sibility of the influenza virus, the seriousness of the disease and the impact of influenza on healthcare resource utilisation (referred to as transmissibility, seriousness of disease and impact, in the subse- quent sections). By assessing severity from multiple dimensions, this encourages countries to establish surveillance at different levels of the healthcare system to create a holistic picture of an influenza ep- idemic or pandemic. Early severity assessment of pandemic influenza is helpful for guid- ing pandemic response actions. However, during the 2009 H1N1 pandemic, severity assessment was not standardised across coun- tries, making it difficult to evaluate the local or global situation as the pandemic evolved.1 The lack of a consistent measure of severity also posed a challenge to calibrate pandemic response, which is de- pendent on geographical spread, clinical severity and public interest, among other factors.1 Using virological and surveillance data from different sources, the severity of each indicator can be represented by more than one type of data, or parameter. The choice of parameters may vary across countries due to different data availability, of which some require substantial resource to collect. While the challenge of data com- parison remains, PISA plays an essential role—to promote enhanced Through the lessons learnt from the 2009 H1N1 pandemic, the World Health Organization (WHO) has developed a framework for pandemic influenza severity assessment (PISA).2 PISA is a struc- tured way of tracking influenza epidemics or pandemics. The three TA B LE 1 Parameters considered for assessing severity of influenza TA B LE 1 Parameters considered for assessing severity of influenza TA B LE 1 Parameters considered for assessing severity of influenza Indicator Singapore parameters considered Data source WHO recommended parameters Transmissibility How many people in a population get sick from influenza on a weekly basis Average daily attendance for ARI 20 government primary care clinics Weekly ILI or MAARI cases as a propor- tion of total visits or incidence rates. Weekly percentage of respiratory pathogen samples testing positive for influenza. Abbreviations: ARI: acute respiratory infection; ED: emergency department of a government hospital; ICU: intensive care unit of a government hospi- tal; ILI: influenza‐like illness; MAARI: medically attended acute respiratory illness; SARI: severe acute respiratory infection. 8 government acute hospitals SARI/ARI or ILI ratio Cumulative death: hospitalisation ratio (ideally for confirmed influenza) Cumulative ICU: hospitalisation ratio (ide- ally for confirmed influenza) Weekly or monthly number or proportion of SARI cases with percentage flu‐posi- tive among SARI cases Weekly excess pneumonia & influenza (P&I) or all‐cause mortality stratified by age. Weekly number of confirmed influenza cases admitted to ICU, or weekly number of confirmed influenza cases admitted to hospital. 1.1 | Influenza surveillance in Singapore Singapore, a city‐state in South East Asia, is a major global travel hub with over 18 million tourist arrivals3 and a population of over 5.6 million in 2018.4 It has a high population density of over 8000 peo- ple per square kilometre, which may facilitate the spread of contact transmissible and airborne diseases such as influenza. Locally, influenza A (H1N1) pdm09, A (H3N2) and influenza B vi- ruses circulate year‐round. Following the 2009 influenza pandemic, we expanded our influenza surveillance network and encouraged government and private primary care clinics to participate in the National Influenza Surveillance Programme. To date, 20 government primary care clinics, providing about 20% of primary healthcare ser- vices in the population,5 and 30 sentinel clinics spread across the country out of 1400 private primary care clinics are enrolled in the programme. Our influenza surveillance network also comprises eight acute government hospitals, providing about 80% of all acute care hospital services in the population5 and the National Public Health Laboratory (NPHL). A time series plot of each parameter was used to illustrate the parameter's variability during each seasonal epidemic and surveil- lance artefacts arising from reporting changes. These two factors were considered in the final selection of parameters used for PISA reporting. In this paper, we document Singapore's experience in developing and evaluating the PISA indicators and parameters, and this would provide other countries with suggestions that they can use in devel- oping their own indicators. 2.1 | Data sources For a parameter, we calculated the percentile rank or the percen- tile of an observed value with respect to the previous 2‐year histori- cal data (eg the percentage of data from January 2016 to December 2017 that were equal or lower than a weekly parameter data col- lected in 2018). We limited the comparison to 2‐year historical data due to recent changes in data extraction methods. Let xw denote the observed value of a parameter and pw denote the percentile of that observed value in week w of a year. Also, let h denote the historical data in the previous 2 years. A wide range of parameters were reported weekly to the Ministry of Health (MOH) and considered for PISA (Table 1). Influenza transmis- sion in the community was monitored using the average daily at- tendance for acute respiratory infection (ARI) and the average daily attendance for influenza‐like illness (ILI) at the government primary care clinics. An ARI diagnosis was made when a case had at least one acute respiratory symptom such as cough, sore throat and co- ryza, while an ILI diagnosis was made when a case had a fever of ≥38.0°C and cough, with onset within the last 10 days. The average daily attendance for ARI and average daily attendance for ILI at the government primary care clinics were used, instead of the weekly attendances, to offset the effect of public holidays and clinic closure on weekends. pw ⎧ ⎪ ⎪ ⎨ ⎪ ⎪⎩ = 0 if xw <min (h) = CF+0.5f n =100 if xw >max (h) Consent was sought for the collection of respiratory sam- ples from all patients if they received outpatient consultation at a government or private primary care clinics that are enrolled in the National Influenza Surveillance Programme and presented with ILI. These samples were routinely submitted to the NPHL and tested using the FilmArray Respiratory Panel and/or real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) to de- tect respiratory viruses. The weekly number of samples was small as not all identified patients participated in the surveillance, and where CF is the number of values in h that is below xw (ie cumulative frequency). f is the number of values in h that is equal to xw (ie fre- quency). n is the number of values in h. surveillance and increase information sharing among public health officials during an influenza epidemic or pandemic. hence, we pooled the results across four weeks and monitored the proportion of respiratory samples positive for influenza over a 4‐weekly moving interval. As not all ILI attendances at the government primary care clin- ics were attributed to influenza, we explored using the product of the average daily attendance for ILI and weekly proportion of respi- ratory samples positive for influenza to estimate the average daily number of influenza‐positive ILI cases at the government primary care clinics. We also collect parameters from the eight acute govern- ment hospitals comprising of the weekly number of ARI Emergency Department (ED) attendances and admissions, the weekly number of pneumonia ED attendances and admissions, and the weekly number of laboratory‐confirmed influenza cases admitted to the intensive care unit (ICU) or died. The former two parameters were collected through MOH’s healthcare utilisation database while the latter was compiled by a team of healthcare professionals in each hospital and forwarded to MOH. 2.2 | Assessing the transmissibility and seriousness of disease indicators’ level of activity As the transmissibility and seriousness of disease indicators were represented by more than one parameter, an overall measure of each indicator's level of activity and the confidence of the indicator was necessary for weekly reporting. 4  |     1 | BACKGROUND Composite (product) of weekly ILI or MAARI and weekly percentage positivity rates for influenza Average daily attendance for ILI Proportion of respira- tory samples positive for influenza over a 4‐weekly moving interval 20 government and 30 pri- vate primary care clinics Estimated average daily number of influenza‐posi- tive ILI cases Seriousness of disease How severely sick an individual gets when infected with the influenza virus Weekly number of ARI ED attendances 8 government acute hospitals SARI/ARI or ILI ratio Cumulative death: hospitalisation ratio (ideally for confirmed influenza) Cumulative ICU: hospitalisation ratio (ide- ally for confirmed influenza) Weekly number of ARI ED admissions Weekly proportion of ARI ED attendances resulting in admission Weekly number of pneu- monia ED attendances Weekly number of pneu- monia ED admissions Weekly proportion of pneumonia ED at- tendances resulting in admission Impact How the influenza epidemic or pandemic affects the healthcare system (and society) Weekly number of labora- tory‐confirmed influenza cases admitted to ICU or died 8 government acute hospitals Weekly or monthly number or proportion of SARI cases with percentage flu‐posi- tive among SARI cases Weekly excess pneumonia & influenza ( ) Seriousness of disease How severely sick an individual gets when infected with the influenza virus SARI/ARI or ILI ratio Cumulative death: hospitalisation ratio (ideally for confirmed influenza) Cumulative ICU: hospitalisation ratio (ide- ally for confirmed influenza) Weekly or monthly number or proportion of SARI cases with percentage flu‐posi- tive among SARI cases Weekly excess pneumonia & influenza (P&I) or all‐cause mortality stratified by age. Weekly number of confirmed influenza cases admitted to ICU, or weekly number of confirmed influenza cases admitted to hospital. Weekly or monthly number or proportion of SARI cases with percentage flu‐posi- tive among SARI cases Weekly excess pneumonia & influenza (P&I) or all‐cause mortality stratified by age. Weekly number of confirmed influenza cases admitted to ICU, or weekly number of confirmed influenza cases admitted to hospital. Impact How the influenza epidemic or pandemic affects the healthcare system (and society) Abbreviations: ARI: acute respiratory infection; ED: emergency department of a government hospital; ICU: intensive care unit of a government hospi- tal; ILI: influenza‐like illness; MAARI: medically attended acute respiratory illness; SARI: severe acute respiratory infection. 5 PUNG and LEE 2.1 | Data sources To quantify an indicator's level of activity, we calculated the av- erage percentile of all the parameters of an indicator. On a scale from zero to 100, percentile values of 70 and 90 were used as cut‐offs 6  |     PUNG and LEE (ie alert thresholds) to provide three classifications of an indicator's level of activity depending on where the average percentile value lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the cut‐offs percentiles provided a measure of confidence—the further away, the average percentile is from an alert threshold, the greater the confidence in the assessment of an indicator's level of activity and vice versa. 2.3 | Assessing the impact indicator's level of activity The weekly number of laboratory‐confirmed influenza cases who were admitted to the intensive care unit (ICU) or died is the only im- pact parameter, and we used data from January 2011 to December 2017 for threshold setting due to the absence of reporting artefacts over the years. The discrete data had a small range of observed values, and hence, we used a different approach to set the alert thresholds and to ensure that alert thresholds had integer values. A sustained high (moderate) influenza activity is said to occur when the impact parameter values remain above the high (moderate) alert thresholds for 2 weeks after the first alert week. We set alert thresh- olds at predefined values and tested two different scenarios. In the first scenario, the moderate and high alert thresholds were set at three and six, respectively. In the second scenario, they were revised to four and six, respectively. We evaluated key performance met- rics of sensitivity, specificity and positive predictive value (PPV) of a threshold to assess the threshold's ability to provide early warn- ing prior to the peak of an influenza season.6 The sensitivity was the proportion of sustained high influenza activity with a moderate alert raised in at least one of the 2 weeks prior to crossing the high alert threshold. The specificity was the proportion of weeks with no alerts during the baseline influenza periods. The PPV for high (mod- erate) influenza activity was the proportion of true high (moderate) alerts among all high (moderate) alerts. 2.1 | Data sources 3 | RESULTS 3.1 | Parameters selected for PISA reporting Time series plots of the parameters in Table 1 are shown in Figure 1. The average daily attendance for ARI at the government primary care clinics (Figure 1A) exhibits a multimodal distribution as it is in- fluenced by the activity of other respiratory viruses, and the sea- sonal peaks of these viruses might not be in sync with the influenza seasons. While the average daily attendance for ARI at government primary care clinics is less representative of the local influenza trans- missibility as compared to its ILI counterpart, it is still important to track it as influenza with low clinical severity may appear more fre- quently as ARI From 2011 to 2015, the average daily attendance for ILI at the government primary care clinics declined (Figure 1B) and this could be attributed to gradual underreporting after the 2009 H1N1 pan- demic. In 2016, ILI case definition was reiterated to all government primary care clinics leading to a rise in the measure. The estimated average daily number of influenza‐positive ILI cases at the gov- ernment primary care clinics was not chosen for as a parameter for transmissibility eventually as it is a repeated representation of its individual components and any variations caused by reporting artefacts will affect its interpretation. Additionally, the proportion of total visits attributed to ARI or ILI, as recommended by WHO, was not monitored as it represented the burden of influenza com- pared to other diseases instead of the transmissibility of the virus. The proportion of respiratory samples positive for influenza over a 4‐weekly moving interval was the only laboratory‐confirmed in- fluenza parameter for the transmissibility indicator. Higher local in- fluenza activity was observed from May to July and from November to January, and generally coincides with the winter in the Southern and Northern Hemisphere, respectively (Figure 1C). Seasonal fluctu- ations were observed in the weekly attendances and admissions at the emergency department (ED) of acute government hospitals for both ARI and pneumonia (Figure 1E‐J). During the 2009 influenza pandemic, the weekly number of ARI ED attendances (Figure 1E) and admissions (Figure 1F) and, consequently, the weekly proportion of ARI ED attendances resulting in admission (Figure 1G) indicated a clear spike. One‐off adjustment in 2016 was also observed in the pneumonia parameters (Figure 1H‐J) due to change in the disease classification and coding systems of some hospitals. 2.1 | Data sources The weekly proportion of ARI or pneumonia ED attendances resulting in admis- sion (Figure 1G,J) was selected as parameters for the seriousness of disease as it indicated the extent to which individual gets sick and required hospital care. The weekly number of laboratory‐confirmed influenza cases who were admitted to the ICU or died described the impact of influenza on healthcare resource utilisation and was the only parameter for the impact indicator. Influenza mortality was a component of the pa- rameter as the management of critically ill patients in general wards could also be resource intensive (eg manpower needed for frequent monitoring a patient's progress and calibration of treatment). Sharp peaks in this parameter were typically observed during May to July, coinciding with winter in the Southern Hemisphere (Figure 1K). 3.2 | Performance of the impact parameter alert thresholds The weekly number of laboratory‐confirmed influenza cases that were admitted to ICU or died ranged from 0 to 24 (Figure 1K). When the moderate and high alert thresholds were predefined at an inte- ger value of 3 and 6, respectively, 29 moderate alerts and 19 high alerts were raised from 2011 to 2017 Of these alerts nine moder (A) (B) (C) (D) (E) (F) (G) (H) PUNG and LEE | (ie alert thresholds) to provide three classifications of an indicator's level of activity depending on where the average percentile value lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the cut‐offs percentiles provided a measure of confidence—the further away, the average percentile is from an alert threshold, the greater the confidence in the assessment of an indicator's level of activity and vice versa. 2.3 | Assessing the impact indicator's level of activity (A) (ie alert thresholds) to provide three classifications of an indicator's level of activity depending on where the average percentile value lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the cut‐offs percentiles provided a measure of confidence—the further away, the average percentile is from an alert threshold, the greater the confidence in the assessment of an indicator's level of activity and vice versa. 2.1 | Data sources Seasonal fluctu- ations were observed in the weekly attendances and admissions at the emergency department (ED) of acute government hospitals for both ARI and pneumonia (Figure 1E‐J). During the 2009 influenza pandemic, the weekly number of ARI ED attendances (Figure 1E) and admissions (Figure 1F) and, consequently, the weekly proportion of ARI ED attendances resulting in admission (Figure 1G) indicated a clear spike. One‐off adjustment in 2016 was also observed in the pneumonia parameters (Figure 1H‐J) due to change in the disease classification and coding systems of some hospitals. The weekly proportion of ARI or pneumonia ED attendances resulting in admis- sion (Figure 1G,J) was selected as parameters for the seriousness of disease as it indicated the extent to which individual gets sick and required hospital care. The weekly number of laboratory confirmed influenza cases who (A) (B) (C) (D) (E) (F) pared to other diseases instead of the transmissibility of the virus The proportion of respiratory samples positive for influenza ove a 4‐weekly moving interval was the only laboratory‐confirmed in fluenza parameter for the transmissibility indicator. Higher local in fluenza activity was observed from May to July and from Novembe to January, and generally coincides with the winter in the Southern and Northern Hemisphere, respectively (Figure 1C). Seasonal fluctu ations were observed in the weekly attendances and admissions at the emergency department (ED) of acute government hospitals fo both ARI and pneumonia (Figure 1E‐J). During the 2009 influenza pandemic, the weekly number of ARI ED attendances (Figure 1E) and admissions (Figure 1F) and, consequently, the weekly proportion o ARI ED attendances resulting in admission (Figure 1G) indicated a (D) The weekly number of laboratory‐confirmed influenza cases who were admitted to the intensive care unit (ICU) or died is the only im- pact parameter, and we used data from January 2011 to December 2017 for threshold setting due to the absence of reporting artefacts over the years. The discrete data had a small range of observed values, and hence, we used a different approach to set the alert thresholds and to ensure that alert thresholds had integer values. A sustained high (moderate) influenza activity is said to occur when the impact parameter values remain above the high (moderate) alert thresholds for 2 weeks after the first alert week. We set alert thresh- olds at predefined values and tested two different scenarios. 2.1 | Data sources In the first scenario, the moderate and high alert thresholds were set at three and six, respectively. In the second scenario, they were revised (C) d to The p (D) ed to o The pr (D) clear spike. One‐off adjustment in 2016 was also observed in the pneumonia parameters (Figure 1H‐J) due to change in the disease classification and coding systems of some hospitals. The weekly proportion of ARI or pneumonia ED attendances resulting in admis- sion (Figure 1G,J) was selected as parameters for the seriousness of disease as it indicated the extent to which individual gets sick and required hospital care. The weekly number of laboratory‐confirmed influenza cases who were admitted to the ICU or died described the impact of influenza on healthcare resource utilisation and was the only parameter for the impact indicator. Influenza mortality was a component of the pa- rameter as the management of critically ill patients in general wards could also be resource intensive (eg manpower needed for frequent monitoring a patient's progress and calibration of treatment) Sharp (F) to four and six, respectively. We evaluated key performance met- rics of sensitivity, specificity and positive predictive value (PPV) of a threshold to assess the threshold's ability to provide early warn- ing prior to the peak of an influenza season.6 The sensitivity was the proportion of sustained high influenza activity with a moderate alert raised in at least one of the 2 weeks prior to crossing the high alert threshold. The specificity was the proportion of weeks with no alerts during the baseline influenza periods. The PPV for high (mod- erate) influenza activity was the proportion of true high (moderate) alerts among all high (moderate) alerts. 3 | RESULTS (E) rics o a thr ing p the p alert alert alert erate alert 3 | f sens eshold (E) sio dise req we on the ram cou monitoring a patient s progress and calibration of treatment). Sharp peaks in this parameter were typically observed during May to July coinciding with winter in the Southern Hemisphere (Figure 1K). 3.2 | Performance of the impact parameter alert thresholds The weekly number of laboratory‐confirmed influenza cases tha were admitted to ICU or died ranged from 0 to 24 (Figure 1K). 2.1 | Data sources 2.3 | Assessing the impact indicator's level of activity From 2011 to 2015, the average daily attendance for ILI at the government primary care clinics declined (Figure 1B) and this could be attributed to gradual underreporting after the 2009 H1N1 pan- demic. In 2016, ILI case definition was reiterated to all government primary care clinics leading to a rise in the measure. The estimated average daily number of influenza‐positive ILI cases at the gov- ernment primary care clinics was not chosen for as a parameter for transmissibility eventually as it is a repeated representation of its individual components and any variations caused by reporting artefacts will affect its interpretation. Additionally, the proportion of total visits attributed to ARI or ILI, as recommended by WHO, was not monitored as it represented the burden of influenza com- (A) (B) From 2011 to 2015, the average daily attendance for ILI at t government primary care clinics declined (Figure 1B) and this cou be attributed to gradual underreporting after the 2009 H1N1 pa demic. In 2016, ILI case definition was reiterated to all governme primary care clinics leading to a rise in the measure. The estimate average daily number of influenza‐positive ILI cases at the go ernment primary care clinics was not chosen for as a paramet for transmissibility eventually as it is a repeated representation its individual components and any variations caused by reporti artefacts will affect its interpretation. Additionally, the proportio of total visits attributed to ARI or ILI, as recommended by WH was not monitored as it represented the burden of influenza com (B) From 2 (B) (ie alert thresholds) to provide three classifications of an indicator's level of activity depending on where the average percentile value lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the cut‐offs percentiles provided a measure of confidence—the further away, the average percentile is from an alert threshold, the greater the confidence in the assessment of an indicator's level of activity and vice versa. 2.3 | Assessing the impact indicator's level of activity The weekly number of laboratory‐confirmed influenza cases who were admitted to the intensive care unit (ICU) or died is the only im- pact parameter, and we used data from January 2011 to December 2017 for threshold setting due to the absence of reporting artefacts over the years. 2.1 | Data sources The discrete data had a small range of observed values, and hence, we used a different approach to set the alert thresholds and to ensure that alert thresholds had integer values. A sustained high (moderate) influenza activity is said to occur when the impact parameter values remain above the high (moderate) alert thresholds for 2 weeks after the first alert week. We set alert thresh- olds at predefined values and tested two different scenarios. In the first scenario, the moderate and high alert thresholds were set at three and six, respectively. In the second scenario, they were revised to four and six, respectively. We evaluated key performance met- rics of sensitivity, specificity and positive predictive value (PPV) of a threshold to assess the threshold's ability to provide early warn- ing prior to the peak of an influenza season.6 The sensitivity was the proportion of sustained high influenza activity with a moderate alert raised in at least one of the 2 weeks prior to crossing the high alert threshold. The specificity was the proportion of weeks with no alerts during the baseline influenza periods The PPV for high (mod From 2011 to 2015, the average daily attendance for ILI at the government primary care clinics declined (Figure 1B) and this could be attributed to gradual underreporting after the 2009 H1N1 pan- demic. In 2016, ILI case definition was reiterated to all government primary care clinics leading to a rise in the measure. The estimated average daily number of influenza‐positive ILI cases at the gov- ernment primary care clinics was not chosen for as a parameter for transmissibility eventually as it is a repeated representation of its individual components and any variations caused by reporting artefacts will affect its interpretation. Additionally, the proportion of total visits attributed to ARI or ILI, as recommended by WHO, was not monitored as it represented the burden of influenza com- pared to other diseases instead of the transmissibility of the virus. The proportion of respiratory samples positive for influenza over a 4‐weekly moving interval was the only laboratory‐confirmed in- fluenza parameter for the transmissibility indicator. Higher local in- fluenza activity was observed from May to July and from November to January, and generally coincides with the winter in the Southern and Northern Hemisphere, respectively (Figure 1C). 2.1 | Data sources When the moderate and high alert thresholds were predefined at an inte ger value of 3 and 6, respectively, 29 moderate alerts and 19 high alerts were raised from 2011 to 2017. Of these alerts, nine moder ate alerts and four high alerts preceded sustained moderate or high (H) 3.1 | Parameters selected for PISA reporting Time series plots of the parameters in Table 1 are shown in Figure 1. The average daily attendance for ARI at the government primary care clinics (Figure 1A) exhibits a multimodal distribution as it is in- fluenced by the activity of other respiratory viruses, and the sea- sonal peaks of these viruses might not be in sync with the influenza seasons. While the average daily attendance for ARI at government primary care clinics is less representative of the local influenza trans- missibility as compared to its ILI counterpart, it is still important to track it as influenza with low clinical severity may appear more fre- quently as ARI. (G) FI G U R E 1 ks in t ciding (H) FI G U R E 1 |  7 |  7 PUNG and LEE | (I) (K) (J) FI G U R E 1 Time series plots of (A) average daily attendance for ARI at government primary care clinics, (B) average daily attendance for ILI at government primary care clinics, (C) proportion of respiratory samples positive for influenza over a 4‐weekly moving interval, (D) estimated average daily number of influenza‐positive ILI cases, (E) weekly number of ARI ED attendances, (F) weekly number of ARI ED admissions, (G) weekly proportion of ARI ED attendances resulting in admission, (H) weekly number of pneumonia ED attendances. 4 | DISCUSSION Influenza surveillance in Singapore spans all acute government hos- pitals, all government and some private primary care clinics. PISA indicators representing the transmissibility of influenza virus, seri- ousness of disease or the impact of influenza on healthcare resource utilisation highlight different aspects of influenza activity. This pro- vides comprehensive surveillance of the severity of a current in- fluenza season and allows the ministry to determine the extent of public health responses required to manage the transmission and to protect vulnerable populations. Influenza surveillance in Singapore spans all acute government hos- pitals, all government and some private primary care clinics. PISA indicators representing the transmissibility of influenza virus, seri- ousness of disease or the impact of influenza on healthcare resource utilisation highlight different aspects of influenza activity. This pro- vides comprehensive surveillance of the severity of a current in- fluenza season and allows the ministry to determine the extent of public health responses required to manage the transmission and to protect vulnerable populations. 2.1 | Data sources cont'd (I) weekly number of pneumonia ED admissions, (J) weekly proportion of pneumonia attendances at the ED resulting in admission, (K) weekly number of laboratory‐confirmed influenza cases admitted to ICU or died (I) (J) (J) (I) (I) (K) FI G U R E 1 Time series plots of (A) average daily attendance for ARI at government primary care clinics, (B) average daily attendance for ILI at government primary care clinics, (C) proportion of respiratory samples positive for influenza over a 4‐weekly moving interval, (D) estimated average daily number of influenza‐positive ILI cases, (E) weekly number of ARI ED attendances, (F) weekly number of ARI ED admissions, (G) weekly proportion of ARI ED attendances resulting in admission, (H) weekly number of pneumonia ED attendances. cont'd (I) weekly number of pneumonia ED admissions, (J) weekly proportion of pneumonia attendances at the ED resulting in admission, (K) weekly number of laboratory‐confirmed influenza cases admitted to ICU or died influenza activity (ie 31.0% and 21.1% PPV for moderate and high alert threshold, respectively). In all four of the observed sustained high influenza activity, a moderate alert was made known at least one week prior to trigger of the high alert (ie 100% sensitivity). No alerts were made in 83 of the 103 weeks of baseline influenza activ- ity (ie specificity of 80.6%). meaningful cut‐offs, and hence, a separate assessment scale was created (Figure 2B) based on the results in the previous section, Performance Matrices for the Impact Parameter Alert Threshold. Table 2 illustrates the weekly PISA results from E‐week 1 to 10 of 2018. In the second scenario, the moderate alert threshold was in- creased to a value of 4, and 20 moderate alerts were raised from 2011 to 2017. Of these alerts, three resulted in sustained mod- erate influenza activity (ie PPV of moderate alert threshold of 15.0%). The sensitivity remained at 100%. No alerts were made in 138 of the 156 weeks of baseline influenza activity (ie specificity of 88.5%). 3.3 | Assessment scale for indicators Figure 2A shows the assessment scale used to qualitatively clas- sify the level of activity of the transmissibility and seriousness of disease indicators. The coloured scale showed gradual transition from dark green to dark red signifying increasing levels of activ- ity of an indicator. The small range of discrete values observed in the single impact parameter limits our ability to provide multiple, Of the three indicators, transmissibility has the widest variety of parameters customised for each country's setting. In Singapore, data on the ARI and ILI attendance at government primary care clin- ics are conveniently extracted from various healthcare surveillance PUNG and LEE 8 8 FI G U R E 2 Assessment scale for (A) transmissibility and seriousness of disease indicators, (B) impact indicator. Severity of an indicator is classified as L: low, M: moderate, H: high. Confidence level of an indicator is classified as 1: low, 2: medium, 3: high FI G U R E 2 Assessment scale for (A) transmissibility and seriousness of disease indicators, (B) impact indicator. Severity of an indicator is classified as L: low, M: moderate, H: high. Confidence level of an indicator is classified as 1: low, 2: medium, 3: high technological and manpower capability, critical care could be pro- vided in general wards. An improved measure would be the ratio of cases fulfilling the definitions of complicated or severe influenza11 to the number of influenza‐positive admission. However, the feasibility of measuring this is dependent on the healthcare system's ability to integrate laboratory and epidemiological data. platforms for weekly reporting. In other countries, the number of callers to public health hotline reporting ILI7,8 or prescription re- cords9,10 were also explored as means to characterise the extend of spread when complemented with data sources from healthcare institutions. Key challenges remain in achieving a representative indicator for seriousness of disease in Singapore. The weekly proportion of ARI or pneumonia ED attendances that were hospitalised were chosen to illustrate the severity of each condition, but the absence of hospital laboratory surveillance data limits our ability to verify the infection status of each patient. Spikes in the weekly proportion of ARI ED at- tendances that were hospitalised (Figure 1G) could be attributed to changes in health‐seeking behaviour, reporting habits of physicians and higher tendency to admit a patient during a pandemic, though extent of influence has yet to be studied. integer values. The PPV of the thresholds was poor and implied that in many occasions, there was no sustained moderate or high influ- enza activity occurring after a moderate or high alert was triggered. The moderate threshold was eventually set at four as about 70% of the historical data was below this value, and a moderate alert was triggered before the onset of all sustain high influenza activity. abnormalities needs to be highlighted and the interpretation of the average percentile under such conditions should be done with caution. Rachael Pung  https://orcid.org/0000-0002-1188-8231 Rachael Pung  https://orcid.org/0000-0002-1188-8231 Rachael Pung  https://orcid.org/0000-0002-1188-8231 The quantification of an indicator's level of activity is achieved by averaging the percentile rank of all the parameters representing an indicator with the assumption that all parameters were equally informative. However, ARI parameters can be influenced by the ac- tivity of other respiratory viruses. As such, there may be occasions where the average daily attendance for ARI was high but the same was not observed for ILI surveillance data. However, it is still import- ant to track the ARI attendances at the government acute hospitals and primary care clinics as it potentially informs us of any changes in the clinical representation of influenza cases. One possible way of overcoming this challenge is to assign weights to each parameter based on its importance in assessing the local influenza situation. The weighted average percentile rank could be computed to repre- sent an indicator's level of activity. 5 | CONCLUSION In this paper, we also presented an assessment scale, which pro- vides a combined measure of an indicator's level of activity and the confidence level of the assessment. With more than one parame- ter serving as proxies for an indicator, the method of providing an aggregated assessment for an indicator remains undocumented in PISA. Furthermore, the confidence of an indicator's assessment is part of PISA reporting, but its interpretation is multifaceted. It is dependent on, but not limited to, reporting biases, timeliness and agreement between the parameters. The first two factors are re- lated to the reliability of the information provided at various sen- tinel sites and can be improved with a structured data collection process. On the contrary, the agreement between the parameters is intrinsic to the influenza activity of a season. Each parameter is a unique proxy of an indicator and might be influenced by the activity of other respiratory viruses. Thus, a high agreement between the parameters provides greater certainty to the measure of an indica- tor's level of activity. We share Singapore's practices in the weekly assessment of PISA indicators. For indicators represented by multiple parameters, a collective assessment of the indicator's level of activity and the confidence level of this assessment were necessary. Here, we have introduced an assessment scale to accomplish both objectives. We placed priority in creating a simple collective assessment for a com- plex indicator. The choice of parameters, sampling criteria and case definitions were regularly reviewed and updated to ensure consist- ent performance of our surveillance system. Our method of PISA reporting could be applied in other countries, with parameters cho- sen based on the resources of the country, and the assessment scale customised to the local setting. 3.3 | Assessment scale for indicators Severity assessment has been largely focused on developing different methods to establish alert thresholds that signal the start or the end of an influenza season. Based on the characteristics of a parameter,12 a variety of methods such as the Moving Epidemic Method (MEM) or cumulative sum control charts (CUSUM) have been developed for early epidemic detection. For Singapore, thresh- olds setting methods were chosen based on the data characteristics. Regular review and enhancement of data extraction methods helps to improve accuracy of the parameters but inevitably creates arte- facts in the historical surveillance data and limits the feasibility of using methods that require long history of surveillance data. Hence, for transmissibility and seriousness of disease parameters, the mod- erate and high alert thresholds of a year were set using the 70th and 90th percentiles of the past 2‐year data. For the impact parameter, the moderate and high alert thresholds were set using predefined The cumulative number of patients tested positive for influenza admitted to ICU is a component to some WHO recommended pa- rameters in Table 1. This component is limited by the number of ICU beds, and ICU admission of a severe influenza case is subjected to competing requirements of other non‐influenza‐positive patients depending on severity. Furthermore, depending on a hospital's TA B LE 2 Weekly PISA results PISA reporting: Transmissibility Seriousness of disease Impact Year E‐week Risk Confidence Risk Confidence Risk Confidence 2018 1 Moderate Low   Low Medium   High Medium   2018 2 Moderate Low   Low Medium   High Low   2018 3 High Low   Moderate High   Moderate Low   2018 4 High Low   Low High   Low High   2018 5 High Low   Low High   Low High   2018 6 High Low   Low High   High Low   2018 7 Moderate Low   Low Low   Low High   2018 8 Moderate High   Moderate Low   Moderate Low   2018 9 Moderate Low   Low Low   Low High   2018 10 Low Low   Moderate Medium   Moderate Low |  9 PUNG and LEE REFERENCES 1. World Health Organization. Strengthening response to pandem- ics and other public‐health emergencies: report of the Review Committee on the Functioning of the International Health Regulations (2005) and on Pandemic Influenza (H1N1) 2009. https​ ://apps.who.int/iris/bitst​ream/handl​e/10665/​75235/​97892​41564​ 335_eng.pdf;jsess​ionxm​l:id=6C57B​89071​8C3C6​65CC1​B36C7​ 0BC3A​15?seque​nce=1 Accessed 15 July, 2019. 2. World Health Organization. Pandemic Influenza Severity Assessment (PISA): a WHO guide to assess the severity of influenza epidemics and pandemics. https​://apps.who.int/iris/bitst​ream/ handl​e/10665/​25939​2/WHO-WHE-IHM-GIP-2017.2-eng.pdf?se- que​nce=1 Accessed January 5, 2019. 3. Singapore Tourism Board. International visitor arrival statistics. https​://www.stb.gov.sg/conte​nt/dam/stb/docum​ents/stati​stics- marke​ting-insig​hts/inter​natio​nal-visit​or-arriv​als/pdf/visit​or-arriv​ als-2018.pdf Accessed February 20, 2019. In addition, when a parameter is higher (or lower) than the histor- ical maximum (or minimum), the percentile of that parameter's data was capped at 100 (or zero). Taking the average percentile values of all parameters of an indicator then helps to ensure that the extreme results of one parameter would not dominate the measure of an in- dicator but allows it to skew the measure towards a higher (or lower) classification of the indicator's level of activity. 4. Singapore Department of Statistics. Population and population structure. https​://www.sings​tat.gov.sg/find-data/search-by- theme/​popul​ation/​popul​ation-and-popul​ation-struc​ture/lat- est-data Accessed February 20, 2019. 5. Lee CE, Satkunanantham K. Singapore’s Health Care System: What 50 Years Have Achieved. Singapore: World Scientific Publishing Co., Pte. Ltd.; 2016:18. The confidence assessment did not consider the number of parameters used to represent an indicator. It is possible for an in- dicator's parameter to reflect a very different level of activity com- pared to the rest of the parameters. In situations where there are few parameters representing an indicator, the extreme parameter is likely to skew an indicator's level of activity towards an extreme. The effect of the extreme parameter on the indicator's level of activity would attenuate when the number of parameters repre- senting an indicator increases. Also, any sustained occurrence of 6. Hashimoto S, Murakami Y, Taniguchi K, Nagai M. Detection of epi- demics in their early stage through infectious disease surveillance. Int J Epidemiol. 2000;29(5):905‐910. 7. Vette K, Bareja C, Clark R, Lal A. Establishing thresholds and pa- rameters for pandemic influenza severity assessment, Australia. Bull World Health Organ. 2018;96(8):558‐567. 8. Public Health England. Surveillance of influenza and other re- spiratory viruses in the UK: winter 2017 to 2018. https​://as- sets.publi​shing.servi​ce.gov.uk/gover​nment/​uploa​ds/syste​m/ uploa​ds/attac​hment_data/file/74060​6/Surve​illan​ce_of_influ​ 8. Public Health England. Surveillance of influenza and other re- spiratory viruses in the UK: winter 2017 to 2018. https​://as- sets.publi​shing.servi​ce.gov.uk/gover​nment/​uploa​ds/syste​m/ uploa​ds/attac​hment_data/file/74060​6/Surve​illan​ce_of_influ​ 10 PUNG and LEE 10  | 12. World Health Organization. Global epidemiological surveillance standards for influenza. https​://www.who.int/influ​enza/resou​rces/ docum​ents/WHO_Epide​miolo​gical_Influ​enza_Surve​illan​ce_Stand​ ards_2014.pdf Accessed January 5, 2019. enza_and_other_respi​ratory_virus​es_in_the_UK_2017_to_2018. pdf Accessed January 5, 2019. 9. Sugawara T, Ohkusa Y, Ibuka Y, Kawanohara H, Taniguchi K, Okabe N. Real‐time prescription surveillance and its application to mon- itoring seasonal influenza activity in, Japan. J Med Internet Res. 2012;14(1):e14. How to cite this article: Pung R, Lee VJM. Implementing the World Health Organization Pandemic Influenza Severity Assessment framework—Singapore's experience. Influenza Other Respi Viruses. 2020;14:3–10. https​://doi.org/10.1111/ irv.12680​ 10. Chen JH, Schmit K, Chang H, Herlihy E, Miller J, Smith P. Use of medicaid prescription data for syndromic surveillance –‐ New York. MMWR Morb Mortal Wkly Rep. 2005;54(Suppl):31‐34. How to cite this article: Pung R, Lee VJM. Implementing the World Health Organization Pandemic Influenza Severity Assessment framework—Singapore's experience. Influenza Other Respi Viruses. 2020;14:3–10. https​://doi.org/10.1111/ irv.12680​ 11. World Health Organization. WHO guidelines for pharmacologi- cal management of pandemic influenza A(H1N1) 2009 and other influenza viruses. https​://www.who.int/csr/resou​rces/publi​catio​ ns/swine​flu/h1n1_guide​lines_pharm​aceut​ical_mngt.pdf Accessed January 5, 2019.
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5.3.1 Start der Verhandlungen um Dublin III
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88 David Lorenz: Die umkämpfte Dublin-Verordnung Damit verschob sich der Schwerpunkt in der Anwendung Dublins weg von der Verteilung der Verantwortung für Asylverfahren zwischen den Mitgliedstaaten hin zur Verhinderung mehrfacher Asylverfahren. Das bedeutet auch, dass die mit Dublin intendierte Umverteilung der Zuständigkeit für Asylverfahren an die Grenzstaaten im Zeitraum dieser Untersuchung der Kommission nicht realisiert wurde: »Contrary to a widely shared supposition that the majority of transfers are directed towards the Member States located at an external border, it appears that the overall allocation between border and non border countries is actually rather balanced.« (Ebd., S. 51) Für das Jahr 2005 veröffentlichte die Europäische Kommission (ebd., S. 50–52) umfassende Zahlen zu Überstellungen (siehe Tabelle 3 im Anhang). Deutschland ist weiterhin der in Bezug auf Überstellungen aktivste Mitgliedstaat. Allerdings halten sich im Unterschied zu den Daten von 1998 – 1999 die Überstellungen nach und von Deutschland fast die Waage. Der Umstand, dass Mitgliedstaaten sowohl ausgehende als auch ankommende Überstellungen haben, führt zu einer neuen statistischen Größe: den Netto-Überstellungen beziehungsweise der Überstellungsbilanz. Netto-Überstellungen und die Überstellungsbilanz bezeichnen die Differenz aus ausgehenden und ankommenden Überstellungen. Netto-Überstellungen geben Auskunft darüber, welche Mitgliedstaaten durch Dublin-Überstellungen mehr Asylverfahren durchführen müssen und welche weniger. Während Deutschland zwar mit 2.748 ausgehenden und 2.716 ankommenden Überstellungen der bezogen auf Überstellungen aktivste Mitgliedstaat ist, machen die 32 zusätzlichen Überstellungen, die aus der Differenz resultieren, kaum einen Unterschied für die Gesamtzahl der Asylverfahren in Deutschland. Eine positive Zahl an Netto-Überstellungen bedeutet, die ausgehenden Überstellungen sind in der Überzahl, eine negative Zahl steht für eine Überzahl der ankommenden Überstellungen. Durch die Osterweiterung der EU 2004 hat Polen Deutschland als das Land abgelöst, in das in der Bilanz mit -1.048 die meisten Asylsuchenden überstellt werden. Polen hat nicht nur die meisten ankommenden Netto-Überstellungen, sondern auch mit fast 20 % einen relativ hohen Anteil der Überstellungen an den gestellten Asylanträgen. Das Vereinigte Königreich überstellte in der Bilanz die meisten Asylsuchenden an andere Mitgliedstaaten. 5.3.1 Start der Verhandlungen um Dublin III Mit der Evaluation von Dublin II durch die Kommission startete der politische Prozess um die Überarbeitung der Dublin-Verordnung zu Dublin III. Wie beschrieben war die Kommission trotz aller Probleme in ihrer Evaluation der Meinung, dass Dublin II »zufriedenstellend angewandt« (Europäische Kommission 2007a, 5 Kämpfe um Gestaltung: Geschichte Dublins S. 6) wurde und die Durchführung der Dublin-Verfahren »funktioniert« (ebd., S. 6). Diesem positiven Urteil widersprach das Europäische Parlament: Im Ausschuss für bürgerliche Freiheiten, Justiz und Inneres, dem sogenannten LIBEAusschuss, wurde am 25. Juni 2008 mit 49 Ja-Stimmen, einer Nein-Stimme und einer Enthaltung (vgl. Europäisches Parlament 2008, S. 16) ein Bericht angenommen, der das Dublin System in seiner damaligen Form als unbefriedigend unter technischen und humanitären Gesichtspunkten und sowohl für Asylsuchende als auch Mitgliedstaaten ungerecht beschreibt (vgl. ebd., S. 6). Neben verschiedenen Forderungen, die im Wesentlichen auf eine verstärkte »Lastenverteilung« zwischen den Mitgliedstaaten und einen verbesserten Schutz der betroffenen Asylsuchenden ausgerichtet sind, enthält der Bericht des Parlaments die Forderung nach einem Aussetzungsmechanismus für Überstellungen in Dublin: »[Das Europäische Parlament] ersucht die Kommission, verbindliche Mechanismen vorzusehen, die die Überstellung von Asylbewerbern in Mitgliedstaaten verhindern, welche keine umfassende und faire Bearbeitung ihres Antrags garantieren, und systematisch Maßnahmen gegen diese Staaten zu ergreifen;« (Ebd., S. 7) Diese Forderung des Parlaments steht im Kontext einer beginnenden europaweiten Kampagne verschiedener NGOs gegen Überstellungen von Asylsuchenden nach Griechenland (siehe Kapitel 5.3.2). Der eigentliche Gesetzgebungsprozess startete mit dem Entwurf der Kommission für Dublin III im Dezember 2008 (Europäische Kommission 2008). Im Vorfeld konsultierte die Kommission neben Repräsentant*innen der Mitgliedstaaten auch Sachverständige des UNHCR, von NGOs, sowie Rechtsanwält*innen, Richter*innen und Parlamentarier*innen des Europaparlaments. Die Kommission stellt die Ergebnisse dieser Konsultationen folgendermaßen dar: Die Mehrzahl der Mitgliedstaaten würden die Grundprinzipien der Verordnung schätzen, wollten allerdings die Leistungsfähigkeit des Systems verbessern. Der größte Teil der NGOs und der UNHCR würden die Grundprinzipien des aktuellen Systems ablehnen und ein System bevorzugen, welches die Zuständigkeit auf der Basis des Ortes des ersten Asylantrags verteilt. In Anbetracht der für diese Forderung ungünstigen politischen Kräfteverhältnisse würden diese Akteur*innen dazu aufrufen, den Schutz der Asylsuchenden in der neuen Verordnung zu stärken. Letzterer Forderung würde sich das Europäische Parlament anschließen. Ihr eigener Entwurf für Dublin III trage laut Kommission all diesen verschiedenen Forderungen Rechnung – an der Grundstruktur von Dublin II solle festgehalten werden und gleichzeitig solle die Leistungsfähig des Systems und der Schutz der Asylsuchenden verstärkt werden. (vgl. ebd., S. 5) Wie vom Europaparlament gefordert schlägt die Kommission ein Verfahren vor, mit dem Überstellungen in einen Mitgliedstaat zeitweise ausgesetzt werden können: 89 90 David Lorenz: Die umkämpfte Dublin-Verordnung »Um zu vermeiden, dass Mitgliedstaaten mit begrenzten Aufnahme- und Absorptionskapazitäten, die einem besonderen Druck ausgesetzt sind, durch Überstellungen im Rahmen des Dublin-Systems zusätzlich belastet werden, wird ein neues Verfahren eingeführt, mit dem Überstellungen in den zuständigen Mitgliedstaat auf der Grundlage des Dublin-Verfahrens ausgesetzt werden können. Von diesem Verfahren kann auch in Fällen Gebrauch gemacht werden, in denen Bedenken bestehen, dass die Überstellung zur Folge hat, dass einem Antragsteller im zuständigen Mitgliedstaat kein angemessenes Schutzniveau, insbesondere im Hinblick auf Aufnahmebedingungen und Zugang zum Asylverfahren, geboten wird.« (Ebd., S. 9) Artikel 31 des Entwurfs für Dublin III regelte den Aussetzungsmechanismus. Er sah vor, dass Mitgliedstaaten in Notsituationen einen Antrag auf temporäre Aussetzung der Überstellungen an die Kommission stellen können. Im Falle einer Aussetzung auf der Grundlage eines nicht angemessenen Schutzniveaus für Asylsuchende hätte entweder ein anderer Mitgliedstaat einen Antrag an die Kommission richten oder die Kommission eigeninitiativ tätig werden können. Die Kommission würde daraufhin eine Entscheidung bezüglich der Aussetzung der Überstellungen treffen, die innerhalb eines Monats durch eine anderslautende Entscheidung mit qualifizierter Mehrheit des Rates korrigiert werden könnte. Die Dauer der Aussetzung der Überstellungen hätte einen Zeitraum von sechs Monaten nicht überschreiten sollen und hätte um weitere sechs Monate verlängert werden können. Die Zuständigkeit für die betroffenen Asylverfahren wäre dann auf die Mitgliedsstaaten übergegangen, in denen sich die entsprechenden Asylsuchenden aufhalten. (vgl. ebd., S. 50–52) Auf den Entwurf der Kommission reagierte das Parlament am 29.04.2009 mit einem Bericht, der den Entwurf der Kommission unterstützte. Es fügte allerdings im Entwurf für Artikel 31, den Aussetzungsmechanismus, einen neuen Absatz ein. In diesem wird ein System zur Umverteilung anerkannter Flüchtlinge zwischen den Mitgliedstaaten gefordert: »ein System zur Umverteilung von Personen, die Anspruch auf internationalen Schutz haben, von Mitgliedstaaten, die einer spezifischen und unverhältnismäßigen Belastung ausgesetzt sind, an andere Mitgliedstaaten im Benehmen mit dem Amt des Hohen Flüchtlingskommissars der Vereinten Nationen, wobei gewährleistet wird, dass die Umverteilung nach nichtdiskriminierenden, transparenten und eindeutigen Regeln erfolgt.« (Europäisches Parlament 2009, S. 26–27) Ein Umverteilungsmechanismus für anerkannte Asylsuchende war bis dahin nicht in der Dublin-Verordnung geregelt worden. Damit konkretisierte das Parlament seine Forderung aus dem Bericht von 2008 (Europäisches Parlament 2008) nach einer »Lastenteilung« unter den Mitgliedstaaten und ergänzte damit den »Aussetzungsmechanismus« um einen sogenannten »Solidaritätsmechanismus«. Die 5 Kämpfe um Gestaltung: Geschichte Dublins Stimmung im Parlament im Hinblick auf die Dublin-Verordnung beschrieb eine für das Parlament an den Verhandlungen beteiligte Person als kritisch: »I think if you would have a vote in the parliament on whether or not to have the Dublin system or to go for something else, I think you would have a majority for something else to stop the Dublin system because it is very, very criticised in the parliament. I’m not sure there would be a majority but it would be almost fiftyfifty. But the Dublin system is very criticised within the parliament.« (Interview mit einem Mitglied des Europaparlaments, Verhandlungsgruppe Dublin III, 2012) Das ist insofern erstaunlich, als im Europaparlament nach der Europawahl 2004 die rechtskonservativen Fraktionen stärker vertreten waren als linke. Die christlichkonservative EVP war mit 268 von insgesamt 732 Sitzen mit Abstand die stärkste Fraktion und gleichzeitig diejenige Fraktion, deren Verhandlungsposition der konservativen Position des Rates am nächsten kam. Die Verhandlungsposition der rechten Parteien war durch eine Spaltung geschwächt: Die Positionen der konservativen Abgeordneten der Zielstaaten und derjenigen der Kernstaaten waren entgegengesetzt – griechische EVP Abgeordnete hatten zum Beispiel eine sehr andere Position zu Dublin als deutsche EVP Abgeordnete. Den einflussreichen Posten des Schattenberichterstatters der EVP Fraktion für die Verhandlungen um Dublin III besetzte ein griechischer Abgeordneter. Dies hatte Folgen für die Dynamik innerhalb der Gruppe der Berichterstatterin und Schattenberichterstatter*innen: »For example, on Dublin we had a Greek shadow from the EVP group, and his positions where not very much in line with those of the German delegation within the EVP, that is the big delegation. So it is up to the shadow to control, sort of speak on behalf of the group and not just for his own interests. But there you could see some conflicts sometimes. The fact that he was the shadow made the group of shadows coming closer together, because his position was closer to the one of the Liberals, the SPE, the Greens and the GUE because were in favour of this solidarity system. And he of course – because of the Greek perspective, they are having these problems. But that made it a bit difficult for him to get the rest of his group on board. And I think in many of the other asylum files we have a German EVP shadow and they can see that it is really a difference between the EVP group on the one hand and the other groups on the other hand.« (Ebd.) Abgesehen von dieser Spaltung der konservativen Fraktion wurden in Hintergrundgesprächen oder Interviews weitere Erklärungsansätze angedeutet. So wurde in einem Interview betont, dass die Parlamentarier*innen im Europaparlament im Gegensatz zu den Akteur*innen des Rates eine eher europäische als nationalstaatliche Perspektive einnehmen und sich damit auch sehr weit von den Positionen ihrer Kolleg*innen in den nationalen Parlamenten oder Regierungen entfernen können (vgl. 91 92 David Lorenz: Die umkämpfte Dublin-Verordnung ebd.). Zweitens wurde der zuständige Innenausschuss des Parlaments als – für die Verhältnisse des Parlaments – linksliberal geprägt beschrieben (vgl. Interview mit einem Mitglied des Europaparlaments, LIBE-Ausschuss, 2012). Drittens wurde in einem Hintergrundgespräch mit einer Mitarbeiterin einer Parlamentarierin der liberalen Parlamentsfraktion die Rolle von Expert*innen für die Arbeit der Parlamentarier*innen betont. Während der Rat auf die Expertise der Beamt*innen der nationalen Verwaltungen zurückgreifen könne, sei das Parlament auf die Expertise externer Expert*innen angewiesen – und diese Rolle wurde im Fall der Dublin Verhandlung in erster Linie durch die Expert*innen und Lobbyist*innen der linksliberalen NGOs im Asylbereich ausgefüllt, nicht nur für die linken Fraktionen des Europaparlaments. Vor diesem Hintergrund lässt sich vielleicht auch die ungewöhnliche Einigkeit verstehen, die in Bezug auf Dublin zwischen den Fraktionen der Liberalen, Sozialdemokrat*innen, Grünen und Linken (vgl. Interview mit einem Mitglied des Europaparlaments, Verhandlungsgruppe Dublin III, 2012) bestand. Während das Parlament den Vorschlag der Kommission unterstützte war der Rat skeptisch. Eine für die deutsche Delegation an den Verhandlungen im Rat beteiligte Person sagte dazu auf meine Frage, welche Punkte der Rat abgelehnt habe: »Ein Punkt war der sogenannte Aussetzungsmechanismus. […] Die große Mehrheit der Mitgliedsstaaten hat diesen Vorschlag nicht mitgetragen, und es war auch einer der Punkte, die verschiedentlich auf politischer Ebene von den Ministern selber isoliert verhandelt wurden. Das EP hat dagegen den KOM-Vorschlag mehrheitlich unterstützt, das EP hatte relativ schnell seine Stellungnahme abgegeben, so dass erkennbar war, wie die Positionierung des EP ist. Aber der Rat hat den Aussetzungsmechanismus abgelehnt und dann musste dann ausgelotet werden, wie es weitergeht.« (Interview mit einem Mitarbeiter des Bundesinnenministeriums (BMI), deutsche Verhandlungsdelegation zu Dublin III im Rat der EU, 2012) In Bezug auf Dublin war der Rat gespalten. Auf der einen Seite standen die Delegationen der eher reicheren Staaten im Kern der EU und an den westlichen und nördlichen Rändern, die tendenziell das Ziel von Binnenmigration in der EU waren. Zu dieser Gruppe zählten laut Interviews und Verhandlungsdokumenten zumindest die Delegationen aus Österreich, Belgien, Deutschland, Frankreich, Großbritannien, den Niederlanden, Dänemark, Finnland und Schweden (vgl. Interview mit einem Mitglied des Europaparlaments, LIBE-Ausschuss, 2012) (BMI 2012). Auf der anderen Seite standen die Delegationen einiger Transitstaaten an den südlichen Grenzen, die sich nicht mit der Rolle als Türsteher*innen des Schengenraums zufrieden geben wollten. Dies waren die Delegationen aus zumindest Zypern, Griechenland, Italien, Spanien und Malta (vgl. Interview mit einem Mitglied des Europaparlaments, LIBE-Ausschuss, 2012) (vgl. Innenminister*innen von Zypern, Griechenland, Italien und Malta 2009; Innenminister*innen von Zypern, Griechenland, 5 Kämpfe um Gestaltung: Geschichte Dublins Italien, Malta und Spanien 2011). Die Zielstaaten konnten sich in allen zentralen Punkten gegen die Transitstaaten durchsetzen und damit die Verhandlungsposition des Rates prägen. Das ohnehin bestehende Machtgefälle zwischen diesen beiden Staatengruppen wurde durch die parallel stattfindenden Eurokrisenprozesse verschärft. Vier der fünf Transitstaaten waren deshalb von Krediten und Bürgschaften der anderen Mitgliedstaaten, der EU und des IWF abhängig. Der Rat entschied im Fall der Verhandlungen um Dublin III mit qualifizierter Mehrheit: Für eine Annahme brauchte es mindestens eine einfache Mehrheit der Mitgliedstaaten und gleichzeitig eine Mehrheit von mindestens 255 der insgesamt 345 Stimmen im Rat. Keine der beiden Gruppen konnte eine einfache Mehrheit der Mitgliedstaaten verhindern. Die Gruppe der Zielstaaten, die gegen den Aussetzungsmechanismus verhandelten, hatte allerdings insgesamt 132 Stimmen – Österreich 10, Belgien 12, Deutschland 29, Frankreich 29, Großbritannien 29, die Niederlande 13 und Schweden 10. Die kleinere Gruppe der oppositionellen Transitstaaten kam dagegen gemeinsam nur auf 75 Stimmen – Italien 29, Spanien 27, Griechenland 12, Zypern 4 und Malta 3. In den Verhandlungen hätte dementsprechend die Gruppe der Zielstaaten eine qualifizierte Mehrheit verhindern können, die Gruppe der Transitstaaten nicht.10 Dazu kam, dass die Zielstaaten die Grundzüge des Status Quo verteidigten, also im Zweifelsfall den Aussetzungsmechanismus auch durch ein Scheitern der Verhandlungen und eine fortgesetzte Anwendung von Dublin II hätten verhindern können. Demgegenüber waren die Transitstaaten für die Durchsetzung ihrer Verhandlungsziele auf die Zustimmung einer Reihe von anderen Staaten und auf einen erfolgreichen Abschluss der Verhandlungen angewiesen.11 In dieser Konstellation, in der auf der einen Seite die Kommission mit dem Parlament für einen Aussetzungsmechanismus und der Rat, trotz seiner inneren Spaltung, gegen den Aussetzungsmechanismus verhandelten, zogen sich die Verhandlungen bis Ende 2010 hin, ohne dass die verschiedenen Positionen sich annäherten oder eine Einigung in Sicht gewesen wäre. Etwa zwei Jahre nach dem Entwurf der Kommission waren die Verhandlungen festgefahren. Oder, in den Worten eines Interviewpartners: »In dem Fall bedeutete das tatsächlich eine Änderung an dem Dublin-System vorzunehmen, indem man diesen Notfall-Mechanismus einbauen wollte, […] mit dem man dann die Transfers aussetzen kann. Das war von der Kommission von Anfang an unterstützt worden eigentlich. Das Parlament hat das auch 10 11 Diese Stimmgewichtung entspricht dem Entscheidungsverfahren nach qualifizierter Mehrheit vor 2017, also auf Basis des Vertrags von Nizza in der EU-27. Für eine ausführlichere Analyse der politischen Konstellation im Rat siehe Lorenz (2013, S. 71–82). 93 94 David Lorenz: Die umkämpfte Dublin-Verordnung unterstützt, der Rat hatte da ein Problem und das ist natürlich der Kernpunkt gewesen und auch von vorneherein der Punkt, wo die Verhandlungen gestartet sind, aufeinandergeprallt und eingefroren.« (Interview mit einem Mitglied des Europaparlaments, LIBE-Ausschuss, 2012) 5.3.2 Rechtskämpfe um Überstellungen nach Griechenland Der im Kapitel zum Dubliner Übereinkommen geschilderte Rechtsstreit um effektiven Rechtsschutz gegen Überstellungen spitzte sich in Deutschland unter Dublin II wegen der Situation in Griechenland zu. Die griechische Verwaltungspraxis bot viele Ansatzpunkte für Klagen gegen Dublin-Bescheide. So wurde 2005 ein Bericht über die Auswirkungen eines griechischen Gesetzes veröffentlicht, welches es den Behörden erlaubte, ein Asylverfahren einzustellen, wenn die asylsuchende Person eigenmächtig den ihr zugewiesenen Wohnort verlässt (Papadimitriou und Papageorgiou 2005, S. 308). Diese Regelung wurde von den griechischen Asylbehörden dazu genutzt, in Griechenland eröffnete Asylverfahren von Personen einzustellen, die nach der Eröffnung des Verfahrens in andere Dublin-Staaten reisten. Nach der Überstellung zurück nach Griechenland hatten diese Personen dann keinen legalen Aufenthaltstitel und waren von einer Abschiebung in ihren Herkunftsstaat bedroht, ohne dass ihr Asylverfahren in einem der Mitgliedstaaten jemals geprüft worden wäre (ebd., S. 310). Die Situation des griechischen Asylsystems verschärfte sich in den darauf folgenden Jahren immer weiter. Während der ersten Jahre der globalen Finanzkrise ab 2007 stieg die ohnehin schon hohe Staatsverschuldung Griechenlands und der griechischen Staat stürzte in eine Staatsschuldenkrise. Um einen Staatsbankrott abzuwenden beantragte der damalige griechische Ministerpräsident Papandreou im April 2010 Finanzhilfe. Diese wurden einen Monat später nach Verhandlungen mit der Troika genannten Trias aus IWF, EU und EZB unter der Auflage einer strengen Austeritätspolitik gewährt. Zeitgleich zu dieser Staatsschuldenkrise, in der die Politik Griechenlands in weiten Teilen gemäß den Interessen und entsprechend den Forderungen der Gläubigerinstitutionen gestaltet wurde (vgl. Kotzias 2016), entwickelte sich Griechenland zum Tor nach Europa für illegalisierte Migration. Die Haupteinreiserouten in die EU verschoben sich aufgrund der grenzpolitischen Kooperationen von Spanien mit Senegal und Mauretanien sowie von Italien mit Libyen vom zentralen Mittelmeer zum östlichen Mittelmeer. Die neue Haupteinreiseroute in die EU führte von der Türkei nach Griechenland (vgl. Frontex 2010, S. 12). 2009 wurden 75 % der »irregulären Grenzübertritte« in die EU an den griechischen Grenzen festgestellt, Ende 2010 waren es 90 % (vgl. European Union Agency for Fundamental Rights 2011, S. 12). 2010 reisten Asylsuchende, die ohne die Möglichkeit der legalen Einreise auf dem Weg in das Territorium der EU waren, fast immer über Griechenland. Von dort setzten sie ihre Reise laut Analysen von Frontex
https://openalex.org/W2052535981
https://www.scielo.br/j/rac/a/KVwWXfp7KNTbGmjrvkVsVBC/?lang=pt&format=pdf
Portuguese
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Fotografias como um recurso de pesquisa em marketing: o uso de métodos visuais no estudo de organizações de serviços
RAC. Revista de Administração Contemporânea
2,007
cc-by
9,602
RESUMO Argumenta-se que a adoção de métodos visuais de coleta e análise de dados em pesquisas de marketing de serviços oferece riqueza de informações aos estudos na área, pois o uso de fotos (ou vídeos) pode proporcionar objetividade e credibilidade, na medida em que uma imagem fornece prova tangível da existência de determinado fenômeno. Para ilustrar o uso dos métodos visuais, foram selecionadas e analisadas fotos de um hospital e de um hotel, extraídas dos sites das empresas com o objetivo de verificar como o gerenciamento de impressões leva um hospital a usar estratégias para intencionalmente se parecer com um hotel. Para dar respaldo ao trabalho, utilizou-se como base teórica os métodos visuais na pesquisa, o papel do cenário nos encontros de serviços, o gerenciamento de impressões e o marketing de serviços. E, visando demonstrar a possibilidade de uso da metodologia em discussão, apesar das possíveis dificuldades da transposição dos métodos visuais, que são adotados na Sociologia e na Antropologia, acredita-se que a discussão metodológica e a ilustração do uso das técnicas de coleta e análise de dados irão contribuir para o enriquecimento do debate e para a busca do aprimoramento das ferramentas metodológicas em administração e, especificamente, em marketing. Palavras-chave: pesquisa; métodos visuais; marketing de serviços; cenários de serviços. Fotografias Como um Recurso de Pesquisa em Marketing: o Uso de Métodos Visuais no Estudo de Organizaçőes de Serviços Fotografias Como um Recurso de Pesquisa em Marketing: o Uso de Métodos Visuais no Estudo de Organizaçőes de Serviços J. Ricardo C. de Mendonça Maria de Lourdes de Azevedo Barbosa André Falcão Durão ABSTRACT It is argued that the visual methods of data gathering and analysis in service marketing research yield a wealth of valuable information, as the use of pictures (or videos) imparts objectivity and credibility to the researchers’ findings. In this article, pictures of a hospital and of a hotel were taken from their official web sites, and subsequently analyzed, to show how the recourse to impression management by both the hospital and the hotel has led the former to intentionally reproduce some features of the latter. The theoretical basis of the paper lies on the use of the visual methods; on the emphasis on the role of the scenery in service production, and on the literature on impression management and service marketing. In spite of the possible difficulties involved in the use of visual methods in marketing research, (and not only in Sociology and Anthropology, as is usually done), the contention is made that this article contributes to the enrichment of the debate, and for the quest for improvement of methodological tools in management and, specifically, marketing. Key words: research; visual methods; service marketing; service set. 57 RAC, v. 11, n. 3, Jul./Set. 2007: 57-81 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão INTRODUÇĂO A administração, desde os seus primórdios, vem beneficiando-se de perspectivas e metodologias de outras áreas da ciência como, por exemplo, a sociologia, a antropologia, a ciência política, a psicologia. Entretanto várias abordagens e métodos dessas disciplinas permanecem desconhecidos ou pouco utilizados nos estudos em administração. Uma dentre essas abordagens utiliza os métodos visuais para coleta e análise de dados da vida social. Os métodos visuais têm suas origens na Sociologia e na Antropologia. Harper (1988) coloca que a abordagem da Sociologia Visual contribuiu para muitos estudos nas décadas iniciais da sociologia americana, mas desapareceu e ressurgiu durante a década de sessenta. Para o autor “é justo dizer que de 1920 a 1960 não existiu Sociologia Visual” (p. 58). Segundo Flick (2004), a discussão sobre uma sociologia visual, centrada em torno de fotografias e de filmes, é recente. Entretanto para a Antropologia a inclusão de fotos e filmes em estudos é mais significativa (Harper, 1988). Tanto é que Lindlof e Taylor (2002) chamam a atenção para o fato de que fotografias, filmes e vídeos têm sido utilizados como ferramentas comuns na coleta de dados na Antropologia há mais de 70 anos, o que é confirmado por Gagliardi (2002), quando coloca que a antropologia visual faz uso etnográfico do filme ou das fotografias para registrar, por meio de imagens, fenômenos culturais. A abordagem visual é um enfoque metodológico em que o pesquisador ao estudar um fenômeno social, utiliza as imagens como fonte de dados, sejam elas estáticas (fotografias) ou em movimento (filmes e vídeos). Como método de pesquisa, a abordagem visual leva em consideração aspectos como a subjetividade do indivíduo que registra as imagens, bem como aquilo que ele escolheu incluir ou excluir da fotografia que foi capturada. Harper (1988) salienta o crescente reconhecimento do potencial dos métodos visuais para estudos tanto em laboratório, quanto em ambientes naturais (estudos de campo). Caulfield (1996) também ressalta o interesse dos cientistas sociais pelo método visual e a diversidade das imagens visuais. De acordo com Caulfield (1996, p. 56) os dois principais interesses da abordagem visual são “imagens produzidas pelo pesquisador (ou seus sujeitos) no curso de um estudo de campo (...) e imagens produzidas pelos atores sociais no contexto da vida cotidiana”. A capacidade da imagem fotográfica de conter a informação de maneira 58 RAC, v. 11, n. 3, Jul./Set. INTRODUÇĂO 2007 Fotografias Como um Recurso de Pesquisa em Marketing econômica e confiável torna possível uma catalogação mais eficiente de dados nas pesquisas. Porém os métodos visuais são pouco adotados nos estudos em administração e raramente têm sido usados em pesquisas qualitativas em marketing. A pouca utilização do método nos estudos em administração pode ser constatada por meio da colocação de Gagliardi (2002, p. 128), que afirma: “O estudo dos artefatos e dos espaços coorporativos emergiu recentemente como uma das mais interessantes correntes dentro da abordagem geral das organizações entendidas como cultura”. Seguindo essa perspectiva, o marketing de serviços, especificamente, pode valer-se do registro das evidências físicas para o envio de mensagens fortes que ajudam a estabelecer a imagem(1) da empresa e a afetar as expectativas dos consumidores (Hutton & Richardson, 1995). Nesse caso, o cenário aparece como um dos elementos que influenciam a percepção do público em relação aos encontros de serviços, aspecto que é referendado por Hutton e Richardson (1995), quando apontam que os elementos tangíveis costumam ser citados pelos consumidores com muito mais freqüência do que os demais atributos determinantes da satisfação e da qualidade do serviço prestado. A adoção de métodos visuais de coleta e análise de dados em pesquisas de marketing de serviços, bem como a inclusão das imagens coletadas nos resultados dos estudos oferece grande riqueza de informações, pois se acredita que pode proporcionar objetividade e credibilidade, na medida em que uma imagem fornece a prova tangível da existência de determinado fenômeno. Assim, neste ensaio, são abordados os métodos visuais, a importância do ambiente físico nos encontros de serviços e mostra-se um caso-exemplo de como se pode adotar este método na pesquisa de marketing. Para tanto são utilizadas quatro fotos (capturadas via internet), duas de um hospital e duas de um hotel, que retratam os mesmos espaços (apartamento e fachada) encontrados nos dois tipos de empresa. OS MÉTODOS VISUAIS DE PESQUISA A pesquisa qualitativa, onde se enquadram os métodos visuais, apesar de ser adotada de forma regular e legítima por sociólogos e antropólogos há várias décadas, só começou a ganhar espaço na área da administração a partir de 1970 (Godoy, 1995). O termo ‘pesquisa qualitativa’ é conceito amplo, que cobre várias formas de investigação, porém o pressuposto filosófico-chave é que todos os tipos de pesquisa qualitativa se baseiam em uma visão da realidade como 59 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão socialmente construída pelos indivíduos em suas interações. Assim, as pesquisas qualitativas procuram entender e explicar o significado dos fenômenos sociais, causando o menor distúrbio possível no ambiente natural (Merriam, 1998). Nesse sentido, Collier (1973) defende que o registro fotográfico contribui como fator de controle para a observação visual direta e acrescenta: “As fotografias são registros preciosos da realidade material” (p.7). Em complementação, Caulfield (1996) afirma que as imagens apresentam interesse sociológico pelo menos por três razões: 1) as imagens refletem o mundo vivo e as relações sociais entre os participantes; 2) fotografias são, freqüentemente, elementos formativos da vida social; e 3) as imagens podem reter informações documentais sobre os seus sujeitos. Ao discutir as fotografias como instrumento e objeto de pesquisa, Flick (2004, p 161) destaca que “as fotografias, os filmes e o vídeo são cada vez mais utilizados como formas genuínas e fontes de dados”. Porém vale ressaltar a discussão de Harper (1988) sobre as razões que levam a Sociologia Visual e os métodos visuais a serem discriminados por muitos pesquisadores. Conforme o autor, isso ocorre precisamente porque qualquer um pode tirar fotografias; “os métodos sociológicos, as pessoas tendem a acreditar, devem ser misteriosos e difíceis. (...) É inaceitável para muitos que algo tão simples possa ser útil como uma ferramenta sociológica” (Harper, 1988, p. 60). Para Harper (1988) os sociólogos visuais sabem que tirar fotografias com importância teórica não é mais fácil do que realizar uma observação ou identificar variáveis significativas. Lindlof e Taylor (2002) afirmam que existem muitas características da mídia visual que apóiam o seu uso em pesquisas científicas. De acordo com Collier (1973), os seres humanos, por diversas razões, não são bons observadores. Assim, a grande capacidade de penetração da câmera fotográfica pode ajudar a ver mais e de forma mais acurada. OS MÉTODOS VISUAIS DE PESQUISA Com base em Flick (2004), algumas vantagens no uso de câmeras na pesquisa social podem ser apontadas: 1) as câmeras permitem registros (gravações) detalhados de fatos; 2) proporcionam apresentação mais abrangente e holística de estilos de vida e de condições sociais; 3) possibilitam o transporte de artefatos e a apresentação destes como retratos; 4) permitem a transgressão de limites de tempo e espaço; 5) podem capturar fatos e processos que sejam muito rápidos ou complexos para o olho humano; 6) permitem registros (gravações) não-reativas das observações; 7) são menos seletivas que as observações; e 8) as fotografias ficam à disposição de outras pessoas para serem reanalisadas. Collier (1973, p. 6) destaca que a câmera é um instrumento; “ela documenta mecanicamente, mas a sua mecânica não limita, necessariamente, a sensibilidade 60 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing do observador humano; ela é um instrumento que exige extrema seletividade”. Para Duffield (1998), os métodos visuais de pesquisa têm potencial para cristalizarem dados e alcançarem audiências, de um modo que nenhum outro método pode, pois praticamente todos os aspectos da vida humana usam o sentido da visão, ou seja, a capacidade de ver. Apesar da maior credibilidade conferida às imagens, o seu significado é negociável e subjetivo. Harper (2000) ressalta que, apesar de as imagens representarem dados empíricos, elas não representam “a verdade”, pois o próprio ato da observação escolhe um ponto de vista(2). Em pesquisas em que este método de coleta de dados é adotado, pode-se fazer uso de imagens preexistentes ou o pesquisador pode ele mesmo produzir as imagens que serão analisadas. Segundo Harper (1988) a Sociologia Visual apresenta várias abordagens, em que os pesquisadores fazem uso de fotografias para descrever ou analisar fenômenos sociais. Essas abordagens se subdividem em duas áreas principais: a) a primeira que utiliza a fotografia no sentido convencional de coleta de dados, sendo os métodos visuais normalmente adotados em trabalhos relacionados a um problema de pesquisa e a uma teoria específica; b) e a segunda, em que os sociólogos visuais utilizam fotos produzidas, ou seja, preexistentes na cultura estudada (Harper, 1988). Harper (1988) descreve ainda, uma categorização de modos para a utilização de métodos visuais em pesquisas: o modo científico; o modo narrativo; o modo reflexivo; e o modo fenomenológico. No modo científico, as fotos podem ser usadas em pesquisas empíricas de várias formas. OS MÉTODOS VISUAIS DE PESQUISA Diversas características de um fenômeno observável podem ser mais bem entendidas se “congeladas” em imagem fotográfica. As fotografias podem ser “lidas” no sentido de entender matizes da interação, da apresentação do self e as relações entre as pessoas e seu ambiente material. Além disso, como as imagens fotográficas isolam um momento no tempo, podem-se avaliar mudanças por meio de novas fotografias do mesmo fenômeno social ou de fenômeno similar. Ainda de acordo com Harper (1988), nas narrativas visuais(3), que são utilizadas no modo narrativo da abordagem visual, as fotos estáticas assumem papel similar às narrativas etnográficas em filme ou de etnografias, que são construídas com base na vida social como ela naturalmente se desenvolve; já o modo reflexivo exibe uma relação diferente entre o pesquisador, a imagem e o sujeito da pesquisa. Nas palavras de Harper (1988, p. 65): No método fotográfico reflexivo, o sujeito compartilha a definição dos significados; deste modo as definições são “refletidas de volta(4)” a partir do sujeito. Apesar disso ser difícil de fazer em uma pesquisa convencional, é mais fácil fazê-lo por meio de métodos fotográficos. O pesquisador pode começar da mesma maneira, ou seja, fotografando um sujeito ou um ambiente; mas 61 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão então ele retorna ao sujeito para a definição do significado da imagem. Nesse processo, que tem sido chamada de “elicitação fotográfica”(5), os papéis do pesquisador e do sujeitos são alterados. Por fim, o modo fenomenológico, sugere que fotografias podem expressar equivalência espiritual ou psicológica. Para Harper (1988, p. 66) fotografias podem literalmente descrever, mas nos deixam imóveis; outras imagens podem inspirar nossas emoções, mas não ser úteis sociologicamente (ou até falsas). Algumas fotos, entretanto, fazem o oposto; ou seja, comunicam insights sociológicos de uma maneira artisticamente estimulante. Segundo o autor a abordagem fenomenológica é, em essência, a elicitação do próprio conhecimento de alguém por meio da análise de fotografias que tenham significado pessoal. Além dos aspectos mencionados, deve-se considerar também, ao se discutir atualmente o uso dos métodos visuais, a tecnologia digital. E para ilustrar as vantagens tecnológicas do uso das modernas câmeras digitais, Lindlof e Taylor (2002, p. OS MÉTODOS VISUAIS DE PESQUISA 115) destacam que as máquinas fotográficas e câmeras de vídeo digitais apresentam vantagens em relação às máquinas fotográficas tradicionais, pois são extremamente compactas e leves; funcionam silenciosamente; fotografam em situações de baixa iluminação e não requerem processos caros; são facilmente carregadas em arquivos de computador, onde podem ser editadas e manipuladas das mais variadas formas, combinadas com outras mídias para colocação em sites na Web e eletronicamente compartilhadas com usuários distantes. A ilustração da utilização do método visual neste artigo é composta por fotos de duas organizações, retiradas de sites na web - um Hospital e um Hotel - que são caracterizados por Zeithaml e Bitner (2003) como organizações de serviços interpessoais, em que o uso do cenário de serviço ocorre tanto por parte dos clientes, quanto por parte dos funcionários. Em ambos os tipos de organização, a complexidade do cenário de serviço é considerada elaborada. Nesse contexto, acredita-se ser necessária e apropriada uma maior discussão sobre o cenário de serviços para o atendimento do objetivo proposto neste ensaio. O PAPEL DO CENÁRIO NOS ENCONTROS DE SERVIÇOS Um encontro de serviço é um período de tempo durante o qual os empregados e clientes de uma organização interagem diretamente (Lovelock & Wright, 2001). Vale destacar que esta interação não se limita à dimensão interpessoal, mas engloba 62 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing todos os aspectos da empresa com os quais o consumidor pode relacionar-se, incluindo, além do fator pessoal, as facilidades físicas e outros elementos visíveis (Grove & Fisk, 1989). Reafirmando que os encontros de serviços não se limitam às relações entre pessoas, Gagliardi (2002, p. 135) coloca que “em uma organização, fins são perseguidos, energias são investidas e idéias são concretizadas em máquinas, produtos e lugares”. O autor evidencia ainda que nas organizações contemporâneas os espaços físicos estão vinculados a artefatos, pois as organizações, em geral, são receptivas aos recursos que possam melhorar a sua eficiência. Para alguns tipos de serviços, a evidência física(6) ou o cenário é parte vital do sistema de prestação do serviço, porque, dentre todas as variáveis à disposição da organização, os elementos tangíveis do serviço são potencialmente aqueles que podem ser mais prontamente controlados pelos gestores (Hutton & Richardson, 1995). Ao discutirem as evidências físicas dos serviços, Zeithaml e Bitner (2003, p. 233) apontam que “os elementos genéricos de evidência física incluem todos os aspectos das instalações físicas da organização (cenários), bem como outras formas de comunicação tangíveis, quer sejam atributos exteriores quer sejam atributos interiores”. Bitner (1992) apresenta contribuições importantes para a compreensão dos sistemas de prestação de serviços, explorando o conceito de Servicescape, que diz respeito aos ambientes sociais e físicos que envolvem experiência, transação ou evento de serviço. Inicialmente, o modelo Servicescape baseou-se exclusivamente na noção de ambiente físico. Contudo, considerando- se que as pessoas em um ambiente podem influenciar as noções relacionadas ao espaço, o ambiente social foi inserido no modelo (Zeithaml & Bitner, 2003). Analisando as dimensões ambientais do Servicescape, os fatores que constituem o ambiente físico referem-se às condições ambientais (temperatura, barulho, odor, música, dentre outros), espaço/funcionalidade (layout, equipamentos, mobília etc.) e sinais, símbolos e artefatos: sinalização interna, decoração, uniforme dos funcionários etc. Já os elementos do ambiente social envolvem as interações sociais que ocorrem entre funcionários da empresa, clientes e qualquer outra pessoa que esteja presente no mesmo ambiente físico. O PAPEL DO CENÁRIO NOS ENCONTROS DE SERVIÇOS O conjunto de elementos ambientais irá determinar as respostas ou reações cognitivas, emocionais e físicas, tanto de clientes quanto de funcionários da empresa. Aqui, cabe destacar a noção de que as dimensões do ambiente de uma organização afetam tanto os comportamentos relacionados aos clientes, quanto aqueles relacionados aos funcionários da organização (Bitner, 1992). Gagliardi (2002) aponta a importância do estudo dos artefatos para o estudo das organizações como sistemas de significados simbólicos. Segundo o autor, os artefatos, constituem fenômeno cultural primário que influencia a vida 63 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão organizacional de duas formas: os artefatos tornam materialmente possível favorecer, obstruir ou mesmo prescrever a ação organizacional; e os artefatos, de forma mais geral, podem influenciar a percepção da realidade, chegando até a delinear crenças, normas e valores culturais. Além disso, “símbolos são concretizações dos sentidos e as coisas constituem seu domicílio mais usual e natural” (Gagliardi, 2002, p. 132). O cenário de serviço afeta a percepção e o comportamento dos clientes e, conseqüentemente, sua satisfação e avaliação da qualidade; por isso é importante conhecer quais as características do ambiente de serviços que podem influenciar a percepção dos consumidores. Nesse sentido, Zeithaml e Bitner (2003) identificaram quatro tipos de papéis desempenhados pelos cenários de serviços: embalagem; facilitador; socializador; e diferenciador. A embalagem do cenário é aquilo que uma organização mostra para o exterior e, por isso, pode ser fundamental na formação das impressões(7) iniciais ou para a construção das expectativas dos clientes (...)” (ênfase adicionada), fornecendo às organizações a oportunidade de transmitir determinada imagem para seu público-alvo por meio da configuração do cenário apresentado. A forma como o cenário é projetado, segundo Zeithaml e Bitner (2003) (layout e arquitetura), pode ser um aspecto facilitador do processo de serviço e da interação do prestador de serviço como consumidor. Portanto o cenário deve ser planejado de acordo com o tipo de serviço a ser prestado e com as operações a serem executadas e podem ser melhorados quando o cenário de serviços é projetado com tal enfoque. Nas palavras de Zeithaml e Bitner (2003, p. 237), “o projeto do cenário de serviços auxilia na socialização tanto de funcionários quanto de clientes, no sentido de que colabora para formar papéis, comportamentos e relacionamentos desejados”. O PAPEL DO CENÁRIO NOS ENCONTROS DE SERVIÇOS Os aspectos físicos do palco onde será desempenhado o serviço proporcionam ‘pistas’ para os atores envolvidos no encontro de serviço (funcionários e clientes) de como eles devem comportar-se nas interações. Por fim, o cenário também pode ser caracterizado como tendo papel diferenciador para a organização de serviços, quando o seu projeto apresenta características distintas daquelas de seus concorrentes. Além disso, o cenário pode comunicar exatamente qual o segmento de mercado para o qual o serviço está direcionado, bem como para reposicionar a organização de serviços, buscando novos nichos de mercado (Zeithaml & Bitner, 2003). Berry (1981), Gronroos (1984) e Lovelock e Wright (2001) consideram tanto o contexto social quanto o contexto físico das trocas no serviço como aspectos fundamentais. Reforçando esse pensamento, Parasuraman, Zeithaml e Berry 64 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing (1988) evidenciam que das 10 dimensões identificadas por eles como determinantes de qualidade de serviços, a maioria relaciona-se a aspectos ligados ao cenário social e ao cenário físico do serviço. (1988) evidenciam que das 10 dimensões identificadas por eles como determinantes de qualidade de serviços, a maioria relaciona-se a aspectos ligados ao cenário social e ao cenário físico do serviço. Os aspectos do cenário, quando bem gerenciados, podem potenciar todo o processo de serviço, agregando valores à sua execução, os quais afetarão de forma favorável o comportamento dos empregados e dos consumidores. Assim, acredita-se que os métodos visuais podem oferecer importantes insights para o estudo dos cenários em serviços. Nesse artigo, serão considerados para a análise os elementos das instalações interiores e exteriores dos cenários de serviço, especificamente aqueles que podem comunicar aspectos do serviço por meio da observação dos dados visuais selecionados. Vergara, Carvalho e Gomes (2004, p. 16), ao discutirem a espacialidade do teatro das organizações, argumentam que: “nos palcos teatrais, o desenvolvimento de iluminação cenográfica consolidou o controle do olhar do espectador. Analogamente, nas organizações, as ‘luzes’ se acendem para a produção, canalizando a atenção do indivíduo para os resultados organizacionais”. Observa-se que, ao discutir as interações dos funcionários e seus clientes na organização de serviços como um teatro, parte-se para um enfoque dramatúrgico, que é aceito na literatura especializada em marketing de serviços. Essa perspectiva leva à discussão sobre a relação entre o gerenciamento de impressões (GI), o marketing de serviços e a metáfora dramatúrgica. O Gerenciamento de Impressőes e o Marketing de Serviços Percebe-se crescente utilização, na literatura de marketing de serviços, da metáfora teatral, no sentido de entender e explicar os encontros de serviços. Um dos principais focos de análise da dramaturgia é como as pessoas criam e gerenciam impressões diante de uma audiência (Grove & Fisk, 1989). A partir do enfoque de Goffman (1959), autores como Grove e Fisk (1989) e Grove, Fisk e Dorsch (1998) apontam que o teatro (os elementos da dramaturgia) passa a ser utilizado como ferramenta analítica para se estudarem os encontros de serviços. Sob esta perspectiva, os prestadores de serviços, como os atores em uma peça de teatro, apresentam elementos comuns no desempenho de seus papéis. Tanto atores, quanto os prestadores de serviços devem procurar criar e manter impressões(8) favoráveis nas suas audiências, movimentando-se por cenário que é montado em palco, contando com ambiente de bastidores para lhes dar suporte e buscando, tal como o elenco de um espetáculo de teatro, o ‘aplauso’ (a satisfação) do seu público (Carvalho, 1998). 65 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão A perspectiva de dramaturgia se baseia na visão do comportamento como metáfora dramática e retrata as interações sociais como teatrais, oferecendo o meio de analisar e descrever os comportamentos. Um dos principais focos de análise da dramaturgia é como as pessoas criam e gerenciam impressões diante de uma audiência (Grove & Fisk, 1989). Conforme Goffman (1959), o gerenciamento de impressões (GI) envolve a tentativa de estabelecer o significado ou o propósito de interações sociais, que dirigirão as ações das pessoas, ajudando a projetar as expectativas de papéis, ou seja, o que se espera de cada um. De acordo com Alvesson (1990), o objetivo do ‘gerenciamento da imagem’ é produzir um ‘retrato’ (uma ‘descrição’), com certo apelo, de uma organização para vários públicos - empregados, consumidores, acionistas, governo etc. - e posicioná-lo de um modo positivo. A imagem é escolhida por alguém dentro de um conceito específico, definindo alvo e significado para a ação, de forma a marcar o ‘emissor’ que tenta projetar determinada impressão para audiência (Alvesson, 1990). Mendonça (2003), de forma mais ampla, define o GI como todas as atividades que um ator social (indivíduo, grupo ou organização) desenvolve no sentido de influenciar o modo como outros o vêem, ou seja, a impressão ou a imagem que desenvolvem ao seu respeito. O Gerenciamento de Impressőes e o Marketing de Serviços O estudo do GI, de acordo com Ornstein (1989), tem se focado principalmente em comportamentos verbais; entretanto a exploração de comportamentos não- verbais - tais como aqueles exibidos por meio da decoração de ambientes físicos - devem ser considerados. Grove, Fisk e Bitner (1992, como citado em Grove, Fisk, & Dorsch, 1998), apresentam um modelo da experiência do serviço como drama, incorporando quatro componentes teatrais: (1) os atores, ou o pessoal do serviço cujos comportamentos e presença ajudam a determinar o serviço; (2) a audiência, ou os consumidores que são os receptores do serviço; (3) o cenário, ou o ambiente físico em que o serviço ocorre; e (4) a performance, ou a entrega do serviço. Para Goffman (1959, p.15) a performance é “toda a atividade de um determinado participante, em uma dada ocasião, que serve para influenciar, de algum modo, quaisquer dos outros participantes”. Conforme Gardner e Martinko (1988), no processo de gerenciamento de impressões, o ambiente proporciona o cenário e o contexto em que os atores realizarão suas performances para suas audiências. RAC, v. 11, n. 3, Jul./Set. 2007 66 Fotografias Como um Recurso de Pesquisa em Marketing ILUSTRAÇĂO DO USO DE MÉTODOS VISUAIS NO ESTUDO EM MARKETING DE SERVIÇOS ILUSTRAÇĂO DO USO DE MÉTODOS VISUAIS NO ESTUDO EM MARKETING DE SERVIÇOS Na realização desta análise de imagem com o objetivo de identificar o seu significado dentro de uma representação social, precisou-se estar atento para as condições que permitiram a imagem representar o objeto, porque, antes de proceder à análise de uma foto, é necessário, verificar os vários sentidos que ela é capaz de propiciar. Essas várias leituras, conhecidas como o caráter polissêmico da imagem, não caracteriza que uma foto possui vários sentidos, mas que seu sentido explícito cria classes de correspondências que permitem múltiplas interpretações. Essa característica é fruto da relação existente entre a imagem, o objeto e o observador. Assim é que se procurou o sentido e a interpretação dos pesquisadores envolvidos neste estudo, para verificar como o gerenciamento de impressões leva o hospital, intencionalmente, a se parecer com o hotel, investindo nos elementos físicos de seus cenários de serviços para criar apelo visual atrativo para os consumidores. Considera-se neste trabalho que a fotografia é a interpretação de determinado recorte, sendo um relato da empresa sobre alguns cenários de serviços (apartamento e fachada) e fruto das representações de um indivíduo ou de um grupo. Foram selecionadas para esse caso-exemplo duas fotos de hospital e duas de hotel, extraídas dos sites das empresas, que foram escolhidas por apresentarem similaridades de posicionamento de produto/serviço e por estarem na mesma categoria, considerada luxo ou ‘cinco estrelas’, estimando-se as duas como empresas do ramo da hospitalidade. Para ilustrar os procedimentos adotados na análise de imagens paradas foram escolhidas fotos de dois cenários físicos comuns às duas empresas: apartamento (ambiente interno) e fachada (ambiente externo). A partir da análise das fotos, procurou-se verificar os aspectos comuns e diferenciados existentes nas imagens dos ambientes selecionados, tomando-se por base os itens constantes da Tabela 1 (4), e identificar em quais deles o hospital está teatralizando sua oferta de forma a se parecer com hotel. RAC, v. 11, n. 3, Jul./Set. 2007 67 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão Tabela 1 (4): Critérios Adotados Para a Análise das Condições Ambientais Fonte: baseado em Zeithaml e Bitner (2000), Hoffman e Bateson (2003). Fonte: baseado em Zeithaml e Bitner (2000), Hoffman e Bateson (2003). A análise foi realizada por três avaliadores com perfis diferenciados para oferecer maior consistência aos dados. ILUSTRAÇĂO DO USO DE MÉTODOS VISUAIS NO ESTUDO EM MARKETING DE SERVIÇOS Um com experiência profissional na área de hospitalidade, outro especialista na área de organizações e um terceiro pesquisador da área de marketing. O processo de análise constou de três etapas. 1) Primeira etapa: cada pesquisador, isoladamente, realizou a análise das fotos; 2) Segunda etapa: os três pesquisadores, em conjunto, estabeleceram comparações entre os elementos identificados por cada um deles na etapa anterior, formando o quadro básico que contemplou os aspectos comuns e diferenciados encontrados; 3) Terceira etapa: partiu-se para uma análise confirmatória das informações obtidas por meio das imagens, realizada de forma conjunta, a partir do quadro extraído da segunda etapa da análise, para verificar quais eram os elementos que permitiam identificar aspectos do cenário físico do hospital que possuíam uma identidade com os dos hotéis para, finalmente, levantar proposições sobre elementos do gerenciamento de impressões que vem sendo utilizado pelos hospitais como estratégia mercadológica para atrair e reter clientes. A Análise Grande parte da literatura recente tem analisado a mudança do eixo competitivo relacionado ao advento dos serviços agregados aos produtos e à melhoria da imagem da empresa na busca da criação de valor junto aos seus clientes. É nessa direção que os hospitais parecem estar redesenhando estratégias que vêm sendo empregadas para torná-los mais atrativos aos olhos dos clientes e do mercado. Uma das bases desse novo desenho indica uma ligação entre a gestão de saúde e a gestão hoteleira, resultando em um campo mais amplo denominado de 68 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing hospitalidade. Assim sendo, um estudo comparativo entre imagens pareceu adequado e interessante para captar esses aspectos. As fotos foram analisadas considerando-se as semelhanças com o seu referente, em que a imagem adquire uma relação como o objeto de sua representação por meio dos diversos sentidos incorporados. Assim é que, para efeito do caso ilustrativo desenvolvido neste estudo, se optou por abordar a imagem no plano da representação (o que ela mostra), no plano de conteúdo (o que ela significa) e no plano do significante (a realidade exterior a que ela faz referência). Ou seja, buscou-se com este caso-exemplo identificar as semelhanças ou diferenças com a realidade exterior a que a imagem remete. A tentativa de gerenciar impressões por meio do cenário nos hospitais, com a intenção de fazê-los parecer-se com hotéis, está em função das relações de serviços em um hospital ocorrerem nas fases que se desenvolve um tratamento médico, que incluem diversos relacionamentos entre setores e entre pessoas, caracterizando, principalmente, um contato pessoal de alta complexidade que inclui a relação paciente/médico, bem como do pessoal de enfermagem, fisioterapeutas, nutricionistas, psicólogos e outros funcionários, como porteiros e recepcionistas, até o pessoal de limpeza e higiene. Além desses aspectos, mais do que em qualquer outra organização, os funcionários de contato se defrontam com pessoas fragilizadas pela doença e em processo de regressão psicológica, bem como familiares sofridos e desgastados (Gonçalves, 1998). Na tentativa de criar ambiente mais atrativo para gerenciar os efeitos negativos dos aspectos anteriormente citados, os hospitais estão buscando apoio na indústria hoteleira. Ou seja, algumas estratégias de gerenciamento de impressões começam a ser incorporadas aos serviços hospitalares. Tal estratégia mercadológica já vinha sendo empregada pelos hotéis, na medida em que sua função foi sempre a de vender ‘sonhos’ e ‘experiências agradáveis’. A Análise Feitas essas ressalvas, cada um, dentre os critérios estabelecidos na Tabela 1 (4), mereceu algumas considerações, de modo que a análise levou aos seguintes resultados. Ambiente Interno São analisados neste tópico os ambientes internos das organizações (conforme as Figuras 1.4 e 2.4) e de acordo com quadro de referência constante na Tabela 1.4 e a teoria que dá suporte a este estudo. 69 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão Apartamento ra 1(4): Quarto do Hospital - http://www.hospitalaleinstein.com.b Figura 2(4): Quarto do Hotel - http://www.novotel.com.br Figura 1(4): Quarto do Hospital - http://www.hospitalaleinstein.com.br Figura 2(4): Quarto do Hotel - http://www.novotel.com.br Arquitetura Interna: Apartamento Arquitetura Interna: Apartamento Arquitetura Interna: Apartamento Os aspectos relacionados à arquitetura interna do apartamento permitem ao cliente comparar o que é ofertado pelas empresas. Na medida em que a competição aumenta em uma determinada área do setor de serviços, oportunidades para diferenciar a empresa tornam-se cada vez mais difíceis de encontrar (Bateson & Hoffman, 2001). A evidência física e o tratamento dado aos encontros de serviços podem exercer impacto direto sobre os consumidores, estabelecendo uma diferenciação no serviço prestado. Verificando-se o apartamento do hospital, percebe-se uma transformação de sua concepção. Antes frios e brancos; hoje com cortinas coloridas para dar vida ao ambiente; as paredes em tons pastéis para dar tranqüilidade e sensação de aconchego, o que faz com que os apartamentos do hotel e do hospital sejam bastante semelhantes. Esse aspecto refere-se ao papel de embalagem do cenário, Zeithaml e Bitner (2003, p. 236), ou seja, o cenário de serviços (apartamento) é aquilo que o hospital e o hotel mostram para o exterior e, por isso, é fundamental na formação das impressões(9) iniciais ou para a construção das expectativas dos clientes. Os elementos da evidência física do serviço permitem transmitir determinada imagem para seu público-alvo por meio da configuração do cenário apresentado. Analisando a arquitetura interna dos apartamentos (do hospital e do hotel), observa-se um traço específico que caracteriza a diferença entre os serviços de 70 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing saúde e os serviços hoteleiros, a cama hospitalar, que tem design específico para esse tipo de atendimento. Essa característica, entretanto, não impede que a aparência geral dos dois apartamentos seja semelhante, pois os espaços, as cores e o restante do mobiliário apresentam aspectos similares. Até mesmo as cortinas já estão sendo utilizadas em hospitais, o que constituía restrição comum em alguns deles por questões de higiene e saúde. Verifica-se então, uma aproximação entre os aspectos físicos do apartamento de hospital na direção do ambiente encontrado no quarto de hotel. Layout: Apartamento Em virtude de que os ambientes de serviços são constituídos para atender às necessidades e interesses específicos dos clientes, o layout do espaço tem importância fundamental. A relevância do layout das instalações é especialmente evidente nos cenários de serviços, o que pode influenciar a satisfação dos clientes, o desempenho da empresa e o comportamento de busca de alternativa pelo consumidor (Zeithaml & Bitner, 2003). Exemplos nesse sentido são encontrados, no caso dos hotéis, em que investimentos em apartamentos para deficientes físicos ou estruturas específicas para clientes da terceira idade, enquanto os hospitais apresentam layout próprio para a movimentação de pacientes, médicos e enfermeiros, mas com respeito e valorização do aspecto estético e aparência geral do apartamento. Existe um layout padrão nos apartamentos, em que os móveis (camas e acessórios) estão dispostos de modo a oferecer espaço amplo e adequado à categoria dos produtos/serviços ofertados, permitindo no hospital e no hotel uma boa circulação de pessoas amplitude e conforto, até mesmo no caso de haver acompanhante, em ambos os casos. Iluminação: Apartamento Comparando-se o espaço do teatro com o das organizações, verifica-se que no teatro a iluminação do cenário é capaz de controlar o olhar do espectador. O mesmo pode ocorrer nas organizações, cujas luzes podem ser trabalhadas a favor da produção do serviço, focando a atenção do consumidor para os resultados dos encontros de serviços (Vergara, Carvalho, & Gomes, 2004). Os apartamentos, ambos com iluminação direta e indireta, possuem finalidades específicas. No caso do hospital, o uso de iluminação indireta próxima à cama para possibilitar cuidados com o paciente durante a noite, sem o incômodo da luz direta. Para o hotel esse tipo de iluminação facilita a leitura individual ou alguma outra necessidade do hóspede. A composição da luz na montagem dos dois cenários é semelhante na concepção. Móveis e Equipamentos: Apartamento A evidência física é parte vital do sistema de prestação do serviço, tanto que Hutton e Richardson (1995) colocam que, dentre todas as variáveis à disposição da organização, os elementos tangíveis do serviço são potencialmente aqueles que podem ser mais prontamente controlados pelos gestores. Considerando-se também que os espaços físicos estão vinculados a artefatos nas organizações contemporâneas, essas são receptivas aos recursos que possam melhorar a sua eficiência (Gagliardi, 2002) e a escolha dos móveis e equipamentos, bem assim a sua disposição no ambiente podem influenciar o resultado dos serviços. Os móveis guardam semelhanças na qualidade percebida das peças, design e 71 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão tipo de material, harmonia da localização em função da necessidade de cada espaço e das cores, em combinação com o conjunto da decoração. As imagens transmitem bem-estar, conforto, requinte, beleza, harmonia de espaços e cores apropriadas, com a finalidade de tentar criar uma imagem de qualidade e cuidado com os clientes. Cores: Apartamento As cores, suas tonalidades e combinações geram reações emocionais e comportamentos, às vezes inesperados, afetando, por exemplo, o humor das pessoas (Zeithaml & Bitner, 2003). No caso do apartamento do hospital, percebe- se uma transformação de concepção, com a inclusão de cortinas coloridas, para dar vida ao ambiente e as paredes em tons pastéis para dar tranqüilidade e sensação agradável, o que deve colaborar na recuperação do paciente. No caso do hotel, os tons claros nas paredes e detalhes de decoração com cores diferenciadas (quadros e colcha) parecem indicar harmonia e beleza. Esse tipo de mudança caracteriza o que Thomas (2002) definiu como movimento holístico da saúde, que estabelece o importante papel do meio ambiente no processo terapêutico. Essa perspectiva leva a constatar a adoção, por algumas empresas de saúde, de conceitos trazidos da área de hotelaria. Sinais, Símbolos e Objetos: Apartamento Sinais, Símbolos e Objetos: Apartamento O estudo dos artefatos é importante para as organizações como sistemas de significados simbólicos, pois estes constituem fenômeno cultural primário que 72 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing influencia a vida organizacional de duas formas: tornam materialmente possível favorecer, obstruir ou mesmo prescrever a ação organizacional; e, de forma mais geral, podem influenciar a percepção da realidade, chegando até a delinear crenças, normas e valores culturais. Além disso, “símbolos são concretizações dos sentidos e as coisas constituem seu domicílio mais usual e natural” (Gagliardi, 2002, p. 132). Nesse sentido, os sinais expostos no interior de uma estrutura funcionam como comunicador explícito. Podem ser usados como etiquetas (ex. nome da empresa, nome do departamento), para orientação (ex. saída, entrada, toilletes) e para comunicar as regras de comportamento (ex. não fumar, silêncio). Entretanto os símbolos e objetos do ambiente também têm papel fundamental para os usuários dos serviços de saúde e de hotelaria, pois fornecem indicativos explícitos aos usuários acerca do significado do local e as normas e expectativas sobre o comportamento nesse local. Materiais de qualidade usados na construção, obras de arte, tapetes e certificados expostos podem comunicar significados simbólicos e criar uma impressão estética genérica (Zeithaml & Bitner, 2003), o que pode ser constatado nas fotos analisadas. RAC, v. 11, n. 3, Jul./Set. 2007 Ambiente Externo 73 RAC, v. 11, n. 3, Jul./Set. 2007 Figura 3(4): Fachada do Hospital - http://www.hospitalaleinstein.com.br Figura 4(4): Fachada do Hotel - http://www.novotel.com.br Arquitetura Externa: Fachada O desafio de muitas empresas no momento de ofertar serviços é o de planejar o espaço e a evidência física, de modo que possam sustentar as necessidades e Figura 3(4): Fachada do Hospital - http://www.hospitalaleinstein.com.br Figura 3(4): Fachada do Hospital - http://www.hospitalaleinstein.com.br Figura 4(4): Fachada do Hotel - http://www.novotel.com.br Arquitetura Externa: Fachada Figura 4(4): Fachada do Hotel - http://www.novotel.com.br Figura 4(4): Fachada do Hotel - http://www.novotel.com.br Arquitetura Externa: Fachada Arquitetura Externa: Fachada Arquitetura Externa: Fachada O desafio de muitas empresas no momento de ofertar serviços é o de planejar o espaço e a evidência física, de modo que possam sustentar as necessidades e 73 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão preferências do consumidor. Por meio da aparência externa de um prédio, por exemplo, o cliente pode avaliar o posicionamento da empresa, o tipo de serviço e o valor a ser pago pelo que é ofertado. O cenário é aquilo que uma organização mostra para o exterior e, por isso, pode ser fundamental nas impressões iniciais ou para a construção das expectativas dos clientes – trata-se de uma metáfora visual para um serviço intangível. Uma instalação bem projetada ou mal projetada do ponto de vista do cliente pode ser uma experiência prazerosa ou não (Zeithaml & Bitner, 2003). Os dois prédios apresentam similaridades na sua estrutura, por terem características de modernidade, materiais com novas tecnologias de construção (vidros arredondados, pinturas especiais). Com formas arquitetônicas diferenciadas, mas elaboradas, ambos buscam transmitir idéia de organização, limpeza, beleza e sobriedade. Os prédios são imponentes e com traços elaborados, o que anteriormente constituía característica própria dos hotéis e está sendo adotada pelos hospitais para gerenciar impressões. Vale salientar que cenários de serviços podem ser interpretados como forma de comunicação não-verbal da empresa, transmitindo, por meio sua características físicas, significados específicos (Zeithaml & Bitner, 2003). Nesse caso, as fotos (3.4 e 4.4) transmitem imagens de locais sofisticados, podendo ser considerados como prédios de luxo e, se fosse realizada a análise das fotos das duas fachadas sem a identificação das empresas, provavelmente não seria possível ao observador distinguir qual, dentre elas, é efetivamente hospital ou hotel. Sinais, Símbolos e Objetos: Fachada Em determinado ambiente, os símbolos, sinais e objetos podem transmitir significados de forma direta ou indireta aos clientes, sendo aspectos fundamentais na formação das impressões sobre o serviço. Esses critérios fornecem comunicadores explícitos, como a entrada de um local, que tem o propósito de orientar o cliente no ambiente a que se dirige. Oferecem também indicativos implícitos relacionados à qualidade do material utilizado na construção das instalações, as cores, os móveis e o layout que transmitem mensagens que agregam valor na percepção dos clientes acerca do serviço que estão comprando (Zeithaml & Bitner, 2003). As características arquitetônicas dos dois prédios propiciam sinais de organização, manutenção eficiente, higiene, cuidados com a aparência e com os jardins, verificando-se claramente a relação entre a embalagem para o produto e o cenário externo para os serviços, pois as características relacionadas aos símbolos, sinais e objetos permitem ao cliente formar pela imagem da “parte de fora” o que deve estar dentro da empresa (Zeithaml & Bitner, 2003). Vale destacar que não se observou a existência de diferenças extremas entre os cenários analisados, ratificando a utilização de elementos do gerenciamento de impressões. Paisagismo: Fachada Com base em Edwards e Shackley (1992), pode-se argumentar que os jardins externos, comunicam significados simbólicos e criam impressão estética genérica da empresa Pelas fotos verifica-se que o hotel e o hospital realizaram investimentos semelhantes no aspecto paisagístico, criando harmonia entre a estrutura dos prédios e espaços disponíveis, oferecendo aspecto mais humanizado ao ambiente. Cores: Fachada As cores utilizadas nas fachadas dos dois prédios se assemelham pela tonalidade clara utilizada na pintura. Isto permite considerar que os matizes de cores utilizadas nas áreas externas do hospital, também observadas no hotel, buscam descaracterizar o aspecto mais austero, neutro e sem atrativos, que, ao longo dos tempos, foi sendo associado aos hospitais. De acordo com os psicólogos ambientais, tanto cenários naturais quanto aqueles criados pelo homem ocasionam reações emocionais (Mintz, 1956; Maslow & Mintz, 1956 como citado em Turley & Milliman, 2000). Pode-se, portanto, argumentar que as cores utilizadas exercem função tão importante para a criação de imagem desses dois tipos de negócios quanto a estrutura e o estilo arquitetônico dos edifícios. 74 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing Tamanho do Prédio: Fachada Os clientes freqüentemente procuram indicadores do ambiente que os auxiliem a qualificar o local para, então, formar suas expectativas sobre a qualidade do serviço (Sharma & Stafford, 2000). Um destes indicadores é o tamanho do prédio, que fornece sinais de imponência, segurança, status, consolidação da empresa no mercado, dentre outros. Completada a análise, algumas considerações finais e proposições merecem ser feitas em relação ao tema proposto neste estudo, visando colaborar com futuros trabalhos voltados para a utilização dos métodos visuais na pesquisa em administração. Entrada: Fachada As entradas do hospital e do hotel apresentam similaridades quanto à aparência e forma, pois possibilitam uma visualização e identificação clara de sua utilidade, o que é fator importante, tanto para o acesso de pacientes como de hóspedes. As duas entradas sociais principais apresentam traços de arquitetura diferentes, mas não demonstram divergências aparentes quanto ao seu uso, o que pode levar o cliente a avaliar a fachada do hospital como a de um hotel, criando expectativas positivas sobre a empresa. 75 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão PROPOSIÇŐES EXTRAÍDAS A PARTIR DA ANÁLISE DAS IMAGENS Como resultado das análises realizadas, pôde-se chegar a algumas proposições relativas à questão da utilização do gerenciamento de impressões a partir dos cenários de serviços, como estratégia mercadológica para atrair e reter clientes. a) P1 Nas áreas comuns aos hospitais e restaurantes (apartamento e fachada) não se percebe diferenças marcantes na sua estrutura física e decoração. b) P2 Os hospitais estão utilizando ferramentas do gerenciamento de impressões para se tornarem mais parecidos com os hotéis aos olhos do cliente. c) P3 O investimento na estrutura física e decoração dos hospitais leva o consumidor a criar uma imagem mais positiva dos serviços de saúde. d) P4 O cenário de serviços é ferramenta importante para utilização pelos hospitais, como forma de oferecer aos clientes informações que lhe permitem fazer inferências sobre a qualidade do que é ofertado. e) P5 O cenário de serviços pode ser utilizado como estratégia de diferenciação entre as empresas de serviços de saúde. As proposições extraídas desta análise se caracterizam como insights para o aprofundamento de pesquisas na área de marketing de serviços e para maior utilização dos métodos visuais em estudos dessa natureza. Feitas essas colocações, parte-se para algumas considerações finais do trabalho mais voltadas para a utilização dos métodos visuais na pesquisa em administração. RAC, v. 11, n. 3, Jul./Set. 2007 76 Fotografias Como um Recurso de Pesquisa em Marketing CONSIDERAÇŐES FINAIS CONSIDERAÇŐES FINAIS As imagens analisadas permitiram verificar as similaridades e as divergências existentes entre dois ambientes de duas empresas de serviços, bem como extrair sinais e informações em quantidade e qualidade suficientes para chegar-se às proposições constantes neste trabalho. Essas proposições podem vir a contribuir para o entendimento relativo às mensagens que os cenários físicos transmitem, como proposição das empresas, e influenciam as imagens que são formadas pelos consumidores de serviços, principalmente levando-se em conta que as empresas empregam, nas suas mensagens visuais, táticas relativas ao gerenciamento de impressões. Isso demonstra que a utilização dos métodos visuais como forma de coleta e interpretação de dados, na pesquisa de administração, pode contribuir para o aprofundamento de questões relacionadas ao marketing de serviços, pois as organizações constituem sistemas de significados simbólicos e os seus artefatos acabam por influenciar as ações organizacionais e a percepção da realidade (Gagliardi, 2002). A concretização dos sentidos pode dar-se por meio da análise de imagens, considerando-se que todos os aspectos da vida humana usam o sentido da visão (Duffield, 1998) e, neste estudo, essa concretização ocorreu com a avaliação de fotografias dos cenários de duas empresas de serviços. As informações foram coletadas a partir de imagens estáticas que constituíram dados empíricos passíveis de seleção e interpretação, visto que o cenário, enquanto aspecto tangível do encontro de serviço, solicita profunda análise visual, já que exerce grande influência na percepção do cliente, pois a fotografia é considerada como registro precioso da realidade material (Collier, 1973). Nesse sentido, as empresas estão permanentemente tentando “gerenciar imagens” e produzir “retratos”, com alguns apelos aos seus vários públicos, para posicionar- se de modo positivo (Alvesson, 1990) e, portanto, comportamentos não verbais, como a decoração de ambiente, são fundamentais para alcançar esse objetivo. Com esse intuito, o marketing de serviços utiliza-se da metáfora teatral para entender e explicar os encontros de serviços, procurando verificar como as empresas criam e gerenciam impressões diante da audiência (Grove & Fisk, 1989). O cenário físico ganha importância nesse contexto, e a observação das imagens desse ambiente tem papel importante no aprofundamento da teoria relacionada a esse tema, bem como pode permitir maior desenvolvimento de metodologias, como a empregada neste trabalho com o uso de imagens. 77 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. Artigo recebido em 18.10.2004. Aprovado em 18.10.2005. Artigo recebido em 18.10.2004. Aprovado em 18.10.2005. CONSIDERAÇŐES FINAIS de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão Considerada como uma técnica de observação indireta da realidade social, o método visual, como qualquer outro método de coleta de dados, possui algumas limitações. Ao discutir o problema da observação e a natureza da fotografia, Collier (1973) aponta que uma das limitações da câmera são as próprias limitações de seu usuário. Ainda de acordo com o autor, enquanto os pesquisadores de campo não souberem o que fotografar, quando, quantas vezes e por que fotografar, a câmera não será vista como recurso funcional. Além disso, se os pesquisadores não dispuserem de recursos seguros para penetrar no conteúdo das fotografias, se não souberam o que é evidência de confiança e valor e o que é intangível e estritamente impressionístico, as fotografias não serão utilizadas de forma adequada. Apesar das possíveis dificuldades da transposição dos métodos visuais adotados na Sociologia e na Antropologia, acredita-se que a discussão metodológica e a ilustração do uso de métodos visuais de coleta e análise de dados realizadas neste ensaio contribuíram para o enriquecimento do debate e para a busca do aprimoramento das ferramentas metodológicas em administração, especialmente na área de marketing de serviços. NOTAS 1 O termo imagem utilizado no contexto em que foi citado significa um conceito ou conjunto de opiniões subjetivas de um indivíduo, do público ou de um grupo social, a respeito de pessoa, produto, empresa etc. 2 Para Collier (1973) as fotografias contêm um número suficiente de “verdades” não verbais, que permitem ao observador reconstruir a realidade esquemática e formar conceitos. 3 Conforme Harper (2000, p. 724) “para expandir a idéia de dados empíricos de uma única imagem para uma seqüência de fotos, foi introduzido o conceito de narrativa visual”. 3 Conforme Harper (2000, p. 724) “para expandir a idéia de dados empíricos de uma única imagem para uma seqüência de fotos, foi introduzido o conceito de narrativa visual”. 4 Da expressão original reflect back. 4 Da expressão original reflect back. 5 Da expressão original photo elicitation. 5 Da expressão original photo elicitation. 6 Características físicas que envolvem um serviço e ajudam os clientes a avaliar a prestação do serviço (Hoffman & Bateson, 2003). 6 Características físicas que envolvem um serviço e ajudam os clientes a avaliar a prestação do serviço (Hoffman & Bateson, 2003). 7 A formação de impressões ocorre quando as pessoas descodificam as mensagens e formam impressões e imagens sobre outras pessoas. 8 Idéia, sentimento ou opinião que o indivíduo tem sobre alguém ou algo, ou que alguém ou algo transmite ao indivíduo; pode ainda significar o efeito que uma experiência ou uma pessoa tem sobre alguém ou algo (baseado no Oxford – Advanced learner’s dictionary, 2000). 8 Idéia, sentimento ou opinião que o indivíduo tem sobre alguém ou algo, ou que alguém ou algo transmite ao indivíduo; pode ainda significar o efeito que uma experiência ou uma pessoa tem sobre alguém ou algo (baseado no Oxford – Advanced learner’s dictionary, 2000). 9 A formação de impressões ocorre quando as pessoas descodificam as mensagens e formam impressões e imagens sobre outras pessoas. 9 A formação de impressões ocorre quando as pessoas descodificam as mensagens e formam impressões e imagens sobre outras pessoas. 78 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing REFERĘNCIAS BIBLIOGRÁFICAS Duffield, K. 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Debates en Sociología N° 54, 2022, pp. 5-12 e-ISSN 2304-4584 Presentación: ¿Hacia nuevos contratos sociales para la conservación? Revelando las luchas por legitimidad en las Áreas Protegidas de América Latina Deborah Delgado Pugley1 Mattias Borg Rasmussen2 1 Pontificia Universidad Católica del Perú. Correo electrónico: deborah.delgado@pucp.pe https:// orcid.org/0000-0002-5595-0359 2 Universidad de Copenhague, Dinamarca. Correo electrónico: mbr@ifro.ku.dk https://orcid. org/0000-0002-1869-3525 5 Debates en Sociología N° 54, 2022 / e-ISSN 2304-4584 Vivimos en tiempos de emergencias socioambientales múltiples, en los cuales los umbrales de los sistemas ecológicos planetarios vienen siendo sobrepasados uno a uno. En este contexto, si bien la historia de la conservación no está libre de patrones de violencia y despojo, desde un punto de vista sociopolítico conducir esquemas de preservación de la naturaleza es indispensable para tener un futuro habitable en un mundo de estructuras moldeadas por las relaciones del capital. Este escenario nos presenta el reto de reconciliar los legados de la conservación y responder a la urgencia de encontrar salidas colectivas al aprieto actual. Este número especial nos llevan a preguntarnos cuáles son las posibilidades para articular nuevos contratos sociales de este orden, enfocándonos en las luchas por la legitimidad en las áreas protegidas de América Latina. La literatura sobre la conservación desde las ciencias sociales ha documentado los efectos adversos del régimen de Áreas Naturales Protegidas del mundo (West et al., 2006). Históricamente, la conservación como la conocemos en las Américas ha estado ligada a la conformación de grandes espacios estatales, manejados con una lógica vertical, regidos por un espíritu marcado por el legado colonial y capitalista. Sin embargo, la espacialidad de las iniciativas de conservación y los modos de gestionarla no son algo estático y menos monolítico. El modelo de conservación como fortaleza, dedicada exclusivamente a la protección de la vida «salvaje» y no humana, fue acuñado en los parques nacionales de los Estados Unidos hacia fines del siglo XIX (Taylor, 2016), para luego ser exportado hacia otros continentes. En el contexto norteamericano, los nativos americanos y los blancos tenían puntos de vista diferentes sobre el uso de la tierra y esto provocó muchos conflictos, así como la privación de derechos de los pueblos indígenas. La historia de la formación del movimiento ambiental demuestra la importancia de considerar como centrales las diferencias raciales, de género y de clase (Taylor, 2016). Así también lo demuestra la amplitud de su crítica y el surgimiento de paradigmas de conservación más recientes, como el manejo comunitario de los recursos naturales o los esquemas de pagos por servicios ambientales. Estas corrientes emergieron como intentos de remediar los problemas de ineficiencia en la gestión de la conservación, y, de forma más trascendente, de violación de derechos fundamentales sociales, culturales y humanos de los modelos anteriores. Las buenas intenciones y la incorporación de la crítica a experiencias pasadas, no obstante, presentan ya resultados mixtos. Usando criterios como sensibilización, efectividad en la gobernanza, legitimidad social y capacidad de movilización, los resultados son dispares. Si la conservación tiene un historial deficiente en términos de promoción de derechos fundamentales, en la actualidad enfrenta al menos dos desafíos sustanciales: las áreas protegidas se superponen en su mayoría a las economías de frontera, donde las instituciones públicas y privadas en competencia, el capital legal y las 6 Delgado Pugley y Borg Rasmussen / Presentación economías ilegales luchan por asegurar el acceso a cualquier recurso que tenga valor para ellos (Delgado, 2019; Hecht, 2005; Messina et al., 2006; Larsen, 2015; Norris, 2017; Wilson y Coomes, 2019). En segundo lugar, el proyecto de conservación es inherentemente un dilema espacial: se basa aún en imaginarios coloniales de sedentarismo y de límites estáticos (Adams, 2020; Noe, 2010). Al pretender establecer límites estables para situar lo que concibe como natural, transforma relaciones sociales que podrían ser más adecuadas de gestionar el territorio. Nuevos espacios y respuestas desde las ciencias sociales se abren frente a estos dilemas. La mirada crítica sobre la historia de la conservación sigue vigente, pero hay un giro hacia una mayor atención a la necesidad de crear regímenes e instituciones que velen por la preservación de la naturaleza. Vías para dar un lugar central a las miradas de los pueblos indígenas a problemáticas ambientales globales se van haciendo paso (Wilson et al., 2022). Se sintetizan y discuten aproximaciones que surgen de la práctica de la conservación a partir de la valoración de las prioridades y los activos y no de las carencias de los actores locales (Wali et al., 2017). En los últimos años, han surgido propuestas para la conservación de convivialidad, en particular desde los trabajos de Bram Buscher y Robert Fletcher (2020) y otras investigadoras e investigadores que trabajan de cerca en casos situados en Asia y África (Krauss, 2021). Estos autores proponen una tercera vía entre los intentos del neoconservacionismo de reservar cada vez más territorios, que tuvo su máxima expresión en la ambición del trabajo de O.E. Wilson (2016) llamado Half-Earth, y otras intervenciones conservacionistas basadas en lógicas del mercado capitalista, como los pagos por servicios ambientales o el REDD+. El problema —sostienen los autores— es que ambas soluciones reproducen relaciones coloniales y capitalistas. Para enfrentar esta situación, proponen que busquemos una conservación de convivialidad basada en principios de postcrecimiento y descolonización. Es una propuesta interesante e importante que también demanda contextualización y enraizamiento territorial. En particular, para el caso de América Latina, creemos que vale la pena explorar las diferentes formas y experiencias que ya se están implementando en los territorios de conservación (Paredes y Kaulard, 2021). Estamos interesados en las posibilidades de construir territorios de conservación enmarcados por lo que Astrid Ulloa (2011) llama «autonomías relacionales», pensando en las formas de gobernanza que surgen en los encuentros entre los proyectos de conservación y las autonomías enraizados en territorios vivos. Para Ulloa, debemos comprender los reclamos por autonomía o gobernanza en su contexto particular, considerando sus articulaciones específicas con instituciones estatales. Pensemos la conservación desde los territorios y a través de sus prácticas e interacciones cotidianas y las negociaciones entre diversos actores desde un análisis de su contexto particular. 7 Debates en Sociología N° 54, 2022 / e-ISSN 2304-4584 Los encuentros y desencuentros en torno a la conservación, así como su influencia en la reconfiguración de los territorios, producen debates, enfrentamientos y diálogos sobre el porqué, el quién y el cómo de las prácticas ambientales (Rasmussen, 2022). Como han señalado con mucha claridad los estudios empíricos sobre la conservación, los encuentros de conservación siempre son marcados por asimetrías de poder. Las autonomías relacionales como proceso implican reconocimiento mutuo, una articulación por un contrato social con lo provisorio que esto sea. Es ahí donde ubicamos nuestra preocupación por la legitimidad. Una conservación diferente, basada en las autonomías relacionales, requiere un entendimiento por los procesos de legitimación que autorizan el ejercicio de poder en el control de recursos y territorios. Este número especial de Debates en Sociología discute las posibilidades de tener nuevos acuerdos sociales en torno a la conservación en América Latina. El desafío que aborda es el de observar aquellas pistas que van más allá de la conservación como un proyecto dirigido por estados territoriales y definido de forma externa al territorio en que se impone. Examina las iniciativas de conservación en varios países de América Latina, donde comunidades locales y Pueblos Indígenas han interactuado con los regímenes de conservación, negociándolos y transformándolos mediante el establecimiento de reservas comunales, áreas protegidas privadas y áreas naturales de manejo integrado. En efecto, este número muestra lo difícil —pero posible— que es diferir de la conservación basada en ordenes impuestos de arriba hacia abajo, criticada a fondo en la literatura (Brosius et al., 2005). Esto es posible gracias a oportunidades cuyo potencial legal ha sido apropiado —y, en ocasiones, reformulado— por comunidades de la región amazónica y andina (Orihuela, 2020). Podría decirse que representa una renegociación de los términos de la conservación en la que las iniciativas de conservación están cada vez más dirigidas por la comunidad (Artelle et al., 2019). En este dossier se agrupan trabajos empíricos y teóricos que siguen diferentes metodologías y acercamientos conceptuales usando casos en varios países de América Latina. En esta colección indagamos acerca de diferentes formas de relacionarse con los proyectos territoriales de los sistemas de áreas naturales protegidas. Desde Colombia, Santiago Martínez-Medina, Hanne Cottyn, Ana María Garrido Corredor y Joshua Kirshner discuten de una manera innovadora e interdisciplinaria sobre la conservación a partir del caso de Chingaza, en Colombia. En su artículo, «Osos vaqueros en el páramo incomún: hacia una conservación cosmopolítica del oso andino en el páramo de Chingaza, Colombia», nos proponen una vía incomún para la conservación, confrontando a los expertos en el estudio y la conservación de este animal desde relatos de las comunidades campesinas que refieren la existencia de dos tipos de oso. 8 Delgado Pugley y Borg Rasmussen / Presentación Graciela María Preda, Mercedes Ejarque, Sofía Lammel y Florencia Pasetto, de Argentina, en su artículo «Entre la conservación y la producción: las familias crianceras del Área Natural Protegida El Tromen (Neuquén, Argentina)», traen un enfoque histórico para entender los usos y sentidos múltiples de territorios de conservación. Enfocándose en un grupo de crianceros con prácticas de trashumancia, las autoras reconstruyen sus trayectorias y las prácticas que los enraizan en estos territorios. El Área Natural Protegida El Tromen surge en este contexto como un actor importante para mediar sus relaciones con el entorno. Si bien la normativa no considera las actividades productivas, en las prácticas cotidianas se establecen vínculos y acuerdos para buscar compatibilizar los principios de conservación y de producción. El artículo muestra así la importancia de tener en cuenta las dinámicas de las poblaciones y sus trayectorias históricas en la formulación de políticas de conservación. Ana Watson y Conny Davidsen nos llevan a la intersección entre la agenda extractiva y la protección de áreas en territorios ancestrales de la Amazonía peruana. En su artículo «Conservation Utilitarianism and Indigenous Tactics: Protected Area and Extractive Agendas in the Peruvian Amazon», narran los efectos contradictorios del proyecto hidrocarburífero de Camisea. Muestran cómo las comunidades indígenas y sus líderes intentan navegar entre dos proyectos territoriales. Enfatizan las estrategias indígenas en contextos donde tanto la conservación como la extracción reproducen jerarquías de saberes y de poder de decisión. El artículo muestra con sensibilidad etnográfica cómo la conservación puede ser un aliado en luchas sociales por el territorio en arreglos que son siempre provisorios. Jean Paul Benavides, de la Universidad Católica de Bolivia, nos trae al caso de la comunidad del río Ivirgarzama, que forma parte del pueblo Yuracaré, y su interacción con pueblos indígenas colonos altoandinos dedicados a la producción de hoja de coca en sus territorios ancestrales. En su artículo «Territorios indígenas versus colonizadores campesinos en Bolivia. Estudio de caso de la Comunidad Indígena Yuracaré del río Ivirgarzama - CIYRI», busca fundamentar que el desconocimiento de los derechos reconocidos a los Pueblos Indígenas resulta contraproducente para la sostenibilidad de los bosques y recursos naturales. Esto sucede en un contexto en el cual la identidad indígena en general es reivindicada por el Estado. Muestra que la titulación de territorios no es una medida suficiente para garantizar los medios de vida indígenas y el bosque asociado de acuerdo con su propio proyecto colectivo y reflexiona sobre las perspectivas para la conservación en la Amazonía boliviana. Jorje Zalles discute las reservas de la biosfera desde la experiencia latinoamericana en un ensayo que nos ubica frente a los límites y posibilidades de esta forma de conservación, que fue una de los pioneras en reconocer la importancia de las sociedades aledañas para las reservas. En su ensayo «Territorios sustentables: legitimando 9 Debates en Sociología N° 54, 2022 / e-ISSN 2304-4584 la conservación en reservas de biosfera», demuestra cómo el concepto de reservas de biosfera ha sido adoptado con entusiasmo en América Latina y el Caribe. El artículo examina las condiciones bajo las cuales se evalúa el éxito de una reserva de biosfera y reflexiona sobre la aptitud de este modelo de conservación en la región. Cerramos el número especial con una entrevista a Fermín Chimatani Tayori, líder del pueblo Harakbut y presidente de la Asociación Nacional de Ejecutores de Contrato Administrativo para las Reservas Comunales - ANECAP del Perú. Podemos escuchar de primera mano la diferencia que existe entre un paradigma que prefiere dedicarse a la conservación y otro que ofrece alternativas económicas a los pueblos indígenas que de una u otra manera se ven involucrados en el trabajo intenso que implica la conservación. *** Adicional al dossier de este número especial, contamos con dos artículos agrupados en la sección Varia de la revista. Ambos giran en torno a estudios de sexualidad y género y están centrados específicamente en relaciones homosexuales de España y Argentina. El artículo «Llegó la ruptura: analizando los divorcios de matrimonios del mismo sexo en España», de los autores Diego Becerril, José Jiménez-Cabello, José Luis Paniza e Inmaculada Puertas, ofrece una mirada poco frecuente y muy necesaria a los matrimonios del mismo sexo. Eso por encontrarse al reverso de discursos de aceptación y legalización de la diversidad. Finalmente, el artículo de Maximiliano Marentes, «Seducir, levantar, estabilizar: amor y política entre varones gays argentinos» analiza la intersección entre trayectorias amorosas y políticas, encontrando una profunda imbricación entre ambas esferas. Referencias Adams, W.M. (2020). Geographies of conservation III: Nature’s spaces. Progress in Human Geography, 44(4), 789-801. https://doi.org/10.1177%2F0309132519837779 Artelle, K.A., Zurba, M., Bhattacharyya, J., Chan, D. E., Brown, K., Housty, J. y Moola, F. (2019). Supporting resurgent Indigenous-led governance: A nascent mechanism for just and effective conservation. Biological Conservation (240), 108284. https://doi. org/10.1016/j.biocon.2019.108284 Brosius, P.J., Lowenhaupt Tsing, A. y Zerner, C. (eds.) (2005). Communities and conservation: histories and politics of community-based natural resource management. Altamira Press. Buscher, B. y Fletcher, R. (2020). The conservation revolution: radical ideas for saving nature beyond the Anthropocene. Verso Trade. 10 Delgado Pugley y Borg Rasmussen / Presentación Delgado, D. (2018). Rights, justice, and REDD+: Lessons from climate advocacy and early implementation in the Amazon Basin. En Duyck S., Jodoin S., Johl A. (eds.), Routledge Handbook of Human Rights and Climate Governance (pp. 183-198). Routledge. Krauss, J.E. (2021). Decolonizing, conviviality and convivial conservation: towards a convivial SDG 15, life on land? Journal of Political Ecology, 28(1). https://doi. org/10.2458/jpe.3008 Hecht, S.B. (2005). Soybeans, development and conservation on the Amazon frontier. Development and Change, 36(2), 375-404. https://doi. org/10.1111/j.0012-155X.2005.00415.x Larsen, P. (2015). Post-frontier resource governance: indigenous rights, extraction and conservation in the Peruvian Amazon. Springer. Messina, J.P., Walsh, S.J., Mena, C.F. y Delamater, P. L. (2006). Land tenure and deforestation patterns in the Ecuadorian Amazon: Conflicts in land conservation in frontier settings. Applied Geography, 26(2),113-128. https://doi.org/10.1016/j. apgeog.2005.11.003 Noe, C. (2010). Spatiality and «borderlessness» in transfrontier conservation areas. South African Geographical Journal, 92(2),144-159. https://hdl.handle.net/10520/ EJC93345 Norris, T.B. (2017). Shared Social License: Mining and Conservation in the Peruvian Andes. Antipode, 49(3), 721-741. https://doi.org/10.1111/anti.12300. Orihuela, J. C. (2020). Embedded Countermovements: The Forging of Protected Areas and Native Communities in the Peruvian Amazon. New Political Economy, 25(1),140155. https://doi.org/10.1080/13563467.2019.1570101. Paredes, M. y Kaulard, A. (2020). Fighting the climate crisis in persistently unequal land regimes: Natural Protected Areas in the Peruvian Amazon. Journal of Cleaner Production (265), 121605. https://doi.org/10.1016/j.jclepro.2020.121605 Rasmussen, M.B. (2022). Convivencia negociada y gobernanza ambiental en áreas naturales protegidas del Perú. Íconos. Revista de Ciencias Sociales (72), 161-183. https:// doi.org/10.17141/iconos.72.2022.4953 Taylor, D.E. (2016). The Rise of the American Conservation Movement. Duke University Press. https://doi.org/10.1515/9780822373971 Ulloa, A. (2011). The politics of autonomy of indigenous peoples of the Sierra Nevada de Santa Marta, Colombia: A process of relational indigenous autonomy. Latin American and Caribbean Ethnic Studies, 6(1), 79-107. https://doi.org/10.1080/17 442222.2011.543874 Wali, A., Alvira, D., Tallman P., Ravikumar, A. y Macedo, M. (2017). A new approach to conservation: using community empowerment for sustainable well-being. Ecology and Society, 22(4), 6. https://doi.org/10.5751/ES-09598-220406 West, P., Igoe, J. y Brockington, D. (2006). Parks and peoples: the social impact of protected areas. Annual Review of Anthropology, 35, 251-277. https://doi.org/10.1146/ annurev.anthro.35.081705.123308 11 Debates en Sociología N° 54, 2022 / e-ISSN 2304-4584 Wilson, E.O. (2016). Half-earth: our planet’s fight for life. WW Norton & Company. Wilson, S.J. y Coomes, O. T. (2019). «Crisis restoration» in post-frontier tropical environments: Replanting cloud forests in the Ecuadorian Andes. Journal of Rural Studies, 67, 152-165. https://doi.org/10.1016/j.jrurstud.2019.02.023 Wilson, N.J., Lira, M.G. y O’Hanlon, G. (2022). A systematic scoping review of Indigenous governance concepts in the climate governance literature. Climatic Change, 171, 32. https://doi.org/10.1007/s10584-022-03354-7 12
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Effects of self-reported sensitivity and road-traffic noise levels on the immune system
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RESEARCH ARTICLE Editor: Markus M. Bachschmid, Boston University, UNITED STATES Received: April 4, 2017 Accepted: October 15, 2017 Published: October 30, 2017 Copyright: © 2017 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are included within the paper and its Supporting Information files. Funding: This study was funded by the Korea Ministry of Environment (MOE) as “the Environmental Health Action Program” to CSS and JHS received funding from Korea Ministry of Environment, grant number: 2014001350001 (URL: http://www.me.go.kr/home/web/main.do). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Ahra Kim1, Joo Hyun Sung2, Jin-Hee Bang1, Seung Woo Cho3, Jiho Lee3, Chang Sun Sim3* Ahra Kim1, Joo Hyun Sung2, Jin-Hee Bang1, Seung Woo Cho3, Jiho Lee3, Chang Sun Sim3* 1 Environmental Health Center, University of Ulsan College of Medicine, Ulsan, Republic of Korea, 2 Department of Occupational and Environmental Medicine, Gyeongsang National University Changwon Hospital, Gyeongsang National University School of Medicine, Changwon, Republic of Korea, 3 Department of Occupational and Environmental Medicine, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * zzz0202@naver.com * zzz0202@naver.com Abstract Sensitivity to noise, particularly road traffic noise, can increase cortisol levels and result in changes in immune system biomarkers. Therefore, continuous exposure to noise can have an effect on immune function, hormonal levels, and cardiovascular function, leading to hypertension and stress. The purpose of this study was to investigate the changes in stress- and immune system-related biomarkers according to the self-reported sensitivity to noise and exposure to road traffic noise, to ultimately determine the potential effects of noise on health. A survey was conducted through questionnaire (ISO/TS 15666) sent to 172 female subjects in Korea, including 128 from Ulsan and 44 from Seoul. The average noise level was calculated, and blood samples were collected for measurements of cortisol levels, Natural killer (NK) / Natural killer T (NKT) cell populations, and NK cell activity (through mea- surements of interleukin-12 (IL-12) and interferon-gamma (INF-γ) concentrations). Multivari- ate linear regression analysis of the measured biomarkers according to the road traffic noise level and self-reported noise sensitivity was conducted adjusting for the effects of age, alco- hol status, smoking status, regular exercise, and residence period. IL-12 levels increased, whereas the NKT cell population decreased with increasing noise levels. The results further suggested that cortisol levels are more influenced by the subject’s sensitivity to noise than to the level of chronic road traffic noise. Therefore, noise appears to have the largest effect on IL-12 levels as well as the population and activity of NKT cells. In conclusion, our results suggest that low-level road traffic noise and sensitivity to noise can affect health by causing changes in the immune response through mechanisms other than increased cortisol. OPEN ACCESS Citation: Kim A, Sung JH, Bang J-H, Cho SW, Lee J, Sim CS (2017) Effects of self-reported sensitivity and road-traffic noise levels on the immune system. PLoS ONE 12(10): e0187084. https://doi. org/10.1371/journal.pone.0187084 Citation: Kim A, Sung JH, Bang J-H, Cho SW, Lee J, Sim CS (2017) Effects of self-reported sensitivity and road-traffic noise levels on the immune system. PLoS ONE 12(10): e0187084. https://doi. org/10.1371/journal.pone.0187084 Editor: Markus M. Bachschmid, Boston University, UNITED STATES Editor: Markus M. Bachschmid, Boston University, UNITED STATES Immune response to road-traffic noise [6] and stress [7, 8]. Therefore, there has been much research interest on the potential health effects of exposure to environmental noise. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Through meta- analysis, a small number of studies have shown that noise-induced cardio- vascular disease is associated with a higher risk for men than for women and a higher risk for people aged older than 65 years [9]. Recent studies have also reported that sleep quality and noise sensitivity are not related to vascular function or noise sensitivity, but rather that night noise increases the risk of cardio- vascular disease due to increased blood pressure in patients and controls [10]. Road traffic noise is a particular noise source that affects a large portion of urban populations. Indeed, more than 40% of the populations in European Union countries are exposed to noise of 55 dB or above; 20% are exposed to daytime noise of 65 dB and above, and more than 30% are exposed to nighttime noise of 55 dB or above [1]. The World Health Organization stated that noise has negative effects on health by triggering physiological changes such as impairment of hearing function and increases in stress hormones and sensitivity [11, 12]. The most well-known mechanism mediating the response to such stress is the hypotha- lamic-pituitary- adrenal axis (HPA axis). When the HPA axis receives a signal of a stress response, corticotropin releasing factor is secreted from the hypothalamus, releasing adreno- corticotropic hormone from the pituitary gland. Adrenocorticotropic hormone then promotes the secretion of cortisol from the adrenal cortex through the blood, which triggers responses to various kinds of stress. The secretion of cortisol in response to stress inhibits the function of the HPA axis to disrupt the secretion of neurohormones and neurotransmitters as well as influencing the endocrine system, thereby disturbing homeostasis of the body, which can induce the development of various stress-related diseases[13]. Recently Meyer et al. [14] reported that mental stress and noise exposure could activate inflammatory cytokines such as interleukin (IL)-6 and IL-1β. These cytokines also interact with each other. Extracllular IL-6 induces cortisol from the zona fasciculate of the adrenal cortex, and has been reported to affect the synthesis of cortisol even when the HPA axis is inhibited. Introduction Environmental noise can be defined as the noise emitted from all sources, other than noise at an industrial workplace [1]. Several studies have shown that environmental noise such as that due to traffic, aircrafts, and construction can have physiological and psychological conse- quences [2, 3] such annoyance, sleep disturbance [4], cardiovascular disease [5], hypertension PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 1 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 Thereby, the immune, endocrine and nervous systems are related to each other. Thus, environmental noise has direct effects on health as well as indirect effects through the release of stress hormones such as cortisol [15, 16], dopamine [17, 18] and changes of alpha- amylase levels [19]. Increased cortisol can further cause changes in the immune system such as cellular proliferation, cytokine secretion, antibody production and cytotoxicity [20, 21]. Indeed, one study showed that the activity of natural killer (NK) cells was decreased by increased corti- sol [22]. NK cells are a leukocyte subset and important components of innate immunity. The innate immune system provides a rapid, non-specific host response against foreign agents such as bacteria, viruses, or tumors before triggering the adoptive immune system [23–25]. Innate immunity includes the antigen-presenting cells, monocytes/macrophages, dendritic cells (DCs), NK cells, and NKT cells. Moreover, cytokine signaling is essential for intercellular communication in the immune system to mediate and control immune functions. IL-12 is an important cytokine mediating immune responses, which is mainly produced by monocytes, macrophages, and DCs in response to bacterial products, intracellular pathogens, or upon interaction with activated T cells. IL-12 has been shown to play a critical role in the pathogenesis of a variety of immune- related diseases. This cytokine can induce interferon-gamma (IFN-γ) production [26], cell proliferation, and cytotoxicity mediated by NK cells and T cells. Hence, exposure to environmental noise affects immunity by inducing stress and increasing the secretion of stress hormones. As sensitivity to noise itself will affect the stress response and such sensitivity varies among individuals, the effects and response of exposure to identical environmental noise can show high individual variation. 2 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 Immune response to road-traffic noise However, studies on the negative effects of environmental noise, including road traffic noise, on health are still lacking in Korea. Therefore, the aim of our study was to analyze the immune response against noise generated from road traffic as well as the self-reported sensitiv- ity to noise in two metropolitan cities in Korea. Specifically, the level of exposure to road traffic noise was estimated using the noise map generated for Ulsan Nam-gu and Seoul Yangchun- gu, and the changes of immune response parameters according to road traffic noise and sensi- tivity were statistically analyzed. Questionnaires The questionnaires included age, residence period, education level, monthly income, alcohol status, smoking status, and exercise status. Education level was divided into a high school degree or below and a community college degree or above. Monthly income was divided into less than 3 million Won and above. Smoking status was divided into current smokers and cur- rent non-smokers (past smokers and non-smokers). Alcohol status was divided into current drinker and current non-drinker (past drinker and non-drinker). Exercise status was divided into participants currently exercising regularly and those who were not. To assess the sensitiv- ity to noise, an 11-point visual analogue scale was generated and used in this study based on ISO/TS 15666 [27]. All research procedures were approved by the Ulsan University Hospital Institutional Review Board (UUh 2014-08-008-012). Blood sampling and NK cell preparation and flow cytometry After the subjects were selected, 7 mL of venous whole blood was taken from the respondents between 9.00 am to 12.00 pm. Peripheral blood mononuclear cells (PBMCs) were isolated from all 172 subjects. The NK cell population was analyzed by fluorescence-activated cell sorting (FACs) at Green Cross Lab- Cells Corporation (Seoul, Korea) to identify the proportion of CD45+CD16+CD56+ cells. Cor- tisol levels were measured by Quantikine enzyme-linked immunosorbent (ELISA) analysis using serum from Green Cross LabCells Corporation. The concentrations of IL-12 and INF-γ in the sera were measured using the Quantikine enzyme-linked immunosorbent (ELISA) kit, according to the manufacturer’s instructions (Bio Legend San Diego, CA, USA). Immune response to road-traffic noise the survey was used to calculate the mean noise levels of the buildings the participants reside in as a road traffic noise index. The noise index used in this study was the day-night average sound level (Ldn), which is a qualitative index of the weighted equivalent day/night noise level that divides a 24h period into 6 am–10 pm and nighttime. Actual noise exposure levels were assessed using a sound level meter (Ulsan: NA-28, Rion, Japan; Seoul: B&K 2250, Bru¨el & Kjær, Denmark). Statistical analysis The statistical analysis was performed with SPSS v.21 for Window (IBM SPSS Inc., Chicago, IL, USA). The data regarding immune response parameters such as cortisol, NK cell popula- tion, and cytokines showed positively skewed distributions; thus, logarithmic transformations were performed for these variables to facilitate further statistical analyses under normal data assumptions. Statistical analyses were performed using multiple linear regression and Pearson’s correla- tion analysis. The results were considered statistically significant when P < 0.05. Participants and noise measurement All subjects participated voluntarily and provided written informed consent. We recruited 1,000 people in Yangcheon-gu (Seoul) and Nam-gu (Ulsan), respectively from July 2015 to December 2015. Participants were stratified according to noise exposure level based on noise map data. Although the residents of Yangcheon-gu are exposed to aircraft and road traffic noise, those of Nam-gu are exposed only to road traffic noise. A total of 336 participants each in Nam-gu, Ulsan and Yangcheon-gu, Seoul finally agreed to the blood test and included in the study for analysis (Fig 1). The questionnaires and blood test results of 336 participants were reviewed. Questionnaires were missing for four subjects, 93 subjects were excluded because of underlying chronic dis- eases, and 67 subjects were men; thus, 172 female subjects were finally selected. The male participants were excluded because they tend to have more activity in environ- ments outside of the residence target area, which could influence the sensitivity to the envi- ronmental noise around the residence. Subjects with any chronic illness (hypertension, hyperlipidemia, stroke, myocardial infarction, angina, arrhythmia, diabetes, and other dis- eases) were also excluded, because of a potential interference on the influence of noise on the immune response. To examine the level of environmental noise that the participants were exposed to, a three- dimensional noise map generated in 2014 based on the participants’ addresses confirmed from Fig 1. Location of residents in Ulsan (A, Nam-gu), Seoul (B, Yangcheon-gu) in noise map. It shows the noise level of each area and the location of participants in Nam-gu of Ulsan (A) and Yancheon-gu of Seoul (B). https://doi.org/10.1371/journal.pone.0187084.g001 Fig 1. Location of residents in Ulsan (A, Nam-gu), Seoul (B, Yangcheon-gu) in noise map. It shows the noise level of each area and the location of participants in Nam-gu of Ulsan (A) and Yancheon-gu of Seoul (B). https://doi.org/10.1371/journal.pone.0187084.g001 3 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 Immune response to road-traffic noise Table 1. General characteristics of the subjects. Ulsan Seoul Total N Mean 95% CI N Mean 95% CI N Mean 95% CI Age (years) 128 44.9 42.9–46.8 44 45.6 41.8–49.5 172 45.1 43.3–46.8 20–29 years 11 (8.6) 5 (11.4) 16 (9.3) 30–39 years 29 (22.7) 7 (15.9) 36 (20.9) 40–49 years 40 (31.3) 14 (31.8) 54 (31.4) 50–59 years 39 (30.5) 13 (29.5) 52 (30.2) Over 60 years 9 (7.0) 5 (11.4) 14 (8.1) Residence period (years) 128 9.6 8.3–10.9 42 7.9 5.5–10.3 170 9.2 8.0–10.3 Noise sensitivity 128 5.4 5.1–5.8 44 6.5 5.9–7.2 172 5.72 5.38–6.05 Education level High school and less 62 23 85 (49.4) College and more 66 21 87 (50.6) Income Under 3,000,000 (KWR) 16 19 35 (20.3) Over 3,000,000 (KWR) 112 25 137 (79.7) Alcohol status No 48 28 76 (44.2) Yes 80 16 96 (55.8) Smoking status No 125 44 169 (98.3) Yes 3 0 3 (1.7) Regular exercise No 70 12 82 (47.7) Yes 58 32 90 (52.3) Unit: Number (percentage) https://doi.org/10.1371/journal.pone.0187084.t001 Table 1. General characteristics of the subjects. was more than 60 years in Ulsan and Seoul. In the case of Seoul, the subjects in their 20s also showed the lowest participation rate. The mean age of the participants was 45.1 years and the mean residence period at the current residence was 9.2 years. The mean sensitivity to noise was 5.72. With respect to edu- cation level, 49.4% of the participants had a community college degree or below and 50.6% had a community college degree or above. For monthly income, 79.7% of the participants reported earning 3,000,000 Won or more and 20.3% reported earning less than 3,000,000 Won. The frequency of current and non-drinkers was 55.8% and 44.2% respectively, and the frequency of current and non-smokers was 1.7% and 98.3%, respectively. In addition, 52.3% of the participants reported exercising regularly and 47.7% reported that they did not exercise regularly. As shown in Table 2, road traffic noise and IL-12 were significantly positively correlated (r = 0.2333). Road traffic noise and the NKT cell distribution rate showed a significant negative correlation (r = 0.214; P < 0.01). IL-12 and the NKT distribution were also negatively corre- lated (r = 0.622), whereas IL-12 and INF-γ showed a significant positive correlation (P < 0.01). Results The sociodemographic variables and sensitivity to noise for 128 participants in Ulsan Nam-gu and the 44 participants in Seoul Yangchun-gu are summarized in Table 1. In both Ulsan and Seoul, the distribution rate of participants was highest at 31.3% and 31.8%, respectively. The average age of all participants in Ulsan was 42.9 years and the average age of all participants in Seoul was 45.6 years. The age group with the lowest participation rate PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 4 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 Immune response to road-traffic noise Table 2. Correlation among noise levels, noise sensitivity and immune response. Variables Ldn Noise sensitivity Cortisol NK cells NKT cells IL-12 Noise sensitivity -0.103 Cortisol 0.065 0.138 NK cells 0.147 0.074 0.057 NKT cells -0.214** 0.106 -0.138 -0.120 IL-12 0.233** -0.134 0.149 0.007 -0.620** INF-γ 0.127 0.017 0.041 -0.167 -0.148 0.251** **. Correlation is significant at the 0.01 level (2-tailed), Immunologic profiles were transformed by natural log. https://doi.org/10.1371/journal.pone.0187084.t002 Table 2. Correlation among noise levels, noise sensitivity and immune response. mean value of Ldn and the noise sensitivity interval are shown as correlation curves through the adjusted predicted value of each biomarker. mean value of Ldn and the noise sensitivity interval are shown as correlation curves through the adjusted predicted value of each biomarker. The change in biomarker for Ldn and noise sensitivity are shown using correlation curves in Fig 2. Multivariate linear regression analysis was performed to analyze the relationships among road traffic noise, individual sensitivity, and age-dependent biomarkers, adjusting for the effects of alcohol status, smoking status, regular exercise, and residence period. As shown in Table 3, as the sensitivity level increased by 1 step, the cortisol level increased by 0.032 μg/dL. Moreover, as the road traffic noise increased by 1 dB, the percentage of NKT cells decreased by—0.038%, whereas the IL-12 level increased by 0.006 pg/mL. The percentage of NK cells and INF-γ levels were not significantly associated with road traffic noise or sensitivity (P > 0.05), and therefore were meaningless as regression coefficients. As shown in Fig 3, the 5 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 https://doi.org/10.1371/journal.pone.0187084.t002 Discussion In this study, the correlations among road traffic noise, sensitivity to noise, stress hormones, and immunity-associated factors were investigated. Previous studies have shown that a high level of noise or sensitivity to noise induces sleep disorders [28, 29], hypertension [30–32], and cardiovascular disease [33, 34]. The result of traffic noise, depression, and anxiety are somewhat limited and somewhat controversial; however, Beutel et al. [35] found that strong noise discomfort was related to higher depression and anxiety in the general population. Participants with a mental health problem may also have higher noise sensitivity and report higher discomfort levels [36]. p y g y p g When exposed to heavy nighttime aircraft, patients with coronary heart disease reported chest pain caused by a typical heart disease [37]. Moreover, these consequent increases in the levels of the stress hormone cortisol from noise or sensitivity can have negative effects on health by reducing the activity of NK cells [38, 39]. Duggal et al. [40] suggested that the observed association between NK cell immunesenes- cence and cortisol was more strongly affected by mental stress rather than by physical stress. Munzel et al. [41] suggested a mechanism of the noise-triggered activation of the immune system using a mouse model, indicating that noise exposure not only increased levels of nor- adrenalin (NA), adrenalin (A) and angiotensin II (Ang II), but also increased cortisol thereaf- ter. Increased Ang II has been reported to activate endothelial NADPH oxidase, which causes oxidative stress that can lead to direct scavenging of nitric oxide and enhanced nitric oxide synthatse uncoupling. Since then, the increase in the NADPH oxidase subunit NOX-2 has been reported to increase immune cell activation and infiltration, such as NK cells, myelomo- nocytic cells, leukocytes, and macrophages/monocytes. Therefore, noise stress causes blood pressure and vascular dysfunction associated with oxi- dative stress. In the present study, the IL-12 and INF-γ levels were positively correlated, suggesting that an increase of cortisol decreases the IL-12 level with a consequent decrease of NK cells and INF-γ, a marker of NKT cell activation. Hence, it is predicted that an increase in cortisol levels would reduce immune function by decreasing the activity of NK cells and NKT cells. Although there are few studies on the relationship between NKT cells and noise, these cells can be activated in both antigen-dependent and independent manners. PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 Discussion Furthermore, because PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 6 / 13 Immune response to road-traffic noise Fig 2. Each real correlation curve for biomarker, Ldn and noise sensitivity. (A-E) It shows correlation curve for Ldn and biomarker such as cortisol (A), NK(B) and NKT cells (C), cytokines and it also shows each the real correlation curve for noise sensitivity and biomarker(F-J). https://doi.org/10.1371/journal.pone.0187084.g002 Fig 2. Each real correlation curve for biomarker, Ldn and noise sensitivity. (A-E) It shows each the real correlation curve for Ldn and biomarker such as cortisol (A), NK(B) and NKT cells (C), cytokines (D and E)) and it also shows each the real correlation curve for noise sensitivity and biomarker(F-J). Fig 2. Each real correlation curve for biomarker, Ldn and noise sensitivity. (A-E) It shows each the real correlation curve for Ldn and biomarker such as cortisol (A), NK(B) and NKT cells (C), cytokines (D and E)) and it also shows each the real correlation curve for noise sensitivity and biomarker(F-J). https://doi.org/10.1371/journal.pone.0187084.g002 https://doi.org/10.1371/journal.pone.0187084.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 7 / 13 https://doi.org/10.1371/journal.pone.0187084.t003 Immune response to road-traffic noise Table 3. Immune responses to road traffic noise, noise sensitivity, and stress response. Variables Multivariate analysis Dependent Independent β±SE p-value R2 ln_Cortisol (㎍/dL) Ldn 0.003±0.003 0.319 0.057 Sensitivity 0.032±0.014 0.020 ln_NK cells (%) Ldn 0.006±0.004 0.111 0.193 Sensitivity 0.018±0.016 0.263 ln_NKT cells (%) Ldn -0.033±0.014 0.021 0.079 Sensitivity 0.046±0.063 0.464 ln_IL-12 (pg/mL) Ldn 0.006±0.002 0.010 0.097 Sensitivity -0.010±0.011 0.365 ln_INF-γ(pg/mL) Ldn 0.007±0.005 0.118 0.105 Sensitivity 0.007±0.020 0.750 SE: standard error, Immunologic profiles were transformed by natural log. Linear regression models adjusted for age, alcohol consumption, smoking status, regular exercise, residence period. Table 3. Immune responses to road traffic noise, noise sensitivity, and stress response. Variables Multivariate analysis Dependent Independent β±SE p-value R2 ln_Cortisol (㎍/dL) Ldn 0.003±0.003 0.319 0.057 Sensitivity 0.032±0.014 0.020 ln_NK cells (%) Ldn 0.006±0.004 0.111 0.193 Sensitivity 0.018±0.016 0.263 ln_NKT cells (%) Ldn -0.033±0.014 0.021 0.079 Sensitivity 0.046±0.063 0.464 ln_IL-12 (pg/mL) Ldn 0.006±0.002 0.010 0.097 Sensitivity -0.010±0.011 0.365 ln_INF-γ(pg/mL) Ldn 0.007±0.005 0.118 0.105 Sensitivity 0.007±0.020 0.750 SE: standard error, Immunologic profiles were transformed by natural log. Linear regression models adjusted for age, alcohol consumption, smoking status, regular exercise, residence period. Table 3. Immune responses to road traffic noise, noise sensitivity, and stress response. SE: standard error, Immunologic profiles were transformed by natural log. Linear regression models adjusted for age, alcohol consumption, smoking status, regular exercise, residence period. 4.t003 Fig 3. Adjusted predicted value change of biomarker for Ldn and noise sensitivity interval. The values of cortisol (pg/dL), NK and NKT cells (%), cytokines (pg/ml) were adjusted for age, alcohol consumption, smoking status, regular exercise, residence period. The mean values of cortisol (A), NK/NKT cells (B), cytokines (C) for each interval for Ldn are shown, and D-F shows the mean value of each biomarker for the noise sensitivity interval. 03 Fig 3. Adjusted predicted value change of biomarker for Ldn and noise sensitivity interval. The values of cortisol (pg/dL), NK and NKT cells (%), cytokines (pg/ml) were adjusted for age, alcohol consumption, smoking status, regular exercise, residence period. The mean values of cortisol (A), NK/NKT cells (B), cytokines (C) for each interval for Ldn are shown, and D-F shows the mean value of each biomarker for the noise sensitivity interval. https://doi.org/10.1371/journal.pone.0187084.g003 https://doi.org/10.1371/journal.pone.0187084.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 8 / 13 Immune response to road-traffic noise Fig 4. Schematic representation of the potential immune response by exposure noise levels or noise sensitivity. Low chronic noise affects DCs and Macrophages and increases IL-12 concentration, but noise sensitivity increases the concentration of cortisol in the body. Therefore, we expect to decrease INF-γ activity, NKT and NK cells population by Ldn and noise sensitivity, which are two influencing factors. https://doi.org/10.1371/journal.pone.0187084.g004 Fig 4. Schematic representation of the potential immune response by exposure noise levels or noise sensitivity. Low chronic noise affects DCs and Macrophages and increases IL-12 concentration, but noise sensitivity increases the concentration of cortisol in the body. Therefore, we expect to decrease INF-γ activity, NKT and NK cells population by Ldn and noise sensitivity, which are two influencing factors. https://doi.org/10.1371/journal.pone.0187084.g004 https://doi.org/10.1371/journal.pone.0187084.g004 they have pro-inflammatory and immunoregulatory characteristics, they are known to play an important role in autoimmune diseases [42, 43], viral infections [44, 45] and cancer [46]. In this study, the level of road traffic noise showed a positive correlation with IL-12 levels but did not affect the NK cell population. However, the proportion of NKT cells was negatively correlated with road traffic noise. In addition, multivariate linear regression analysis on the relationships between road traffic noise level or noise sensitivity to immune and stress parameters, controlling for the potentially confounding variables of age [47–49], alcohol status, smoking status, regular exercise, resi- dence period, showed that the increase of noise level increased the IL-12 level but decreased PLOS ONE | https://doi.org/10.1371/journal.pone.0187084 October 30, 2017 9 / 13 Immune response to road-traffic noise the NKT cell distribution rate, similar to results of the correlation analysis. Because cortisol is considered to be more strongly affected by sensitivity than chronic road traffic noise and road traffic noise does not largely affect cortisol unlike the biological response to > 80 dB noise exposure [15], it appears that the IL-12 level and NKT cell frequency and activity are likely regulated by different mechanisms [15]. That is, cortisol increases through the HPA-axis, which is stimulated by a sensitivity response to activate DCs and macrophages, and the chronic noise itself can increase the levels of IL-12, a pro-inflammatory cytokine, as a synergistic effect. However, noise stress will decrease the NKT cell population to induce a pro-inflammatory response, with negative consequences for health [18]. The limitations of this study are as follows. First, as a cross-sectional study, although correla- tions could be determined, the cause-and-effect relationship cannot be established from these data. Acknowledgments The authors would like to thank occupational & environmental medical center, Ulsan Univer- sity Hospital and Korea Ministry of Environment (MOE). The authors also are grateful to the participants of survey. Supporting information S1 Dataset. Full dataset of tables. (PDF) Conclusions Low-level environmental noise and sensitivity to noise are likely to have negative effects on health by triggering changes in the immune response through a mechanism distinct from an increase in cortisol (Fig 4). Second, the effects of extreme noise levels such as occupational noise were not considered. Finally, not all potentially confounding variables were controlled in the analysis. Nevertheless, the clear associations detected in the present study and previous work suggest the importance of carrying out longitudinal studies on the effects of chronic noise on health and to determine the mechanism underlying the immune response to chronic loud noise. 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Monitoring the quality of frozen-thawed venous segments using bioimpedance spectroscopy
Physiological measurement
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PAPER • OPEN ACCESS PAPER • OPEN ACCESS Physiological Measurement Physiological Measurement Monitoring the quality of frozen-thawed venous segments using bioimpedance spectroscopy To cite this article: M Amini et al 2020 Physiol. Meas. 41 044008 Abstract Objective: Storage at temperatures as low as −80 ◦C and below (cryopreservation) is considered a method for long-term preservation of cells and tissues, and especially blood vessel segments, which are to be used for clinical operations such as transplantation. However, the freezing and thawing processes themselves can induce injuries to the cells and tissue by damaging the structure and consequently functionality of the cryopreserved tissue. In addition, the level of damage is dependent on the rate of cooling and warming used during the freezing-thawing process. Current methods for monitoring the viability and integrity of cells and tissues after going through the freezing-thawing cycle are usually invasive and destructive to the cells and tissues. Therefore, employing monitoring methods which are not destructive to the cryopreserved tissues, such as bioimpedance measurement techniques, is necessary. In this study we aimed to design a bioimpedance measurement setup to detect changes in venous segments after freezing-thawing cycles in a noninvasive manner. Approach: A bioimpedance spectroscopy measurement technique with a two-electrode setup was employed to monitor ovine jugular vein segments after each cycle during a process of seven freezing-thawing cycles. Main results: The results demonstrated changes in the impedance spectra of the measured venous segments after each freezing-thawing cycle. Significance: This indicates that bioimpedance spectroscopy has the potential to be developed into a novel method for non-invasive and non-destructive monitoring of the viability of complex tissue after cryopreservation. Monitoring the quality of frozen-thawed venous segments using bioimpedance spectroscopy M Amini1, E Niemi2, J Hisdal3, H Kalvøy4, C Tronstad4, H Scholz2,5, A Rosales6 and Ø G Martinsen1,4 M Amini , E Niemi , J Hisdal , H Kalvøy , C Tronstad , H Scholz , A Rosales and Ø G Mart 1 Department of Physics, University of Oslo, Oslo, Norway 2 Department of Transplant Medicine and Institute for Surgical Research, Oslo University Hospital, Oslo, Norway 3 Vascular Investigations and Circulation Lab, Aker Hospital, Oslo University Hospital, Oslo, Norway 4 Department of Clinical and Biomedical Engineering, Rikshospitalet, Oslo University Hospital, Oslo, Norway 5 Hybrid Technology Hub, Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway 6 Department of Vascular Surgery, Aker Hospital, Oslo University Hospital, Oslo, Norway Keywords cryopreservation, freezing-thawing, non-invasive, venous, bioimpedance spectroscopy © 2020 Institute of Physics and Engineering in Medicine To cite this article: M Amini et al 2020 Physiol. Meas. 41 044008 To cite this article: M Amini et al 2020 Physiol. Meas. 41 044008 View the article online for updates and enhancements. This content was downloaded from IP address 77.18.51.186 on 08/05/2020 at 06:54 This content was downloaded from IP address 77.18.51.186 on 08/05/2020 at 06:54 https://doi.org/10.1088/1361-6579/ab85b7 Physiol. Meas. 41 (2020) 044008 Physiological Measurement Physiological Measurement OPEN ACCESS RECEIVED 29 November 2019 REVISED 11 March 2020 ACCEPTED FOR PUBLICATION 1 April 2020 PUBLISHED 4 May 2020 Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. 1.1. Cryopreservation y p In order to preserve the viability and structural integrity of living cells and tissues for a long period, they are stored under very low (cryogenic) temperatures where all the chemical, biological and physical reactions in living cells would be suspended or dramatically reduced. This process is called cryopreservation (Karlsson and Toner 1996, Pegg 2007, Jang et al 2017). Cryopreservation is usually performed at −80 ◦C and below as traces of unfrozen solution can still be found in tissue above this temperature, and consequently the optimal duration of cryopreservation would play an important role in the prevention of any kind of damage to the cells (Mazur 1984). However, if the cryopreservation is performed at −130 ◦C and below then all biological activities, including biochemical reactions that can lead to cell death, would stop. Furthermore, at −196 ◦C (liquid nitrogen temperature) there would not be enough thermal energy to drive any type of biochemical process (Mazur 1984, Gao and Critser 2000, Woods et al 2016). Under cryogenic temperatures, background ionizing radiation (which causes photophysical reactions) could be the only cause of damage to tissues, and this is not considerable (Woods et al 2016). Thus, in temperatures lower than −130 ◦C the duration of Physiol. Meas. 41 (2020) 044008 M Amini et al Figure 1. Physical changes in the cells under different cooling rates: slow and rapid. Figure 1. Physical changes in the cells under different cooling rates: slow and rapid. cryopreservation does not play a very important role in the viability of the cells, so the cryopreservation period can range from short (Ku et al 1992, Müller-Schweinitzer 1994a) to undefined periods (Elmore et al 1991, Bujan et al 2000). j The advantage of cryopreserved cells and tissues is their constant availability for both research and clinical application, where their quality can be tested for suitability for transplantation purposes (Ibars et al 2016, Jang et al 2017). However, experimental findings have shown that the cryopreservation process can be damaging to the cells (Mazur 1963, Gao and Critser 2000). One of the methods for cryopreservation of cells and tissues is the freezing-thawing procedure (Karlsson and Toner 1996). Although storage under cryogenic temperatures is beneficial for long-term preservation of cells and tissues, the freezing and thawing processes can be detrimental to their viability (Karlsson and Toner 1996, Bia et al 2006, Müller-Schweinitzer et al 2007). 1.1. Cryopreservation However, the challenge is not only to withstand storage at high subzero temperatures but also to withstand the transition phases and to traverse the temperature range of −15◦C to −60◦C. By the time the tissue reaches the temperature of −15 ◦C, ice crystals are already formed in the extracellular matrix but the cell membrane blocks the growth of ice in the intracellular matrix. Consequently, water has started flowing out of the cells osmotically, due to its higher chemical potential in comparison to the water in the extracellular matrix. Therefore, the temperature zone in which both ice crystallization in the extracellular matrix and loss of intracellular water happen is considered a lethal zone for cells, especially when they need to traverse it twice (once during freezing and once during thawing) (Mazur 1963, Gao and Critser 2000, Woods et al 2016). Both cooling (freezing) and warming (thawing) rates play important roles in determining the viability of cells and tissue (Pegg 2007) and should optimally result in the recovery and survival of cell and tissue structure (Karlsson and Toner 1996). The rate of temperature change controls the transport of water out of the cells during freezing and into the cells during thawing, and thus indirectly influences the concentration of the solution surrounding the cells (Mazur 1963, Pegg 2007). At low temperatures, permeability of the cell membrane to water is higher than to the corresponding solute, which makes the cell membrane semipermeable (Lynch and Diller 1981, Karlsson and Toner 1996). In other words, formation of intracellular ice is influenced by cooling rate and what happens to that ice is controlled by warming rate (Pegg 2007). Freezing rates that are too rapid or too slow can both cause damage to the cells, however the damage mechanisms are different (Gao and Critser 2000). On the other hand, if cells are cooled too rapidly (figure 1) the intracellular water volume remains almost the same, as water does not have time to leave the cells fast enough by osmosis to maintain equilibrium (Karlsson and Toner 1996). In this way, icy crystals would be formed inside the cell (which is called ‘Intracellular Ice Formation’ or IIF), which can be lethal to the cells (Mazur 1963, Mazur et al 1972, Gao and Critser 2000). Therefore, damage to the cells during rapid cooling is due to IIF or osmotic rupture (Karlsson and Toner 1996). 1.1. Cryopreservation Changes in temperature and concentrations of intra- and extracellular solutes damage the lipid-protein complexes in the cell membrane (Lovelock 1953, Gao and Critser 2000), causing leakage and also changes in membrane permeability (Pegg 2007, Müller-Schweinitzer 2009). Therefore, an optimal cooling rate (which would be high enough to minimize the solution effect and low enough to avoid IIF) is necessary to maintain the viability and integrity of the cells and their membranes (Mazur 1963, Mazur et al 1972, Gao and Critser 2000). 2 Physiol. Meas. 41 (2020) 044008 M Amini et al Warming or thawing can be as challenging for the survival of the cells as the cooling or freezing process (Mazur 1984, Gao and Critser 2000). Studies have shown that maximum thermal stress to the cells occurs during the warming or thawing process (Zhang et al 2005, Müller-Schweinitzer 2009) as small intracellular ice crystals show different behaviors when they are treated by slow or rapid warming (Pegg 2007). Therefore, the extent to which the warming rate would affect the tissue under cryopreservation actually depends on whether the prior rate of cooling has caused cellular dehydration or intracellular ice formation (Gao and Critser 2000, Woods et al 2016). Slow warming gives enough time for the small intracellular ice crystals to recrystallize, join and grow into harmful ice crystals (Pegg 2007). Therefore, although there is no change in the amount of ice in the cells, what causes injury is the size and shape of the ice. In addition, during slow warming the cells would be exposed to concentrated solutes for a longer period of time, which increases the chances of toxicity caused by the solutes (Woods et al 2016). In contrast, during rapid warming (which is generally applied in a 37 ◦C water bath) (Bellon et al 1999) there is not enough time for the ice crystals to recrystallize, merge and grow bigger, so the ice crystals just melt (Pegg 2007). In this way, rapid warming prevents the formation of large ice crystals that can be damaging to the cells and thus helps rescue them (Mazur 1984, Gao and Critser 2000). 1.2. Cryopreservation of vascular tissue Previous research has shown that blood vessel cells and tissues can be seriously damaged when they are subjected to cooling and warming (freezing and thawing). This damage can range from loss of contractility in the smooth muscle layer to loss of function in endothelial cells (Müller-Schweinitzer 2009). According to some studies, storage of blood vessels at −80 ◦C would provide better preservation than lower temperatures such as −160 ◦C/−190 ◦C (Ku et al 1994, Müller-Schweinitzer 1994a, 1994b, Bujan et al 2000). Storage of vascular tissues in temperatures of around −80 ◦C for a short period of time (3–4 weeks) can preserve the structural integrity of the tissues. For example, studies have shown that the viscoelastic properties of human veins would not change during the cryopreservation period (Bia et al 2006, Müller-Schweinitzer et al 2007). However, storage under these temperatures for a long period would not guarantee the survival of mammalian cells (Müller-Schweinitzer et al 1986, Ku et al 1994). According to previous clinical research, slow warming of arteries and animal vessels (with a rate of 1 ◦C min−1) would cause less damage to the endothelial cell layer in comparison to rapid warming (which can cause fractures in the vascular wall) (Hunt et al 1994, Wassenaar et al 1995, Pegg et al 1997, Bujan et al 2000, Pascual et al 2004, Pasquinelli et al 2006, Müller-Schweinitzer et al 2007). 2. Methods Six ovine jugular veins were subjected to seven cycles of freezing-thawing. Studies have shown that preservation in −80 ◦C freezers provides long-term storage of tissues and cells from weeks to months, and is one of the most commonly used storage methods. The combination of slow freezing and fast thawing methods employed in this study is considered to be an optimal cryopreservation method, which induces less injury and damage to the cells and tissue (Shu et al 2015). For the freezing part, slow cooling down to −80 ◦C with a freezing rate of 1 ◦C min−1 was performed in order to minimize the effects of ice injury to the venous tissue. For the thawing part, fast warming was performed by using a water bath at 37 ◦C in order to reduce recrystallization and ice injury. The venous segments were preserved in the University of Wisconsin (UW) solution during the whole storage and freezing-thawing periods. Bioimpedance spectroscopy measurement was performed on all veins after each freezing-thawing cycle, with at least a 48 h interval between each two consecutive measurements for each vein. A two-electrode bioimpedance spectroscopic measurement setup (figure 2) was applied and the same pair of electrodes was used as both current-carrying and voltage pick-up electrodes. The electrodes consisted of a gold wire going through the glass tubes and the venous segment, and a titanium plate placed at the bottom of the main container. The venous segments were fixed between two glass tubes in the main container. The container, glass tubes and venous segment were filled with saline, with a slightly positive pressure inside the vein to keep it expanded and prevent contact between the electrode and the wall of the venous segment. The measurement device employed was a Solartron complex impedance measurement system (SI 1260/1294, Solartron Group PLC., Hampshire, UK), using a controlled electrical excitation voltage of 500 mV rms to record the impedance modulus and phase of the veins in the frequency range of 1 Hz to 1 MHz (figure 3). The change in impedance over cycles was assessed statistically by selecting the impedance modulus at one frequency of interest and fitting these values from all veins in a linear mixed effects (LME) model. The change in impedance was modeled as a linearly dependent function ofthe number of cycles (as a fixed effect) and with a random intercept for the different veins. 1.3. Monitoring the quality of the frozen tissues tissue are related to their physiological and morphological properties, bioimpedance spectroscopy can b employed for evaluation of the tissue of interest (Dean et al 2008, Grimnes and Martinsen 2015). The aim of the present study was therefore to employ bioimpedance spectroscopy as a non-invasive and non-destructive measurement technique, and to evaluate its ability in monitoring changes in venous segments which have undergone multiple freezing-thawing cycles. 1.3. Monitoring the quality of the frozen tissues It is important to evaluate the viability and structural integrity of the cells and tissues after cryopreservation (freezing-thawing cycle) and before their clinical application (such as transplantation). Some of the monitoring methods that are currently employed for this purpose include microcomputed tomography imaging (Bischof et al 2007); magnetic resonance swift imaging (Zhang et al 2014, Manuchehrabadi et al 2017); FT-IR spectroscopy (Giugliarelli et al 2016); label-free fluorescence imaging with optical fiber (Alfonso-Garcia et al 2018); light and cryomicroscopy, scanning and transmission electrode microscopy imaging (Bujan et al 2000); and general histological evaluation methods (Merdassi et al 2011). In addition there are mechanical tests such as tonometry for evaluating the biomechanical properties and functionality of tissues, such as their viscoelasticity (Bia et al 2006). However, these methods are mostly invasive and destructive to the tissues as they require segmenting the tissue into tiny rings or pieces, and also staining. Therefore, there is a need for a non-destructive method which would be able to monitor the tissue quality after its cryopreservation in a non-destructive manner. Electrical bioimpedance measurements facilitate non-destructive miniaturized methods for real-time evaluation of the state or properties of biological materials. The bioimpedance measurement technique has been used in clinical applications to determine fluid content, electrolytic composition, cellular morphology, organ perfusion, inflammation, body composition, etc. The spatial resolution can range from molecular-size to whole-body, and can be focused by proper development of measurement setup and electrode design (Grimnes and Martinsen 2015). Structural tissue properties are usually highly detectable in a bioimpedance measurement, as are the concentration of cells and their geometry. Destruction of cell membrane and cell death are examples of detectable vital signs in a tissue (Amini et al 2018). Therefore our hypothesis is that a potential change in the viability of the cells due to freezing/thawing will be detectable with a properly designed bioimpedance setup. Bioimpedance spectroscopy is one of the bioimpedance measurement techniques, which records the electrical impedance of a tissue over a frequency range to detect the frequency-dependent electrical properties of the tissues. As electrical properties of the 3 Physiol. Meas. 41 (2020) 044008 M Amini et al Figure 2. Bioimpedance spectroscopy measurement set-up with two-electrode configuration for monitoring veins after each freezing-thawing cycle. Figure 2. Bioimpedance spectroscopy measurement set-up with two-electrode configuration for monitoring veins after each freezing-thawing cycle. 3. Results Comparison of the bioimpedance spectra recorded after each freezing-thawing cycle demonstrated obvious changes in both the modulus and phase angle of the measured bioimpedance. For four of the six monitored veins, this change followed a trend where the impedance modulus decreased systematically from the first to the seventh cycle. For the other two veins, following the measurement protocol and subsequently completing the measurements was impossible. This was due to the presence of tiny holes in the walls of the venous segments and consequent leakages from the walls; this made expanding the venous segment and preventing the venous wall from collapsing and coming into direct contact with the electrode inside the vein impossible. Therefore, measurements from these two veins were not reliable and were excluded from the results. In figure 4 the bioimpedance spectra from all four veins, including the impedance modulus (|Z|) and the phase angle (Theta), are shown. Figure 5 shows the changes in impedance modulus (at the particular frequency of 10 kHz) over the seven cycles of freezing-thawing for all four veins. The reason for choosing 10 kHz was that this frequency is above the frequency range corresponding to contribution from electrode polarization impedance, and below the frequency range were inductive properties are demonstrated. Based on the spectra shown in figure 4, the impedance modulus at 10 kHz (figure 5) was selected for statistical analysis of the change in impedance over cycles. Fitting the 10 kHz impedance to the LME model gave an estimated change of −1.26 Ohms per cycle, with a 95% confidence interval from −1.49 to −1.04 Ohms per cycle (p < 0.001). 2. Methods The LME was implemented using the fitlme() function in Matlab 2018b. 4 Physiol. Meas. 41 (2020) 044008 M Amini et al Figure 3. Block diagram of the bioimpedance spectroscopy measurement system. Figure 3. Block diagram of the bioimpedance spectroscopy measurement system. 4. Discussion Each step of the cryopreservation process can cause injury to the cells and influence the viability and functionality of the cells or tissues, therefore monitoring and evaluating the tissues after cryopreservation and before their clinical application is important. The majority of current monitoring methods are invasive and destructive. As an example, methods for histological evaluation of cells and tissues can be mentioned, which are highly accurate and considered as the gold standard. Thus, the use of an evaluation method that is not destructive to cryopreserved tissue is warranted. Bioimpedance measurement techniques such as bioimpedance spectroscopy have shown their suitability as real-time, non-invasive methods for characterizing tissues and evaluating their viability and integrity in a non-destructive manner (Grimnes and Martinsen 2015). The main finding of the present study, which employed bioimpedance spectroscopy as the monitoring method, was observation of a decreasing trend in the impedance modulus and phase angle in four of the six 5 Physiol. Meas. 41 (2020) 044008 M Amini et al (a) (b) 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 100 101 102 103 104 105 106 101 102 103 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle Theta [degree] Frequency [Hz] IZI [ohm] 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 100 101 102 103 104 105 106 101 102 103 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle Theta [degree] Frequency [Hz] IZI [ohm] (c) (d) 100 101 102 103 104 105 106 101 102 103 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle IZI [ohm] Theta [degree] Frequency [Hz] 100 101 102 103 104 105 106 101 102 103 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle IZI [ohm] Theta [degree] Frequency [Hz] Figure 4. Bioimpedance spectra from four veins recorded after each freezing-thawing cycle, including the impedance modulus |Z| and phase angle Theta of all four veins: (a) vein 1, (b) vein 2, (c) vein 3, and (d) vein 4. 4. Discussion In addition, the results of statistical analysis of impedance changes at 10 kHz, with the confidence interval of 95% and p-value < 0.001, indicate that the decrease in impedance modulus per cycle and for all veins is statistically significant. The observed decrease in impedance measurements between each freezing-thawing cycle can be an indication of a decrease in the structural integrity of the endothelial cell layer. This can be due to solution-effect injury in addition to ice injury damage to the cells and their membranes during thawing, which would consequently affect the functionality of the venous segment. Looking closer at the low frequency part of the plots in figure 4 (in the impedance modulus spectra from the first and second cycles in the frequency range around 1 kHz) a small arc can be detected, which flattens out for the subsequent cycles. The corresponding circle segments are also evident in the phase plot. Both of these findings strongly suggest the presence of an α-dispersion for the first and second cycles. The α-dispersion can usually be seen in the frequency range of 1 Hz to 100 kHz (Grimnes and Martinsen 2010, 2015) and is associated with tissue interfaces, such as cell membranes. Therefore, there is reason to believe that this α-dispersion can be used to detect a change in the properties of cell membranes or intracellular structures after consecutive freezing-thawing cycles (Ivorra et al 2005). g g y However, the arc representing α-dispersion (which is mostly evident from a few hundred Hz) should not be confused with the impedance modulus curve below 100 Hz, which has a large increase in impedance towards 1 Hz due to electrode polarization impedance (EPI). The presence of EPI or electrode impedance at the electrode interface with the tissue/solution in the frequency range below 1 kHz, and its contribution to changes in the impedance modulus and phase angle, is often inevitable — especially when a two-electrode configuration is employed (Grimnes and Martinsen 2015). Generally, electrode material and size, sample impedance, measurement frequency and some other factors affect the influence of EPI in the measurements. Therefore, by being aware of this dependency it is possible to design the measurement setup such that the contribution of EPI would be reduced or minimized (Kalvøy et al 2011, Amini et al 2018). 4. Discussion (a) (b) 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 100 101 102 103 104 105 106 101 102 103 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle Theta [degree] Frequency [Hz] IZI [ohm] 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 100 101 102 103 104 105 106 101 102 103 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle Theta [degree] Frequency [Hz] IZI [ohm] 100 101 102 103 104 105 106 101 102 103 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle IZI [ohm] Theta [degree] Frequency [Hz] Theta [degree] Theta [degree] (c) (d) 100 101 102 103 104 105 106 101 102 103 100 101 102 103 104 105 106 40 20 0 -20 -40 -60 -80 first cycle second cycle third cycle fourth cycle fifth cycle sixth cycle seventh cycle IZI [ohm] Theta [degree] Frequency [Hz] (d) (b) Figure 4. Bioimpedance spectra from four veins recorded after each freezing-thawing cycle, including the impedance modulus |Z| and phase angle Theta of all four veins: (a) vein 1, (b) vein 2, (c) vein 3, and (d) vein 4. Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 0 5 10 15 20 25 |Z| at 10kHz [Ohms] Vein 1 Vein 2 Vein 3 Vein 4 Figure 5. Changes in impedance modulus |Z| at the particular frequency of 10 kHz over seven freezing-thawing cycles for all four veins. Figure 5. Changes in impedance modulus |Z| at the particular frequency of 10 kHz over seven freezing-thawing cycles for all four veins. 6 Physiol. Meas. 41 (2020) 044008 Physiol. Meas. 41 (2020) 044008 M Amini et al measured veins (in the frequency range of 1 Hz to 1 MHz) after each freezing-thawing cycle. In the other two veins it was not possible to follow the measurement protocol and complete the measurements, due to leakage caused by the formation of tiny holes in the blood vessel walls. The plots in figure 5 show that all four veins demonstrate linear changes in the same direction along with the cycles, and strongly suggest that these results are not random changes. 4. Discussion In our measurement setup, which used gold as the electrode material (which demonstrated one of the lowest EPIs among the other electrode materials tested in our pilot measurements) and specific dimensions for the gold electrode (diameter of 1 mm and length covering the whole length of the venous segment), it is expected that the contribution of EPI would be low. In addition, changes in impedance modulus after each freezing-thawing cycle are most evident in higher frequency ranges (above 100 Hz), since the contribution from EPI is inversely proportional to frequency. The positive phase angle seen in the MHz range can be interpreted as if the veins demonstrate inductive properties. However, studies have shown that the existence of inductive properties in tissue samples and cell membranes under stable conditions in passive bioimpedance measurements is not very likely (Cole 1972, Riu 2004). Nevertheless, the positive phase angle does not need to be a sign of measurement error and can be caused by the contribution of self-inductance when measuring low impedance values (<50Ω) at high frequencies, such as in our case. Especially when a two-electrode setup is used, the self-inductance of the measuring lead and wire would be in series with the tissue impedance and in this way contribute to the measurements. Hence, the lower the impedance value is, the larger the inductive influence would be (Grimnes and Martinsen 2007). This study has shown that a bioimpedance spectroscopy technique is capable of detecting changes in venous grafts after freezing-thawing cycles. However, the mechanisms and types of morphological changes in the vascular grafts that have led to these distinct changes in the impedance spectra are unknown and require further investigations. In addition, optimization of the measurement setup and electrode configuration, in order to minimize the contribution of electrode impedance polarization, is recommended. In this way, the arcs representative of α-dispersion, which are the result of structural changes in the cells and the venous segment, would become even more visible. Conflict of Interest The authors do not have any potential conflicts of interest to declare. 5. Conclusion From the results of this study, it can be concluded that bioimpedance spectroscopy has the potential to be developed into a novel method for non-invasive and non-destructive monitoring of the viability of complex tissue after cryopreservation. 7 M Amini et al Physiol. Meas. 41 (2020) 044008 M Amini https://orcid.org/0000-0001-8835-9998 M Amini https://orcid.org/0000-0001-8835-9998 Ethical approval The research related to animal use has complied with all the relevant national regulations and institutional policies for the care and use of animals. 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Bioim 9 142–58 Bellon J M, Gimeno M J Pascual G et al 1999 Arterial damage induced by cryopreservation is irreversible following organ culture Vasc. Endovasc. Surg. 17 136–43 g Bia D, Zocalo Y, Pessana F Armentano R et al 2006 Viscoelastic and functional similarities between native femoral arteries and fresh or cryopreserved arterial and venous homografts Rev. Esp. Cardiol. 59 679–87 Bia D, Zocalo Y, Pessana F Armentano R et al 2006 Viscoelastic and functional similar cryopreserved arterial and venous homografts Rev. Esp. Cardiol. 59 679–87 Bischof J C, Mahr B, Choi J H, Behling M and Mewes D 2007 Use of X-ray tomography to map crystalline and amorphous phases frozen biomaterials Ann. Biomed. Eng. 35 292–304 g Bujan J et al 2000 Rapid thawing increases the fragility of the cryopreserved arterial wall Eur. J. Vasc. Endovasc. 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Surg. 60 165–7 Wassenaar C, Wijsmuller E G, Van Herwerden L A, Aghai Z, Van Tricht C L and Bos E 1995 Cracks in cryopreserved aortic allografts a rapid thawing Ann. Thorac. Surg. 60 165–7 p g g Woods E J, Thirumala S, Badhe-Buchanan S S, Clarke D and Mathew A J 2016 Off the shelf cellular therapeutics: factors to consider during cryopreservation and storage of human cells for clinical use Int. Soc. Cell. Ther. 18 697–711 p g g Woods E J, Thirumala S, Badhe-Buchanan S S, Clarke D and Mathew A J 2016 Off the shelf cellular therapeutics: factors to consider during cryopreservation and storage of human cells for clinical use Int. Soc. Cell. Ther. 18 697–711 Woods E J, Thirumala S, Badhe-Buchanan S S, Clarke D and Mathew A J 2016 Off the shelf cellular thera during cryopreservation and storage of human cells for clinical use Int. Soc. Cell. 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References 26 113–20 (https://www.ingentaconnect.com/content/cryo/cryo/2005/00000026/00000002/art00005) Zhang A, Cheng S, Gao D and Xu L X 2005 Thermal stress study of two different artery cryopreservation methods Cryo Lett. 26 113–20 (https://www.ingentaconnect.com/content/cryo/cryo/2005/00000026/00000002/art00005) ntaconnect.com/content/cryo/cryo/2005/00000026/00000002/art00 Zhang J, Chamberlain R, Etheridge M, Idiyatullin D, Corum C, Bischof J and Garwood M 2014 Quantifying iron-oxide nanoparticles at high concentration based on longitudinal relaxation using a three-dimensional SWIFT look-locker sequence Magn. Reson. Med. 71 1982–8 Zhang J, Chamberlain R, Etheridge M, Idiyatullin D, Corum C, Bischof J and Garwood M 2014 Quantifying iron-oxide nanoparticles at high concentration based on longitudinal relaxation using a three-dimensional SWIFT look-locker sequence Magn. Reson. Med. 71 1982–8 9
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Correction: The population genetics of human disease: The case of recessive, lethal mutations
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1. Amorim CEG, Gao Z, Baker Z, Diesel JF, Simons YB, Haque IS, et al. (2017) The population genetics of human disease: The case of recessive, lethal mutations. PLoS Genet 13(9): e1006915. https://doi. org/10.1371/journal.pgen.1006915 PMID: 28957316 Correction: The population genetics of human disease: The case of recessive, lethal mutations Carlos Eduardo G. Amorim, Ziyue Gao, Zachary Baker, Jose´ Francisco Diesel, Yuval B. Simons, Imran S. Haque, Joseph Pickrell, Molly Przeworski Carlos Eduardo G. Amorim, Ziyue Gao, Zachary Baker, Jose´ Francisco Diesel, Yuval B. Simons, Imran S. Haque, Joseph Pickrell, Molly Przeworski The authors note that the citation of Wright (1937) and Nei (1968) is incorrect in the “Models of mutation-selection balance” section in Results. Wright (1937) not only showed the equilib- rium frequency of a deleterious allele for a population of infinite size (Eq 1) but also derived the full equilibrium frequency distribution of a deleterious allele under a finite effective popu- lation size. He also derived the mean (Eq 2) allele frequency of a fully recessive deleterious allele and gave approximations for large and small effective population sizes. Nei (1968) fur- ther derived the approximate variance (Eq 3) in allele frequency of a fully recessive mutation by using Wright’s general formula. The authors confirm that this error does not affect the con- clusions of the article. OPEN ACCESS Citation: Amorim CEG, Gao Z, Baker Z, Diesel JF, Simons YB, Haque IS, et al. (2018) Correction: The population genetics of human disease: The case of recessive, lethal mutations. PLoS Genet 14(7): e1007499. https://doi.org/10.1371/journal. Reference a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Published: July 2, 2018 Copyright: © 2018 Amorim 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. 1 / 1 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007499 July 2, 2018
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Fane: A Firewall Appliance For The Smart Home
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Proceedings of the Federated Conference on Computer Science and Information Systems pp. 449–458 DOI: 10.15439/2019F177 ISSN 2300-5963 ACSIS, Vol. 18 g Computer Science and Information Systems pp. 449–458 ISSN 2300-5963 ACSIS, Vol. I. INTRODUCTION I N THE the last years, the proliferation of Smart Home installations has gained momentum. Today, the consumer market offers a huge number of different Internet-of-Things (IoT) devices. I We show that it is possible to develop a generic IT- Security concept for IoT devices in a Smart Home installation by making few realistic assumptions, e.g., the IoT network segment is only used by single-purpose IoT devices, which do not fundamentally change their communication profiles. We have implemented this security concept in FANE. Our evaluation indicates that FANE can secure the IoT network segment without requiring the user to possess network-security expertise. Smart thermostats, cameras, speakers and even toothbrushes contain information technology that connects the IoT device over the Internet with cloud services or other IoT devices. For example, IoT devices from the Google Nest family [1] provide a straightforward, user-friendly way to control heating, cooling and home security. Smart speakers like Amazon Alexa [2] allow to control many daily activities via voice control. From the perspective of the manufacturers, the Smart Home concept allows new business models, e.g., to sell new product features as digital upgrades for IoT devices. Paper structure: In Section II, we review related work. In Section III we provide a problem statement. We describe FANE in Section IV, followed by a proof-of-concept imple- mentation in Section V and an experimental evaluation in Section VI. Section VII concludes. On the other hand, consumers might be tempted to overlook that the IoT devices pose an IT-Security risk. For example, the lifetime of a traditional security camera ends when the device is broken. In contrast, the lifetime of an IoT security camera that connects over the Internet should come to an end when its manufacturer discontinues security updates, even if the IoT security camera is still working. Otherwise, the IoT security camera might end up as, say, part of the Mirai bot network, which consisted of approx. 500,000 devices in 2016 [3]. FANE: A Firewall Appliance for the Smart Home Erik Buchmann Hochschule für Telekommunikation in Leipzig Gustav-Freytag-Straße 43-45, 04277 Leipzig, Germany Email: buchmann@hft-leipzig.de Christoph Haar Hochschule für Telekommunikation in Leipzig Gustav-Freytag-Straße 43-45, 04277 Leipzig, Germany Email: haar@hft-leipzig.de Abstract—With the advent of the Internet of Things (IoT), many domestic devices have been equipped with information technology. By connecting IoT devices with each other and with the Internet, Smart Home installations exist that allow the automation of complex household tasks. A popular example is Google Nest that controls cooling, heating and home security. However, Smart Home users are tempted to neglect that such IoT devices pose IT-Security risks. Examples like the Mirai malware have already shown that insecure IoT devices can be used for large-scale network attacks. Thus, it is important to adapt secu- rity approaches to Smart Home installations. In this paper, we introduce FANE, our concept for a Firewall AppliaNcE for Smart Home installations. FANE makes a few realistic assumptions on the network segmentation and the communication profile of IoT devices. This allows FANE to learn firewall rules automatically. Our prototypical implementation indicates that FANE can secure a wide range of IoT devices without requiring network-security expertise from the Smart Home user. explore options to integrate a firewall into typical Smart Home installation that can detect and deter such attacks. This is challenging, since the firewall must be compliant with the typical modes of use of a Smart Home installation, and a consumer cannot be expected to evaluate firewall rules or identify false alarms. On the other hand, the IoT devices used differ from general-purpose devices such as smartphones and desktop computers. This might allow for pre-configuration to some extent. In particular, we make the following contributions: 1) We systematically compare the lifecycle of a classical firewall with the lifecycle of IoT devices in a typical Smart Home installation. 2) We propose FANE, a Firewall AppliaNcE on a Wi-Fi bridge in Smart Home installations. 3) We describe a proof-of-concept implementation of FANE based on a Raspberry Pi, and we evaluate it with three different IoT devices. II. RELATED WORK In this section we provide a brief definition of Internet of Things and Smart Home, and we introduce related work on firewalls, firewall management and approaches to generate firewall rules automatically. PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 450 B. Security Challenges To assess the security properties of Smart Home installa- tions, it is important consider the basic security challenges that occur in installations of IoT devices. One study [6] lists six major security issues: Identity and Authentication: In IoT environments, numer- ous devices need to authenticate each other in order to provide trustable services. Thus, reliable techniques for identification and authentication are needed. Access Control: To create new services it is necessary to aggregate data from different providers. This is challenging, because in typical IoT scenarios each provider has its own access control policy. Design and Selection Policy Definition Implementation and Configuration Plan Firewall Rules Implement Firewall Rules Review Firewall Operations Determine appropriate Actions IT-Security Operations Configuration Management Information Security Management Policy System Architecture Firewall Configuration Firewall Ruleset Activity Document Control Flow Information Flow Fig. 1. Traditional Firewall-Lifecycle Protocol and Network Security: If IoT devices commu- nicate with each other in a distributed network architecture, distributed schemes for key management are needed. Protocol and Network Security: If IoT devices commu- nicate with each other in a distributed network architecture, distributed schemes for key management are needed. distributed schemes for key management are needed. Privacy: The Smart Home concept means that numerous IoT devices monitor the actions of its users in order to devise meaningful responses. Thus, privacy very important from a user perspective. Policy Trust and Governance: In IoT architectures there are two dimensions of trust. The first dimension is between users and their IoT devices. The other dimension is between the IoT devices. Device A needs to trust the accuracy and integrity of the data produced by device B. Data governance goes in the same direction, in a sense of data and access governance. Fault Tolerance: Mechanisms for fault tolerance need to be established to counteract faulty or tampered devices. Other studies [7] list similar challenges. D. Firewall Lifecycle connected cars over smart buildings to connected machinery in an Industry 4.0 setting. The concept "‘Smart Home"‘ narrows down this range to devices that let end users to control, monitor or access everyday objects of the daily routine [5]. Traditionally, a firewall must be part of the IT-Security process, as described by the ISO 270xx standards family [10], the German BSI Grundschutz Standard 200-2 [11] or the ITIL process for security Management [12]. The IT-Security process starts with a IT-Security policy that has been passed by the management. Based on this policy, business objectives, the assets to be protected and a risk classification can be identified. Subsequently, measures can be defined and implemented that restrict the IT-Security risks to acceptable levels. In the follow- ing, the effectiveness of these measures needs to be monitored. Based on this information, corrective actions can be planned and executed [13]. Note that all process steps require a person with IT-Security expertise, which cooperates with various IT experts from the operations department. A. Internet of Things and Smart Homes From the perspective of a consumer without in-depth ex- pertise of network security, it is next to impossible to find out if the IoT devices present in a Smart Home installation are subject to attacks over the Internet. In this paper, we The "‘Internet of Things"’ (IoT) refers to physical appli- ances, which have been equipped with information technology in order to connect them with other devices directly or over the Internet [4]. IoT includes a wide range of appliances, from IEEE Catalog Number: CFP1985N-ART c⃝2019, PTI 449 E. Firewall Rules It is a labor-intensive task for a domain expert to create a rule set for firewalls for manually. One option to obtain firewall rules (semi-)automatically is to use data mining or machine learning on a training set consisting of network packets. This option is based on the assumption all user applications operate as intended while the training set is recorded. Respective approaches [15]–[17] have been proposed for Intrusion De- tection systems, but might be adaptable to firewalls as well. By using k-Means, C4.5 decision tree algorithms, Naive Bayes classifier, Neural Networks or Support Vector Machines, it is possible to derive common characteristics of allowed network connections. Those characteristics could be translated into firewall rules. It is also possible to generate firewall rules by mining the firewall log [18] instead of a dump of network packets. However, all approaches require an IT-Security expert to decide which generated rules are relevant to meet the security requirements, and the quality of the generated rules still needs further research. b) Network Architecture: Firewalls depend on the net- work segmentation. With traditional use cases, a network installation might contain multiple segments protected by multiple firewalls. A prominent example is a perimeter net- work [20], which contains assets such as Web servers that must be accessible from an external network. Two sets of firewall rules protect the perimeter network against the external network and the internal network against the perimeter and the external network. However, the number and architecture of the network segments might be individually different for each network installation. In contrast, a typical Smart Home installation with IoT devices produces three network segments with different se- curity properties: (a) the untrusted Internet, (b) the home network with trusted devices such as the user’s laptop and printer, and (c) an IoT network segment that contains all IoT devices. Since the IoT device and its software comes as an integrated package, the user has little options to influence the security of the IoT device, e.g., by disabling unused network protocols or by removing unused software functionality. Thus, the IoT network segment should be separated from the home network [23], which is used for sensible tasks such as online banking or online shopping. E. Firewall Rules All devices in the IoT network segment can be expected to require an Internet connection, to A different option to generate firewall rules is to deduce them from a formal specification of security requirements by using argumentation logic [19]. This approach allows to auto- matically obtain a detailed, comprehensive set of rules from a high-level specification. However, creating a specification of the security requirements for a certain system architecture still requires expert knowledge in IT-Security. A. Does a Firewall Fit into the Smart Home Concept? To find out in which ways traditional firewall use cases differ from Smart Home use cases, we consider the modes of use, network architecture, application scenario, user roles and information technology used. a) Modes of Use: A firewall is an access control mech- anism that allows or blocks network traffic between two network segments that have different security properties [20], e.g., an internal network and the Internet that is open for anybody. The firewall enforces a set of firewall rules that allow or prohibit network packets to travel from one segment into the other one. The firewall rules depend on the use cases that are executed over both network segments. For example, a business workflow "‘Answer customer requests"’ might require that a set of machines in the internal network is allowed to send and receive email to/from the Internet. Thus, firewall rules must be defined by a network-security expert with domain knowledge. If the workflows, the applications or the segment boundaries are changed, the expert must adapt the firewall rules as well. Traditionally, firewalls are tailored for complex multi-purpose scenarios where the hosts execute numerous different applications that change over time. Smart Home use cases are fundamentally different [21]: A typical IoT device is a physical object that has been extended with information technology to improve its usefulness. For example, a smart toothbrush [22] can tell its user if a tooth has gone unbrushed. Thus, IoT devices are constructed for a single purpose that does not change over time. It only makes sense to install a toothbrush control software on a smart toothbrush. As a result, IoT devices are single-purpose objects. If the device is not needed any more, it will be disposed. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 451 A firewall fits into the IT-Security process [14] as shown in Figure 1. In the Information-Security Management phase, the management defines a security policy based on company-wide security objectives. This policy is independent from technical realities. Based on the security policy, an IT-Security expert designs the architecture of the firewall system and selects the firewall system components. In the implementation and configuration phase, the IT-Security expert adapts the firewall system to the system architecture with its network segments, hosts and applications. This includes a preliminary set of firewall rules that define which network packets are allowed to pass the firewall. In the next step, a plan-do-check-act cycle takes place where the firewall rules are designed, implemented, reviewed and improved in a repetitive way. Typically, this cycle is part of IT-Security operations. It allows to adjust the firewall rules to changes such as new business applications, hosts moving from one network segment to another one, or in case of detecting new attacks. Note that not only the management of firewall rules is a cyclic process, but also the IT-Security process. If the management observes that the security policy is ineffective, this policy can be changed as well, and it has an impact on all design decisions further down the IT-Security process chain. C. Firewalls State of the Art Firewalls are able to control and log the network traffic based on rules set by an administrator or security expert. In literature different firewall generations are distinguished [8]. 1st generation firewalls are known as packet filters which operates on the transport layer. The filtering is based on source and destination IP addresses, ports and protocols. 2nd generation firewalls are also operating on the transport layer and they are known as stateful packet inspection. State tables are used to keep track of the network traffic and filtering is based on state and context of packets. 3rd generation firewalls are operating on the application level and require different proxies for each service. The proxy acts as a middleman between source and destination to reestablish a new session. Current firewall technologies are called next generation fire- walls. These next generation firewalls are looking deep into packets and combine traditional firewall technologies with network filtering capabilities on the application level [9]. However, all these generations have in common that an expert is needed to define rules or check them for correctness which motivates our new approach. mplement Firewall Rules Fig. 1. Traditional Firewall-Lifecycle A. Network Architecture A firewall separates network segments with different secu- rity properties. Typical IoT devices do not allow its user to observe security properties, and to configure security-related aspects, such as disabling unused functions. Furthermore, an IoT device is designed to be used like a classical, non-smart device, i.e., its users are tempted to forget that the device might pose IT-Security risks. For this reason, IoT devices should be placed in network segments that are isolated from all other network segments of the Smart Home. In contrast, an IoT device for a Smart Home usually is pre- configured by the manufacturer for typical use cases. The end user can deploy and configure the IoT device with minimal efforts, does not need to monitor it later on and does not need expert knowledge. e) Information Technology: IoT devices make use of network protocols which have been well established. They use Linux-based operating systems, Cloud resources and Open Source programming libraries. The network security of IoT devices is based on mechanisms for encryption, certification and signatures that have been in use for years. Thus, from a technical point of view, off-the-shelf firewalls can be directly used to control the network traffic of IoT devices. Thus, FANE operates as a Wi-Fi bridge that connects the IoT network segment to the Internet and includes a firewall, as shown in Figure 2. The IoT network segment only contains single-purpose IoT devices, and the Wi-Fi bridge is the only connection of the IoT segment to other network segments and the Internet. We observe that this allows us to specify the security concept in advance. IV. FANE: A FIREWALL APPLIANCE In this section, we introduce FANE, a concept for a Firewall AppliaNcE that is compatible with the Smart Home paradigm. Homes must differ in the following properties from traditional firewalls: On the opposite side, one of the fundamental principles of the Smart Home concept is to let IoT devices use sensors to observe its environment, in order learn appropriate actions with a minimum of user interaction and without requiring the user to scrutinize the operations of the IoT device on a regular basis. For example, the nest thermostat observes the temperature preferences of its user and if he or she is at home, and controls the heating system accordingly. Furthermore, the duration of use of IoT devices is an one-dimensional process that starts with the deployment of a device and ends with it’s disposal, just like non-smart devices [24], i.e., it does not follow a periodic lifecycle where it is constantly monitored and improved. For example, a smart light switch never changes its function, and it cannot be adapted to different needs. We have ruled out a cloud-based approach [25], [26] that externalizes the firewall to a trusted third party on the In- ternet. Although such an approach might fulfil the properties described, it requires a permanent Internet connection. In ad- dition, a cloud-based firewall would transfer security-relevant information into the cloud. Thus, both the Internet connection of the firewall and the trusted third party would be a valuable target for an attacker. d) User Roles: Setting up a traditional firewall typically requires three distinct roles: The role "‘Information Security Management"’ defines a security policy by considering the assets and (business) objectives that are relevant for a certain part of the IT infrastructure. Based on the policy, the role "‘Configuration Management"’ designs a firewall system, se- lects appropriate firewall components, and provides an initial installation and configuration of the system. Finally, a role "‘IT-Security Operation"’ constantly monitors and improves the firewall system, both on the level of the firewall rules and of the firewall hard- and software. PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 452 Homes must differ in the following properties from traditional firewalls: Homes must differ in the following properties from traditional firewalls: provide a service, to obtain updates and upgrades, to allow a remote control via smartphone app, etc. provide a service, to obtain updates and upgrades, to allow a remote control via smartphone app, etc. c) Application Scenario: Firewalls follow the IT-Security lifecycle, as explained in Section II. Based on a general security policy that has been defined from a management perspective, a network-security expert defines the position of the firewall(s) in the network architecture and a set of firewall rules. By using a plan-do-check-act-cycle, the firewall rules as well as the firewall hard- and software must be constantly monitored, evaluated and adapted to changes in the IT infrastructure. c) Application Scenario: Firewalls follow the IT-Security lifecycle, as explained in Section II. Based on a general security policy that has been defined from a management perspective, a network-security expert defines the position of the firewall(s) in the network architecture and a set of firewall rules. By using a plan-do-check-act-cycle, the firewall rules as well as the firewall hard- and software must be constantly monitored, evaluated and adapted to changes in the IT infrastructure. P1 The firewall must be usable without expert knowledge. P2 The firewall must fit to the durations of use of Smart Home components. P3 The firewall must operate in a way that is typical for IoT devices in the Smart Home. P3 The firewall must operate in a way that is typical for IoT devices in the Smart Home. P1 implies not only that the configuration and installation of a firewall in a Smart Home must not require network security expertise. It also means that a user cannot be expected to tell false alarms from real alarms, or to decide if a certain firewall rule is applicable to the home network. From P2 it follows that such a firewall must deal with IoT devices that are bought once for a certain purpose and never change its basic properties until disposal, and it must operate in the same way. Furthermore, the firewall must operate in the same way. P3 means that a firewall in a Smart Home needs to operate without permanent care from the user, i.e., it must monitor the network traffic, deduce meaningful firewall rules and provide appropriate reactions to forbidden network packets. III. PROBLEM STATEMENT In this section, we explore the differences between tradi- tional firewalls and firewalls needed for IoT devices in a Smart Home installation. In addition, we derive requirements for a Smart Home firewall. B. Security Concept From a technical perspective, it would be a simple exercise for a network security expert to set up a firewall that controls the network traffic of an IoT device. However, this procedure conflicts with the general understanding how IoT devices should operate in a Smart Home. Thus, a firewall for Smart From Section III it follows that a traditional firewall ap- proach is complex, because the underlying network segmen- tation and the processes executed over the boundaries of these segments are complex, too, and might change from 453 CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 453 IP Forwarding Internet Router NAT, DNS, DHCP Firewall Wi-Fi Access Point Internet (untrusted) Home Network (trusted) FANE IoT Network (less trusted) Smartphone Cloud Server IoT Device Fig. 2. System Architecture IoT Network (less trusted) Internet (untrusted) Firewall Cloud Server Home Network (trusted) Smartphone Fig. 2. System Architecture 3) An IoT device is allowed to open a connection to devices in other (trusted) network segments of the Smart Home, if this is part of its normal operation. time to time if a new software is installed on a device in the network. With our network architecture, we have reduced this complexity. We only need to consider three kinds of communication activities: 4) A device from a trusted segment is allowed to open connections to the IoT network segment. • An IoT device wants to communicate with a server on the Internet. For example, a smart thermostat wants to communicate with the user’s smartphone, which is mediated over a cloud service. 5) IoT devices are allowed to open connections to other devices in the IoT network segment. 5) IoT devices are allowed to open connections to other devices in the IoT network segment. A. Our FANE prototype We have realized FANE on the basis of a Raspberry Pi, which executes several linux shell scripts to configure and operate an iptables packet filter (see Subsection II-C). Figure 4 illustrates our hardware configuration and the main software packages used. Implement Firewall Rules Smart Home Operations Review Smart Home Operations IP Forwarding Dnsmasq Hostapd IPtables USB Power Raspberry Pi Model B Ethernet Port SD Card Slot HDMI Port Ubuntu Mate OS USB Port USB WiFi Adapter Fig. 4. Our FANE prototype USB WiFi Adapter Ethernet Port Alert Security Expert Activity Control Flow Document Information Flow Fig. 4. Our FANE prototype a) Hardware: From Section IV it follows that FANE must provide a Wi-Fi access point that creates a network segment for IoT devices. The IoT devices might want to communicate with other devices in the same segment, the home network segment and the Internet. Thus, FANE must be connected to the Internet, and its firewall must control all incoming and outgoing network packets of the IoT network segment. V. PROOF-OF-CONCEPT IMPLEMENTATION In this section, we describe the software and hardware components of our FANE prototype, how FANE learns firewall rules and in which way it interacts with the user. PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 454 Implementation and Configuration Learn Firewall Rules Implement Firewall Rules Review Smart Home Operations Re-evaluate Firewall Rules Smart Home Operations Pre-Configuration Management System Architecture Firewall Configuration Firewall Ruleset Activity Document Control Flow Information Flow Alert Security Expert Policy Fig. 3. FANE Operations that cannot be handled automatically by FANE. For example, the IoT network segment might face a denial-of-service attack from the Internet, or an IoT device has been taken over and tries to connect to the attacker’s command and control server on the Internet. In such cases, the user might decide to call the customer support of the IoT device, or ask an IT-Security expert for further investigations. Firewall Configuration C. Smart Home Firewall Operations • An IoT device wants to communicate with a device in another network segment. For example, the user installs a control application on a laptop to configure the smart thermostat. FANE has to meet conflicting requirements: It must meet the expectations provided by Smart Home components (P2). In particular, this means that FANE must operate without constant supervision (P3). At the same time, as a security component it must not neglect the IT-Security process, including a plan- do-check-act cycle to refine firewall rules. However, this must be possible without requiring the user to possess expert knowledge (P1). • An IoT device wants to communicate with another IoT device in the same network segment. For example, our smart thermostat wants to directly communicate with the smart air condition. Since FANE operates as a bridge to the Internet, only the first two kinds of communication have to be monitored, and the security properties of the endpoints of the communication can be specified at production-time of FANE: The open Internet is insecure by default, the IoT devices are less secure, and the devices in other network segments of the Smart Home are trustworthy. This allows to pre-configure the security concept of FANE in advance, i.e., it does not need a user with network- security expertise (Property P1): We circumvent these conflicts, as shown in Figure 3): We distinguish between pre-configuration management and Smart Home operations. Because we restrict FANE to the network architecture described in Subsection IV-A, the policy definition, the firewall design and a baseline configuration of firewall rules can be done at pre-configuration time. Thus, we shift the initial parts of the IT-Security process into the responsibility of the Smart Home firewall manufacturer who possess IT-Security expertise. Furthermore, we propose to automate the configuration and the plan-do-check-act cycle in a way that it’s phases can be started without expert knowledge at operation time. Finally, we define a process step in a way that the user is informed when an IT-Security expert is needed. 1) No device on the Internet is allowed to open a network connection to the IoT network segment. 2) An IoT device is allowed to open a connection to the Internet, if this is part of its normal operation. VI. EXPERIMENTAL EVALUATION In this section, we explore the applicability of FANE with three different Smart Home appliances. Fig. 5. Firewall rules learned from an adjusted packet log Note that this procedure can be extended easily to extended firewall features, e.g., to include the iptables options for stateful inspection. At the end of the rule generation phase, FANE installs the rules and is ready for operation. When FANE is connected to power and Internet for the first time, or if the user wants FANE to learn new rules, it enters the monitoring phase for a certain period of time. In this phase, FANE waits for new IoT devices connecting to the access point, and logs the network packets. We have implemented this phase as follows: If an IoT device is not working properly, if a new IoT device is added to the IoT network segment or if an existing device is used in a way it has never been used before, the user can order FANE to re-evaluate the rule set. In this case, the user has the option to discard rules from preceding learning procedures, and to re-start the monitoring- and rule-generation phase. At boot time, a cron task with the time prefix @reboot starts a script that finds out if the set of firewall rules is the one that has been pre-configured from the security concept (Subsection IV-B). Alternatively, a user command starts the monitoring phase manually. In the monitoring phase, FANE uses the monitoring tool inotify to find out if the dhcp.leases file changes. This indicates new devices using the access point. In this case, inotify executes a script that obtains the IP address of the device from dhcp.leases. At the same time, FANE uses tcpdump to create a log file containing all network packets sent or received during the monitoring phase. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME Firewall rules learned from an adjusted packet log D. User Interaction After having defined the operations of FANE, we can define the user interactions needed. Observe that no interaction requires expert knowledge (Property P1). FANE comes as an IoT device that runs out-of-the-box after being connected to a power outlet and the Internet. When FANE is connected to the Smart Home installation for the first time or if new IoT devices are added, the user can tell FANE to learn new firewall rules by observing the network packets of the IoT devices. Assume an IoT device uses a functionality that has not been used during the learning stage, or the device has been updated and a new network connection is now blocked by FANE. In this case, the user has the option to let FANE re-evaluate the rule set. That is, FANE executes a learning stage on a certain device with the option to discard rules that have been learned before. The rules from the security concept (Subsection IV-B) cannot be discarded. We have implemented this approach on a third-generation Raspberry Pi model B. This is a credit-card sized single board computer containing a quad-core processor with 1.2GHz, 1 GB main memory and various network and connection interfaces. Because the on-board Wi-Fi chip cannot be configured as a Wi-Fi access point, we have connected an external Wi- Fi module via USB. We have used a 32 GB SD Card for permanent storage. We would need only two switches to initiate the learning- and re-evaluation stage of the user interface, and one LED indicating an alert. The IT-Security expert, which might be needed to handle serious attacks on the IoT nework segment, would be able to obtain firewall logs and other information by using an SSH connection. This way, our FANE prototype If FANE blocks a large number of network packets per time- interval, it generates an alert. The alert shows the user that immediate action needs to be taken, i.e., something happens CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME B. Learning Firewall Rules 7 15:25:07 IP 10.200.65.101.8554 > 35.157.255.122.80: 8 iptables -A FORWARD -s 10.200.65.101 -sport 1024:65535 -d 35.157.255.122 -dport 80 -p tcp -j ACCEPT 7 15:25:07 IP 10.200.65.101.8554 > 35.157.255.122.80: 8 iptables -A FORWARD -s 10.200.65.101 -sport 1024:65535 -d 35.157.255.122 -dport 80 -p tcp -j ACCEPT For our FANE prototype, we have used a straightforward ap- proach to learn firewall rules. For more elaborate approaches, see Section II. The learning stage consists of two phases, a monitoring phase and a rule generation phase. We assume that all network traffic recorded during the monitoring phase is allowed, i.e., we assume that no IoT device has been manipulated or attacked before the monitoring phase ends. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 455 costs less than 60 EUR. However, to ease development we have used an external USB keyboard and a LCD monitor. costs less than 60 EUR. However, to ease development we have used an external USB keyboard and a LCD monitor. reduced to unique entries in a second step. The odd lines in Figure 5 show, how the set of addresses and ports looks like after FANE has removed surplus information and duplicats from the log file. In a third step, a shell scripts parses the remaining addresses and ports into firewall rules that allow such packets for the iptables chain "‘FORWARD"’. The odd lines in Figure 5 illustrate this step. We have used the iptables policy "‘DROP"’, i.e., FANE drops all packets that are not allowed by the rules generated. b) Software: We have used the Ubuntu Mate Linux operating system as a basis of our software configuration. On top of a minimal OS installation, we need the following software packages and services: • awk (script language to edit text files) • cron (timed execution of processes) • dnsmasq (DHCP client and DNS cache) • hostapd (Wi-Fi access point) 1 15:23:18 IP 10.200.65.101.1080 > 35.158.162.95.80: 2 iptables -A FORWARD -s 10.200.65.101 -sport 1024:65535 -d 35.158.162.95 -dport 80 -p tcp -j ACCEPT • inotify-tools (monitor changes in files) • iptables (network address translation and firewall) • iptables (network address translation • tcpdump (record network packets) By configuring the Ethernet interface eth0 as a DHCP client, our Raspberry Pi can be connected to any Internet router without further configuration. We have configured the wlan0 interface with a static IP address and subnet mask, and we have configured it as a Wi-Fi access point by using hostapd. Our Smart Home firewall must act as a bridge between eth0 (Internet) and wlan0 (Wi-Fi segment for IoT devices). Thus, we have used iptables and sysctl to activate IP forwarding, including network-address translation and masquerading. With dnsmasq, we have realized a DHCP service. 3 15:23:22 IP 10.200.65.101.8553 > 35.157.158.75.1883: 4 iptables -A FORWARD -s 10.200.65.101 -sport 1024:65535 -d 35.157.158.75 -dport 1024:65535 -p tcp -j ACCEPT 5 15:24:36 IP 10.200.65.101.8653 > 35.156.40.103.1883: 6 iptables -A FORWARD -s 10.200.65.101 -sport 1024:65535 -d 35.156.40.103 -dport 1024:65535 -p tcp -j ACCEPT 7 15:25:07 IP 10.200.65.101.8554 > 35.157.255.122.80: 8 iptables -A FORWARD -s 10.200.65.101 -sport 1024:65535 -d 35.157.255.122 -dport 80 -p tcp -j ACCEPT Fig. 5. Fig. 7. Fragment of the packet log of the IoT relay 1) An electrical IoT relay. C. IoT Power Outlet 2) An IoT power outlet. 2) An IoT power outlet. Our second use case is an IoT power outlet "‘Smart Wi- Fi Socket Model SWA1"’, sold for 18 EUR, produced by Shenzhen Lingan Intelligent Technology [28]. Similarly to the IoT relay, the IoT power outlet can be turned on or off via smartphone app. In addition, it can be controlled with Amazon Alexa or Google Home, which allows to integrate non-smart electrical devices into an elaborate Smart Home concept without requiring a technician. Any command to the IoT power outlet is handled by a cloud service over the Internet. 3) An IoT security camera. 3) An IoT security camera. The IoT devices do not communicate directly with each other, but with the user’s smartphone and the Internet. Thus, for our experiments we do not need to preconfigure rule 5 from our security concept (see Subsection IV-B). We have configured each device for FANE’s IoT network segment. We have used a monitoring phase of 40 minutes, and we have operated each device periodically during this phase. In the following, we briefly introduce each IoT device, and we describe what we have learned by using FANE as described. In our monitoring phase, we have used the IoT power outlet via smartphone in the same way as the relay for 40 minutes. At the end of the monitoring phase, FANE has collected a packet log of approx. 2,600 lines, all of them TCP packets. The rule generation phase has generated rules that allow five 5 13:41:31.669239 IP 52.71.154.91.443 > 10.200.65.109.55161: Flags [P.], ... Fig. 6. Our experimental setup A. Setup Figure 6 shows our experimental setup. FANE is directly connected to the Internet router, and its integrated access point spans a Wi-Fi network segment for IoT devices. The Internet router creates a Wi-Fi home network that connects a smartphone to the Internet. Different cloud services connect the smartphone to the IoT devices. A cloud service might use a load balancer, i.e., the IP addresses the IoT devices connect to might change from time to time. At the end of the monitoring phase, FANE stops tcpdump and enters the rule generation phase. In this phase, FANE parses the log file from tcpdump into firewall rules according to the IP addresses of the IoT devices that have used the access point in the monitoring phase. In particular, FANE uses a sed command to filter the log for incoming and outgoing IP addresses and ports. This set of addresses and ports is We have tested three different devices, which communicate differently with a control app on the user’s smartphone: PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 456 smartphone app, which allows a technician to integrate non- smart electrical devices into a straightforward Smart Home installation. Sending commands from the app to the relay requires an Internet connection, i.e., there is no option to directly connect the smartphone app to the IoT device. After the relay is connected to the access point provided by FANE, and the user has installed the smartphone app, the relay is ready to use. Internet FANE Cloud Server IoT Devices Smartphone of the User eth0 wlan0 IoT Network Internet Router Home Network Fig. 6. Our experimental setup Cloud Server Cloud Server In our monitoring phase of 40 minutes, we have switched the relay on and off frequently via smartphone app for 10 minutes. After that, we have waited for a period of 20 minutes. Finally, we have operated the relay for further 10 minutes. After completing the monitoring phase, FANE has written 1,800 lines in the packet log. All packets followed the TCP protocol and were sent/received to/from one singular IP ad- dress located at a dedicated server leased from Amazon. Thus, the rule generation phase has generated only one rule for in- and outgoing packets. The IoT relay was working properly after FANE has activated the firewall rule set generated. Figure 7 shows an example from the traffic log FANE has recorded from the IoT relay. Internet wlan0 1 13:41:31.551813 IP 10.200.65.109.55147 > 52.71.154.91.443: Flags [F.], ... Home Network 2 13:41:31.551870 IP 10.200.65.109.55145 > 52.71.154.91.443: Flags [F.], ... 3 13:41:31.551914 IP 10.200.65.109.55161 > 52.71.154.91.443: Flags [.], ... 3 13:41:31.551914 IP 10.200.65.109.55161 > 52.71.154.91.443: Flags [.], ... 4 13:41:31.668878 IP 52.71.154.91.443 > 10.200.65.109.55161: Flags [.], ... 5 13:41:31.669239 IP 52.71.154.91.443 > 10.200.65.109.55161: Flags [P.], ... 1 13:41:31.551813 IP 10.200.65.109.55147 > 52.71.154.91.443: Flags [F.], ... VII. CONCLUSION The last years have brought a plethora of Internet-of-Things (IoT) devices dedicated to Smart Home installations. While such IoT devices have numerous practical use cases, observa- tions have shown that many of them come with IT-Security risks. For example, the Mirai botnet consisted of approax. 500,000 baby-phones, security cameras and other insecure IoT devices that were able to execute distributed denial-of- service attacks with 1 Tbit/s network bandwidth. However, typical Smart Home users do not possess the network-security knowledge needed to identify and deter attacks on IoT devices. Furthermore, the Smart Home concept encourages the users to leave IoT devices unattended for long periods of time. We have observed that the IoT security camera was not working properly, after FANE started to filter network packets. Our investigations have shown that this due to a specific load balancer. The IP address of the load balancer was allowed by the firewall rule set generated. But the load balancer referred the IoT security camera frequently to IP addresses unknown to FANE. However, it would be possible to adapt the learning approach to cope with such a load balancer. For example, FANE could detect and accept IP addresses that are close by addresses that are already allowed by the rule set. The packet log has also shown that the IoT security camera first tries to reach the smartphone app in the same network segment directly, via multicast. Thus, even if the IoT security camera makes use of the Internet connection, it might be able to provide its basic functionality without the Internet. From this observation we conclude that there might be options for FANE to distinguish between communication needed for the normal operation of an IoT device, and other communication needed for advertising purposes or usage analytics that can be blocked without undesired side-effects. In this paper, we have introduced FANE, our concept for a Firewall AppliaNcE for Smart Home installations. FANE makes a few realistic assumptions on the network segmen- tation and the communication profile of IoT devices. This allows to pre-configure FANE with a generic security concept. It also enables FANE to learn firewall rules automatically by observing the network traffic of IoT devices. Experiments with a prototypical implementation have pro- vided evidence that FANE can secure ordinary IoT devices without requiring network-security expertise from the Smart Home user. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 457 different IP addresses, all of them in the address range of the Amazon AWS cloud. E. Discussion Our three use cases have provided evidence that a straight- forward learning approach is applicable to many IoT devices used in Smart Home scenarios. Two of our three IoT devices remained fully operative after FANE has monitored the net- work activities of our devices for 40 minutes, and has sub- sequently generated and activated firewall rules. Furthermore, our observations have shown that it would be easily possible to extend our learning approach to consider load balancers. As there is no communication standard for IoT devices, it is problematic to generalize our findings to all IoT devices used in the Smart Home. However, using a cloud service seems to be typical for many use cases. Only network packets can pass FANE that are allowed by a specific rule. Thus, FANE increases the security of the Smart Home installation. The IoT power outlet was fully operational after FANE has started to filter network connections. We have observed that only one of the five addresses in the firewall rule set was actually used to operate the outlet via smartphone app. We assume that some network connections are used only for analyzing customer behavior or similar purposes, i.e., blocking them would not reduce the functionality of the device. D. IoT Security Camera The most complex IoT device tested was a "‘720P HD IP Wireless security camera"’, sold for 37 EUR and manufactured by XinweiYa [29]. The IoT security camera sends a live video stream to the smartphone of the user. Furthermore, the smartphone app allows to restart the IoT security camera, and to rotate it around two axes. After connecting the IoT security camera to a power outlet, it can be configured with a smartphone app to use FANE’s access point. FANE operates without requiring the user to possess expert knowledge, by making three assumptions: First, the network segment created by FANE’s access point contains IoT devices only. This allows to specify a security policy in advance, before FANE is delivered to the user. Second, the IoT devices operate as single-purpose appliances that do not fundamentally change their communication profiles. Due to this assumption, FANE can learn a rule set that remains stable over a long period of time, which makes it compatible with the Smart Home concept. Third, we assume that the IoT devices are working properly during the monitoring phase. This allows FANE to learn firewall rules unattended. During our monitoring phase of 40 minutes, we have restarted the IoT security camera, we have let the IoT security camera sent a live video stream of 10 minutes to the smart- phone, we have waited for 20 minutes, and we have restarted it again for another live stream of 10 minutes. After 40 minutes, FANE has collected 8 MB packet log of approx. 27,000 lines, most of them UDP packets. The rule generation phase produces a rule set of 20 rules for this device. Those rules allow services like Network Time Protocol (NTP) or Domain Name System (DNS) as well as cloud services hosted on Amazon AWS, the Microsoft cloud and the Alibaba cloud. B. 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ACKNOWLEDGMENT We would like to thank Eric Ilgunas for his exceptional work on realizing and evaluating the FANE prototype. We would like to thank Eric Ilgunas for his exceptional work on realizing and evaluating the FANE prototype. VII. CONCLUSION Only one device was not working properly after FANE has activated its firewall rules due to a specific load balancer. However, this problem could be solved by accepting IP addresses close to addresses that FANE already knows. Finally, we have observed that the IoT security camera produces more network load by an order of magnitude than the other IoT devices tested. While this has slowed down the rule generation phase, it did not overstrain the IP forwarding capacity of our Raspberry Pi during normal operation. PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 REFERENCES [22] Procter & Gamble, Oral-b genius electric toothbrushes, https : / / www. oralb. co . uk / en - gb / products / electric - toothbrushes/oral-b-genius, Accessed: 2019-04-25. [8] K. Neupane, R. Haddad, and L. Chen, “Next generation firewall for network security: A survey,” in Southeast- Con 2018, IEEE, 2018, pp. 1–6. [23] N. Gupta, V. Naik, and S. Sengupta, “A firewall for internet of things,” in 2017 9th International Conference on Communication Systems and Networks, IEEE, 2017, pp. 411–412. [9] J. Surana, K. Singh, N. Bairagi, N. Mehto, and N. Jaiswal, “Survey on next generation firewall,” Interna- tional Journal of Engineering Research and Develop- ment, vol. 5, no. 2, pp. 984–988, 2017. [24] J. Stark, “Product lifecycle management,” in Product lifecycle management, Springer, 2015. [10] G. Disterer, “Iso/iec 27000, 27001 and 27002 for infor- mation security management,” 2013. [25] A. R. Khakpour and A. X. Liu, “First step toward cloud- based firewalling,” in 2012 IEEE 31st Symposium on Reliable Distributed Systems, IEEE, 2012, pp. 41–50. [11] Bundesamt für Sicherheit in der Informationstech- nik, “BSI-Standard 200-2, IT-Grundschutz-Methodik,” https://www.bsi.bund.de, 2017. [26] C. Modi, D. Patel, B. Borisaniya, H. Patel, A. Patel, and M. Rajarajan, “A survey of intrusion detection techniques in cloud,” Journal of network and computer applications, vol. 36, no. 1, pp. 42–57, 2013. [12] O. of Government Commerce, Introduction to ITIL, The key to managing IT services. Van Haren Publishing, 2005. [27] ewelink, Sonoff relay, http : / / ewelink . coolkit . cc, Accessed: 2019-04-25. [13] S. Fenz, G. Goluch, A. Ekelhart, B. Riedl, and E. Weippl, “Information security fortification by ontolog- ical mapping of the iso/iec 27001 standard,” in 13th Pacific Rim International Symposium on Dependable Computing, IEEE, 2007, pp. 381–388. [28] lingansmart, Power outlet, http://www.lingansmart.com, Accessed: 2019-04-25. [29] XinweiYa Co.,Ltd., Security camera, http : / / www . cctvgood.com, Accessed: 2019-04-25. [14] S. W. Lodin and C. 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Injection and Combustion Analysis of Pure Rapeseed Oil Methyl Ester (RME) in a Pump-Line-Nozzle Fuel Injection System
Energies
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Article Injection and Combustion Analysis of Pure Rapeseed Oil Methyl Ester (RME) in a Pump-Line-Nozzle Fuel Injection System Flavio Caresana 1 , Marco Bietresato 2 and Massimiliano Renzi 2,* 1 Dipartimento di Ingegneria Industriale e Scienze Matematiche, Università Politecnica delle Marche, I-60131 Ancona, Italy; f.caresana@univpm.it 2 Facoltà di Scienze e Tecnologie, Libera Università di Bolzano, I-39100 Bolzano, Italy; marco.bietresato@unibz.it y p 2 Facoltà di Scienze e Tecnologie, Libera Università di Bolzano, I-39100 Bolzano, Italy; marco.bietresato@unibz.it * Correspondence: massimiliano.renzi@unibz.it; Tel.: +39-0471-0171816; Fax: +39-0471-017009 * Correspondence: massimiliano.renzi@unibz.it; Tel.: +39-0471-0171816; Fax: +39-0471-017009 Abstract: This work suggests an interpretation to the quantitatively higher formation of NOx in a compression ignition (CI) engine when fueled with pure biodiesel (B100). A comparative study about the use of rapeseed oil methyl ester (RME) and diesel fuel mixtures on injection timing, in-chamber pressure, heat release rate, and NOx emissions were carried out using a diesel engine equipped with a pump-line-nozzle injection system. Such engines are still widely adopted mainly in agriculture, as the fleet of agricultural machinery is particularly old (often over 20 years) and the use of biofuels can reduce the environmental footprint of the sector. This work aims to supply some general explanations and figures useful to interpret the phenomena occurring within the fuel line and in the combustion process when using biodiesel, as well as in engines with different construction characteristics and fueling systems. Given the contradictory results available in the literature, the so-called “biodiesel NOx effect” cannot be explained solely by the different physical properties of biodiesel (in particular, a higher bulk modulus). Experimental results show that, with the same pump settings, the start of injection with the RME is slightly advanced while the injection pressure values remain almost the same. With the RME, the pressure in the injection line increases faster due to its greater bulk modulus but the pressure rise starts from a lower residual pressure. The start of combustion takes place earlier, the heat release during the premixed phase is steeper, and a higher peak is reached. The NOx emissions with the RME are at least 9% higher when compared to mineral diesel fuel. The greater amount of the RME injected per cycle compensates for its minor lower heating value, and the brake torque at full load is similar to the two analyzed fuels. Finally, a variation of the pump line timing is evaluated in order to assess the effect of the delay and the advance of the injection on the performance of the engine and on the emissions.   Citation: Caresana, F.; Bietresato, M.; Renzi, M. Injection and Combustion Analysis of Pure Rapeseed Oil Methyl Ester (RME) in a Pump-Line-Nozzle Fuel Injection System. Energies 2021, 14, 7535. https://doi.org/10.3390/ en14227535 Academic Editor: Rocio Maceiras Academic Editor: Rocio Maceiras Received: 27 September 2021 Accepted: 8 November 2021 Published: 11 November 2021 Keywords: compression ignition engine; biodiesel; B100; heat release rate; injection timing advance; injection pressure; biodiesel NOx effect Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Article Injection and Combustion Analysis of Pure Rapeseed Oil Methyl Ester (RME) in a Pump-Line-Nozzle Fuel Injection System A viable and simple solution in the variation of the injection strategy is suggested to counterbalance the biodiesel NOx effect. Keywords: compression ignition engine; biodiesel; B100; heat release rate; injection timing advance; injection pressure; biodiesel NOx effect Citation: Caresana, F.; Bietresato, M.; Renzi, M. Injection and Combustion Analysis of Pure Rapeseed Oil Methyl Ester (RME) in a Pump-Line-Nozzle Fuel Injection System. Energies 2021, 14, 7535. https://doi.org/10.3390/ en14227535 energies energies 1. Introduction Other properties, compared to mineral diesel, include a higher cetane number, lubricity, and flash point, while the toxicity is reduced. Among the detrimental characteristics, it is possible to list its lower volatility, the calorific value, the oxidation stability [10,11], and the higher viscosity [12] and pour point [13] that, however, can be adjusted due to new processes that improve the low temperature performance of biodiesels [5–7]. When the biodiesel is blended with diesel fuel at high percentages, it can also cause reliability problems for the engine components, as it can cause corrosion and chemically attack some plastic components of seals and coatings [14]. g Different oils are used to produce biodiesel fuels depending on the local availability. The most common in the United States is soybean oil (from Glycine max (L.) Merr.); coconut oil (from Cocos nucifera L.) and palm oils (mainly from Elaeis guineensis Jacq.) are used in Malaysia and Indonesia; the Jatropha (Jatropha curcas L.), Karanja (Pongamia pinnata L.), and Mahua trees (Mahua Indica J. F. Gmel) are used as a significant fuel sources in India and southeast Asia; while rapeseed oil-based esters (from Brassica napus L.) are commonly used in many European countries [15,16]. In Austria and Germany, neat biodiesel is widely used, whereas in France, Italy, Spain, Sweden, the Czech Republic, and other European countries, fuel blends of diesel fuel with up to 25–30% rapeseed oil methyl ester (RME) are popular [17]. Some reviews on the effects of biodiesel fuels on compression-ignited (CI) engine performance and emissions report the following main outcomes [18–22]: (i) the fuel consumption increases proportionally to the loss of the heating value of the fuel; (ii) the particulate matter (PM) emissions show a consistent reduction; (iii) a minimal or absent loss of power is reported, except when maximum power is required. As regards the brake thermal efficiency (BTE), its value is not significantly affected with biodiesel, but a slight increase or a reduction in the BTE can be noted depending on the engine load, the type of biodiesel, and the blend with mineral diesel fuel [23]. Improvements in the efficiency are mainly due to the synergic blending effects that could be caused by the reductions in friction losses [24]; instead, lower efficiencies are typically reported at high engine loads and rotational speeds because of the poor spray characteristics, poor mixing, high viscosity, and the lower calorific value of biodiesel [25]. 1. Introduction The role of “alternative” fuels to meet the reduction targets in the impact of the energy production and transport sectors has increased in the last few years. Currently, “bioenergy” is the largest contributor to the global renewable energy supply, especially for thermal energy production, but has not yet reached a large-scale production for liquid biofuels, as this must overcome some complex research uncertainties on the management of those resources, their lifecycle emissions, their effects on water, the soil management, the process efficiencies, as well as facing their potential negative effects [1]. In particular, among the biomass-derived fuels, and despite the existence of some serious problems associated with their application with respect to feedstock requirements and land availability—the “food 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/ 4.0/). https://www.mdpi.com/journal/energies Energies 2021, 14, 7535. https://doi.org/10.3390/en14227535 Energies 2021, 14, 7535 2 of 25 vs. fuel” debate—the so-called “first generation biofuels” have grown mainly thanks to government subsidies and legislative pressures [2]. vs. fuel” debate—the so-called “first generation biofuels” have grown mainly thanks to government subsidies and legislative pressures [2]. Meanwhile, novel biofuels have been introduced that use nonfood biomass, dedicated energy crops, and biomass coproducts and waste from many different sectors, such as agriculture, horticulture, forestry, paper and pulp processing, and wastes (e.g., municipal solid waste (MSW)) [3]. Novel improved technologies are being developed to increase the production yield of qualitatively acceptable biodiesel [4], and new processes have been proposed to improve the low-temperature performance of biodiesels [5–7]. Among the available biofuels, “biodiesel” is one of the candidates in this class and its use is expanding throughout the world [8]. As an example, total biofuel consumption in the EU was around 17.0 Mtoe in 2018, and in terms of energy content, while biodiesel’s share was 82.0%, bioethanol was 17.1%, and biomethane fuel was 0.9%, respectively [9]. The main advantage of the use of biodiesel resides in its renewability, which allows for the reduction of the burdens of greenhouse gases (GHG). From the environmental point of view, it is possible to enumerate other important benefits deriving from the chemical composition of biodiesel, which has a minimal sulfur and aromatic content. 1. Introduction As regards the emissions, all of the authors agree in the reduction of the carbon monoxide (CO), hydrocarbon (HC), and PM emissions due the use of biodiesel [26]. With regard to the nitrogen oxides (NOx) concentration, most of the authors [11,14] report a slight increase with biodiesel; nevertheless, some studies have actually found very different evidence, spanning from “NOx increment only in some operating conditions” up to “there is lack of difference between diesel fuel and biodiesel” or even “NOx emissions decrease with biodiesel”. As it appears from the literature reviews [27–30], despite the great number of research articles available, various topics regarding biodiesel use in diesel engines remain unsolved and need further investigation. In 2007, Lapuerta et al. [27] showed that, although a dominant Energies 2021, 14, 7535 3 of 25 3 of 25 trend could be found in most cases, there are always opposing trends proposed elsewhere by contrast. As possible reasons for this, they indicate: the large number of different engine technologies tested, the various operating conditions or driving cycles followed, the different biodiesel fuels used (i.e., obtained from different feedstocks and with different qualities), and the various measurement techniques and procedures applied. In 2018, Chen et al. [31] titled their article “NOx emission of biodiesel compared to diesel: Higher or lower?”, thus evidencing that the question has remained open. On a deeper analysis, the need for further research appears less surprising, as the literature on the subject consists only of results obtained from different engines, with different injection systems, and in different operating conditions. Moreover, the effect of biodiesel is sometimes addressed by taking the engine as a “black box”; thus, not all the data necessary to analyze the problem are reported (or known by the experimenters). Moreover, in several experiments, it is very complex to isolate the different thermo-chemical phenomena and to control the operating conditions and devices of the engine that can contribute to the variation of the performance and the emissions of a motor fueled with blends of biodiesel. 1. Introduction In general, the main causes of the increase in the NOx formation are [29]: (i) the advancement of the injection (due to the higher bulk modulus of biodiesel) and, therefore, the higher in-cylinder pressure and temperature; (ii) the lower formation of soot and the consequent lower radiative heat transfer, which implies a higher combustion temperature; (iii) the higher oxygen content in the biodiesel composition, which facilitates the combustion and the formation of NOx; (iv) other physical properties of the fuel, like the different viscosity and density, which increase the amount of fuel injected with a consequent higher combustion temperature, lower spray cone angle, and fuel atomization; (v) the higher and advanced heat release rate (HRR) in the premixed phase of the combustion process. The various combinations of the factors cited above interfere with each other to determine NOx emissions: the increasing or decreasing trend depends on the delicate balance due to every change in the fuel and combustion characteristics or engine operating conditions [22]. g g p g However, it is worthwhile to note that there is no shared agreement on the reasons for the higher NOx formation with biodiesel. At first this could seem quite strange, given the number of researchers that have analyzed the effects of these parameters, starting from the first organized studies by the National Renewable Energy Laboratory (NREL) to measure the sound speed of biodiesel and to establish its impact on the injection timing in diesel engines [32]. In their review article, Jinlin Xue et al. [28], in trying to individuate the factors of effect on NOx emissions for biodiesel, indicate the higher oxygen content as the main cause of the NOx emissions increase, but they say that the injection characteristics also have an impact and that a further study is needed to inquire into the effect of the injection timing and pressure. A key role of fuel-based oxygen was also found by Graboski and McCormick [33] after analyzing a large number of test results of various vegetable oil based fuels. They concluded that the main advantage of feeding biodiesels in diesel engines seemed related to their high oxygen content. On the other hand, Lapuerta [34] suggests that the oxygen/fuel mass ratio of biodiesel (equal to 2.92) is lower than the mineral diesel fuel ratio (3.58) and, therefore, the oxygen content cannot be the reason for the higher concentration of NOx. 1. Introduction Some authors have reported that full load torque in unchanged diesel engines does not vary substantially by switching from diesel fuel to the ME biofuels because the actual energy content delivered per each active stroke of the plunger is not substantially lower than with the diesel fuel [44,45]. Indeed, the higher viscosity of the RME reduces the internal leakages of the injection pump, and this increases the volumetric fuel delivery per stroke. For example, Labeckas et al. [39] found that, within an unmodified diesel engine, the volumetric fuel delivery per stroke with the RME increased by about 2.6% with respect to the diesel fuel. y p As far as the injection pressure and an advance in the start of ignition (SOI) is consid- ered, it is necessary to assess in a different way the engines that operate with a common rail system and with a mechanical injection system. In particular, in the mechanical injection systems, the injection timing can be modified only by regulating the fuel compression stroke with some shims. An advanced injection timing leads to enhancements in the turbu- lence of the mixture, to an earlier and rapid combustion, to a longer ignition delay time, and to higher combustion chamber pressures and temperatures [30,46]. On the other hand, a retarded injection timing results in lower chamber pressures and temperatures during the combustion, as well as in a shorter ignition delay time [30]. Several experimental inves- tigations [22,30] have revealed that an advance of the injection timing results in a reduction of CO, unburned HC, and PM emissions. The same studies showed that retarding the fuel injection can reduce the NOx concentration at the exhaust thanks to the reduction of the peak temperature in the engine [47]. As a matter of fact, both the injection timing and the injection pressure are easily controllable only in common rail injection systems, where the SOI event is commanded by the electronic control unit (ECU) and the injection pressure in the rail can be easily regulated. For common rails, one could reasonably conclude that the injection timing is fluid-independent, whilst the pressure in the rail may vary with biodiesel in case the same torque has to be reached [48–50]. Nevertheless, an advance in the SOI and a higher in-cylinder pressure was also reported in common rail engines [51]. 1. Introduction Among the different types of biodiesels and fueling systems, this study focuses on a methyl ester biodiesel used in a pump-line-nozzle (PLN) injection system. The PNL CI engines are still a widespread technology in small-scale engines for cogeneration, for industrial applications, in the agricultural sector, and in off-highway diesel engine applications where more advanced technologies are too expensive and, often, advanced gas-cleaning solutions cannot be adopted. Indeed, in all of these machines, a simple and straightforward solution for the reduction of pollutants and the limitation of greenhouse gas emissions is surely the adoption of alternative (bio)fuels [35]. The engine used here is also particularly representative of the majority of the engines used in agricultural vehicles, and also due to a mere numerical issue: in the most developed countries, the agricultural sector is characterized by a very low turnover of mechanical units (e.g., even less than Energies 2021, 14, 7535 4 of 25 1% in Italy in 2012 [36]) and a consequent high average age (around 25 years in Italy [36]), while developing countries prefer engines/machines with a simple and robust construction due to many economic and environmental conditions. In both scenarios, newer engine technologies (e.g., the common rail) are still struggling to gain market shares. The simplicity of these engines also has a further appreciable effect in an experimental context: due to their intrinsic simplicity, it is possible to avoid any masking of the effects of fuel substitution as a result of the engine’s automatic adjustment mechanisms used in more recent, electronically controlled engines (first and foremost, the automatic injection advance variation system). g j y Diesel engines operating on neat methyl esters (ME) or their blends with diesel fuel have already been tested by various researchers evidencing, in line with the majority of the biodiesels, reductions in CO, HC, and PM emissions and smoke, along with a higher concentration of NOx in the exhausts. Çelikten [37] reported that in all performance modes, RME leads to lower CO and to higher NOx emissions than the fossil fuels. Krahl et al. [38] found similar results on these pollutants, but they could not find a general tendency regarding the HC emissions. Most of the authors agreed that the reduction of the calorific value due to the ME mixing with diesel fuel causes an increase in the brake-specific fuel consumption (BSFC) [39–43]. 1. Introduction However, mechanical injection systems have been shown to be influenced by some fluid properties, mainly the bulk modulus and the viscosity, and it is almost generally accepted that, when substituting biodiesel to diesel fuel: (i) the SOI is advanced and (ii) the injection pressure becomes higher. These conclusions are somehow taken for granted and often used to explain the biodiesel NOx effect, and their combined effect advances the start of combus- tion (SOC) and contributes to a higher combustion rate; both enhance the NOx formation according to the well-documented Zeldovich mechanism [52]. It is worth remembering that only data from experiments at the fixed position of the injection pump should be considered. If the engine was set at the same torque level (for a given rotational speed), a greater amount of biodiesel should be injected to compensate for its lower heating value. In such cases, the injection pump settings should not be the same for the diesel fuel and the Energies 2021, 14, 7535 5 of 25 biodiesel. Moreover, some injection pumps have a fixed end of injection timing and, when additional quantities of fuel are requested to be injected, the start of injection is advanced. biodiesel. Moreover, some injection pumps have a fixed end of injection timing and, when additional quantities of fuel are requested to be injected, the start of injection is advanced. In a previous article [53], the differences in the injection timing and the pressure of the RME as an alternative to diesel fuel in a PLN injection system were discussed. The final considerations suggested that the injection timing due to the higher bulk modulus of biodiesel is less advanced than is commonly believed. The aim of the present work is to study in greater detail the injection characteristics of a CI engine fueled with pure RME (B100) in pump-line-nozzle systems. In such engines, the adoption of modern injection solutions and gas cleaning solutions is necessarily limited due to cost issues, and a lower environmental impact can only be obtained by using alternative (bio)fuels and tuning their management so to optimize the performance and the emissions. This study tries to supply some general explanations and figures that can be helpful to interpret the phenomena occurring in the fuel line and in the combustion process when using biodiesel, as well as in engines with different construction characteristics and fueling systems. 1. Introduction Specifically, the performance variations, the thermal efficiency, and the NOx concentration in a mechanical PLN engine are reported in this work. Further analyses and the consequences on combustion behavior are evidenced, in particular the SOI and the SOC advances are shown, and the trend of the HRR is evaluated with the aim of finding a correlation with the biodiesel, etc. Moreover, a quick and simple modification of the fuel injection timing on the pump line was tested in order to highlight the chance of reducing the NOx increase in this typology of engines without causing any performance decay. Some authors also delved into the RME combustion characteristics [54–61], and their results are addressed in the concluding Section 3.4 and compared with those of the present study. 2. Materials and Methods Quantity Unit Diesel Fuel Biodiesel Bulk modulus MPa 1490 1650 Cetane number - 48 54 C/H/O molar ratio 16:30:0 19:34:2 Density at 15 °C kg·dm−3 0.835 0.885 Lower heating value on a mass basis (LHVm) kJ·kg−1 42,739 37,400 Kinematic viscosity at 40 °C mm2·s−1 2.60 4.50 Carbon content wt % 86.5 77.4 Hydrogen content wt % 13.4 12.0 Oxygen content wt % 0.0 10.5 Sulfur content wt % 0.05 <0.01 Stoichiometric air-fuel ratio - 14.5 12.6 Iodine value g(I2)·(100 g)−1 - 118.2 Pour point °C −26 −12 Flash point °C 72 >150 Biodegradability (after 28 days) % 18 85 Quantity Unit Diesel Fuel Biodiesel Bulk modulus MPa 1490 1650 Cetane number - 48 54 C/H/O molar ratio 16:30:0 19:34:2 Density at 15 ◦C kg·dm−3 0.835 0.885 Lower heating value on a mass basis (LHVm) kJ·kg−1 42,739 37,400 Kinematic viscosity at 40 ◦C mm2·s−1 2.60 4.50 Carbon content wt % 86.5 77.4 Hydrogen content wt % 13.4 12.0 Oxygen content wt % 0.0 10.5 Sulfur content wt % 0.05 <0.01 Stoichiometric air-fuel ratio - 14.5 12.6 Iodine value g(I2)·(100 g)−1 - 118.2 Pour point ◦C −26 −12 Flash point ◦C 72 >150 Biodegradability (after 28 days) % 18 85 EVIEW 7 of 26 Quantity Unit Diesel Fuel Biodiesel Bulk modulus MPa 1490 1650 Cetane number - 48 54 C/H/O molar ratio 16:30:0 19:34:2 Density at 15 °C kg·dm−3 0.835 0.885 Lower heating value on a mass basis (LHVm) kJ·kg−1 42,739 37,400 Kinematic viscosity at 40 °C mm2·s−1 2.60 4.50 Carbon content wt % 86.5 77.4 Hydrogen content wt % 13.4 12.0 Oxygen content wt % 0.0 10.5 Sulfur content wt % 0.05 <0.01 Stoichiometric air-fuel ratio - 14.5 12.6 Iodine value g(I2)·(100 g)−1 - 118.2 Pour point °C −26 −12 Flash point °C 72 >150 Biodegradability (after 28 days) % 18 85 Quantity Unit Diesel Fuel Biodiesel Bulk modulus MPa 1490 1650 Cetane number - 48 54 C/H/O molar ratio 16:30:0 19:34:2 Density at 15 ◦C kg·dm−3 0.835 0.885 Lower heating value on a mass basis (LHVm) kJ·kg−1 42,739 37,400 Kinematic viscosity at 40 ◦C mm2·s−1 2.60 4.50 Carbon content wt % 86.5 77.4 Hydrogen content wt % 13.4 12.0 Oxygen content wt % 0.0 10.5 Sulfur content wt % 0.05 <0.01 Stoichiometric air-fuel ratio - 14.5 12.6 Iodine value g(I2)·(100 g)−1 - 118.2 Pour point ◦C −26 −12 Flash point ◦C 72 >150 Biodegradability (after 28 days) % 18 85 REVIEW 7 of 26 Figure 2. 2. Materials and Methods The experiments were carried out on a single-cylinder Ruggerini RF91 diesel engine (by Kohler Lombardini, Reggio nell’Emilia, Italy [62]), whose main characteristics are described in Table 1. The engine was tested on an APIcom RE100 test stand (Cento, Ferrara, Italy) with commercial diesel fuel and rapeseed biodiesel (Figure 1). At the end of each trial, the fuel was switched back to diesel fuel and the engine was kept running for a while before shut-down to flush out the biodiesel from the fuel line and the injection system. The properties of the two fuels are reported in Table 2. In particular, according to the EASAC report 2012 classification, the biodiesel used during the experiments is a first generation one, produced starting from the oil extracted from rapeseed and passed through refining, transesterification, and subsequent distillation processes [16]. Table 1. Technical characteristics of the tested diesel engine (Ruggerini RF51). Quantity Specification Type Single-cylinder four-stroke direct injection Nr. of valves 2–OHV Bore × stroke 90 × 75 mm Displacement 477 cm3 Compression ratio 18.5 Maximum power 8.1 kW (11 CV) at 3600 rpm Maximum torque 25 N·m (2.5 kgf·m) at 2500 rpm Injection advance 16◦CA before top dead center Injection nozzle opening pressure 210 bar Table 1. Technical characteristics of the tested diesel engine (Ruggerini RF51). Table 1. Technical characteristics of the tested diesel engine (Ruggerini RF51). Quantity Specification Type Single-cylinder four-stroke direct injection Nr. of valves 2–OHV Bore × stroke 90 × 75 mm Displacement 477 cm3 Compression ratio 18.5 Maximum power 8.1 kW (11 CV) at 3600 rpm Maximum torque 25 N·m (2.5 kgf·m) at 2500 rpm Injection advance 16◦CA before top dead center Injection nozzle opening pressure 210 bar Specification Single-cylinder four-stroke direct injection 2–OHV 90 × 75 mm 477 cm3 18.5 8.1 kW (11 CV) at 3600 rpm 25 N·m (2.5 kgf·m) at 2500 rpm 16◦CA before top dead center 210 bar In-cylinder and injection pressures were measured by using piezoelectric transducers, specifically an AVL GU12P (Graz, Austria [63]) fitted on the cylinder head, and a PCB Piezotronics 112A21 (Depew, NY, USA [64]) fitted on the injection pipe about 10 mm apart (Figure 2). An AVL 3066A0” charge amplifier received the outputs of the pressure transducers and fed them to an AVL 619 INDIMETER system. 2. Materials and Methods p Quantity Unit Diesel Fuel Biodiesel Bulk modulus MPa 1490 1650 Cetane number - 48 54 C/H/O molar ratio 16:30:0 19:34:2 Density at 15 °C kg·dm−3 0.835 0.885 Lower heating value on a mass basis (LHVm) kJ·kg−1 42,739 37,400 Kinematic viscosity at 40 °C mm2·s−1 2.60 4.50 Carbon content wt % 86.5 77.4 Hydrogen content wt % 13.4 12.0 Oxygen content wt % 0.0 10.5 Sulfur content wt % 0.05 <0.01 Stoichiometric air-fuel ratio - 14.5 12.6 Iodine value g(I2)·(100 g)−1 - 118.2 Pour point °C −26 −12 Flash point °C 72 >150 Biodegradability (after 28 days) % 18 85 Table 2. Main specifications about the used fuels. Quantity Unit Diesel Fuel Biodiesel Bulk modulus MPa 1490 1650 Cetane number - 48 54 C/H/O molar ratio 16:30:0 19:34:2 Density at 15 ◦C kg·dm−3 0.835 0.885 Lower heating value on a mass basis (LHVm) kJ·kg−1 42,739 37,400 Kinematic viscosity at 40 ◦C mm2·s−1 2.60 4.50 Carbon content wt % 86.5 77.4 Hydrogen content wt % 13.4 12.0 Oxygen content wt % 0.0 10.5 Sulfur content wt % 0.05 <0.01 Stoichiometric air-fuel ratio - 14.5 12.6 Iodine value g(I2)·(100 g)−1 - 118.2 Pour point ◦C −26 −12 Flash point ◦C 72 >150 Biodegradability (after 28 days) % 18 85 EVIEW 7 of 26 Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Table 3 Main specifications about the measuring instruments used in the test campaign Hole to house the in- chamber pressure sensor tip Fuel injection pressure sensor Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. 2. Materials and Methods An AVL 36401 encoder, connected to the engine output shaft, passed its signal to the AVL 619 INDIMETER through Energies 2021, 14, 7535 6 of 25 an AVL 3064Z03 light pulse converter. For each working condition, pressure signals were measured over 50 consecutive cycles with 0.5◦resolution, and then averaged. The main characteristics of the measuring instruments are reported in Table 3. Maximum power 8.1 kW (11 CV) at 3600 rpm Maximum torque 25 N·m (2.5 kgf·m) at 2500 rpm Injection advance 16 °CA before top dead center Injection nozzle opening pressure 210 bar an AVL 3064Z03 light pulse converter. For each working condition, pressure signals were measured over 50 consecutive cycles with 0.5◦resolution, and then averaged. The main characteristics of the measuring instruments are reported in Table 3. Maximum power 8.1 kW (11 CV) at 3600 rpm Maximum torque 25 N·m (2.5 kgf·m) at 2500 rpm Injection advance 16 °CA before top dead center Injection nozzle opening pressure 210 bar an AVL 3064Z03 light pulse converter. For each working condition, pressure signals were measured over 50 consecutive cycles with 0.5◦resolution, and then averaged. The main characteristics of the measuring instruments are reported in Table 3. Maximum power 8.1 kW (11 CV) at 3600 rpm Maximum torque 25 N·m (2.5 kgf·m) at 2500 rpm Injection advance 16 °CA before top dead center Injection nozzle opening pressure 210 bar (b) (a) (a) (b) Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the execution of the tests. Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the execution of the tests. (a) (b) Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the execution of the tests. Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the execution of the tests. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. 2. Materials and Methods Instrument Specifications Dyno brake model (APIcom, Cento (FE), Italy) APIcom Eddy current RE100—X TWIN—4943/02 Torque measurement (APIcom, Cento (FE), Italy) Range 0–500 N·m; Uncertainty ± 0.25% Rotational speed measurement (APIcom, Cento (FE), Italy) Range 0–10,000 rpm; Uncertainty ± 10 rpm Load indicator (APIcom, Cento (FE), Italy) Range 250–6000 W; Uncertainty ± 10 W Temperature indicator Range 0–900 ◦C; Uncertainty ± 1 ◦C Fuel Volume Burette range 1–30 cm3; Uncertainty ± 0.2 cm3 Exhaust gas analyzer Range NO 0–5000 ppm; Uncertainty ± 50 ppm Range HC 0–20,000 ppm; Uncertainty ± 10 ppm Range CO 0–10%; Uncertainty ± 0.03% Pressure transducer (AVL, Graz, Austria) Range 0–110 bar; Uncertainty ± 0.1 bar Crank angle encoder (AVL, Graz, Austria) Range 0–360◦; Uncertainty ± 0.1◦ Specifications APIcom Eddy current RE100—X TWIN—4943/02 Range 0–500 N·m; Uncertainty ± 0.25% Range 0–10,000 rpm; Uncertainty ± 10 rpm Range 250–6000 W; Uncertainty ± 10 W Range 0–900 ◦C; Uncertainty ± 1 ◦C Burette range 1–30 cm3; Uncertainty ± 0.2 cm3 Range NO 0–5000 ppm; Uncertainty ± 50 ppm Range HC 0–20,000 ppm; Uncertainty ± 10 ppm Range CO 0–10%; Uncertainty ± 0.03% Range 0–110 bar; Uncertainty ± 0.1 bar Range 0–360◦; Uncertainty ± 0.1◦ Specifications APIcom Eddy current RE100—X TWIN—4943/02 Range 0–500 N·m; Uncertainty ± 0.25% Range 0–10,000 rpm; Uncertainty ± 10 rpm Range 250–6000 W; Uncertainty ± 10 W Range 0–900 ◦C; Uncertainty ± 1 ◦C Burette range 1–30 cm3; Uncertainty ± 0.2 cm3 Range NO 0–5000 ppm; Uncertainty ± 50 ppm Range HC 0–20,000 ppm; Uncertainty ± 10 ppm Range CO 0–10%; Uncertainty ± 0.03% Range 0–110 bar; Uncertainty ± 0.1 bar Range 0–360◦; Uncertainty ± 0.1◦ Pressure transducer (AVL, Graz, Austria) Crank angle encoder (AVL, Graz, Austria) By using appropriate correlations for the heat losses, the gross HRR would have been derived from the net HRR (NHRR) curve but considering that the aim was comparing the combustion behavior of diesel fuel and B100 in the same engine, the differences in heat losses were considered to be the same in all the fueling conditions, thus not influencing the outcomes of the study. Besides, most of the literature regarding the analysis of RME combustion, in engines similar to that of the present study, report data on either the net or the apparent heat release rate. 2. Materials and Methods The SOC was located in correspondence with the crank angle (CA) at which the first derivative of the in-cylinder pressure with respect to crank angle (dp/dθ) suddenly changes its slope, going from a negative to a positive value, and so presenting a local minimum [65]. p g g g p p g During the experimental campaign, the engine was tested at full load and at rotational speeds spanning from 2000 rpm to 3000 rpm, with steps of 250 rpm. The pump control rack was fixed at its maximum so that the pump operated in the same way independently of the delivered fuel. By doing so, only differences in the fuel properties were responsible for changes in the injection and combustion parameters. Besides, as shown in Section 3.1, the brake mean effective pressures (BMEPs) at full load were almost the same with the two fuels. For this reason, the full-load comparisons are also comparisons at nearly the same BMEP value. 2. Materials and Methods Detailed view showing the positions on the engine head of the piezoresistive sensors by AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Table 3. Main specifications about the measuring instruments used in the test campaign. Hole to house the in- chamber pressure sensor tip Fuel injection pressure sensor Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Hole to house the in- chamber pressure sensor tip Fuel injection pressure sensor Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. 7 of 25 Energies 2021, 14, 7535 Table 3. Main specifications about the measuring instruments used in the test campaign. Table 3. Main specifications about the measuring instruments used in the test campaign. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Figure 3 shows the fuel mass flow rate and the corresponding injection quantities per cycle as a function of the rotational speed. The mass of B100 injected per cycle is 8–10% greater than that of the diesel fuel to compensate for the lower heating value. Consequently, the engine brake torque, as shown in Figure 4, is almost the same with the two fuels, while the BSFC of the RME is slightly higher, in accordance with the higher fuel flow rate injected. These results confirm the outcomes of other authors that studied similar injection systems [44,45]. Figure 5 reports the comparison of NOx emissions with the diesel fuel and the RME. The NOx concentrations are higher with the RME than with the diesel fuel, thus confirming the well-known biodiesel NOx effect, except for the higher rotational speeds, in correspon- dence to which the differences between the two fuels become lower. Figure 6 reports the percentage increase of the NOx concentration in the exhaust with the RME compared to the diesel fuel as a function of the rotational speed. On the same chart, the variations of the heat release peak between the diesel fuel and the RME during the combustion process are also reported. Even though a detailed description of the combustion process is reported in the next section, this interesting figure is anticipated, as it helps to highlight how the increase of the NOx emissions with biodiesel is strongly correlated to the increase of the heat release peak; indeed, the two above-mentioned trends present a similar morphology. This can be explained by considering the main mechanism of NOx formation in a combustion process: Energies 2021, 14, 7535 8 of 25 with the higher the thermal formation mechanism. A higher peak in the heat release is obviously correlated to a higher local temperature and pressure in the combustion chamber, thus to a higher risk of formation of this polluting compound. injection systems [44,45]. the thermal formation mechanism. A higher peak in the heat release is obviously correlated to a higher local temperature and pressure in the combustion chamber, thus to a higher risk of formation of this polluting compound. injection systems [44,45]. g p p , g risk of formation of this polluting compound. gure 3. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two test els. 20 22 24 26 28 30 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 1750 2000 2250 2500 2750 3000 3250 injected quantity (mg cycle-1) fuel consumption (g h-1) engine rotational speed (rpm) fuel consumption Diesel fuel consumption RME injected volume Diesel injected volume RME Figure 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fuels. 4, x FOR PEER REVIEW Figure 4 Engine torque and BSFC as a function of the rotational speed for the two tested fuels 0 50 100 150 200 250 300 350 400 450 500 15.0 17.5 20.0 22.5 25.0 27.5 30.0 1750 2000 2250 2500 2750 3000 3250 brake specific fuel consumption (g/kWh) torque (Nm) rotational speed (rpm) torque Diesel fuel torque RME BSFC Diesel fuel BSFC RME Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. risk of formation of this polluting compound. 20 22 24 26 28 30 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 1750 2000 2250 2500 2750 3000 3250 injected quantity (mg cycle-1) fuel consumption (g h-1) engine rotational speed (rpm) fuel consumption Diesel fuel consumption RME injected volume Diesel injected volume RME , x FOR PEER REVIEW ass flow rate and injected mass per cycle as a function of the engine rotational speed for the tw el mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fue ure 3 Fuel mass flow rate and injected mass per cycle as a function of the engine rotation Figure 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed re 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two . Figure 3. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels 0 20 40 60 80 100 120 140 1750 2000 2250 2500 2750 3000 3250 NOx emissions (ppm) rotational speed (rpm) Diesel fuel RME Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. 0 20 40 60 80 100 120 140 1750 2000 2250 2500 2750 3000 3250 NOx emissions (ppm) rotational speed (rpm) Diesel fuel RME rotational speed (rpm) unction of the rotational sp otat o a speed ( p ) Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. g p fuels. fuels. Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function of the rotational speed (ref diesel fueling) 0% 10% 20% 30% 40% 50% 60% 1750 2000 2250 2500 2750 3000 3250 NOx and max heat release variations (%) rotational speed (rpm) NOx % variations max heat release % variations Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function of the rotational speed (ref. diesel fueling). 0% 10% 20% 30% 40% 50% 60% 1750 2000 2250 2500 2750 3000 3250 NOx and max heat release variations (%) rotational speed (rpm) NOx % variations max heat release % variations Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function of the rotational speed (ref. diesel fueling). Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function of the rotational speed (ref diesel fueling) rotational speed (rpm) Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function of the rotational speed (ref. diesel fueling). Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function of the rotational speed (ref. diesel fueling). of the rotational speed (ref. diesel fueling). 3.2. Injection Pressure Characteristics and Combustion Analysis 3.2. Injection Pressure Characteristics and Combustion Analysis 3.2. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Injection Pressure Characteristics and Combustion Analysis The fuel pressure in the injection pipe was measured by means of a piezoelectric transducer, and the recorded signals were pressure referenced according to the method proposed in [53]. In fact, the piezoelectric transducers can detect rapid pressure variations but, conversely, give no information on the absolute pressure values; therefore, pressure referencing is needed if absolute pressure values are required or different situations must be compared, as in this case. Indeed, an incorrect pressure referencing may lead to errors in the injection advance and the injection pressure estimation [19]. Thus, in the following figures (Figures 7–11) the pressure values are reported as relative to the fuel’s vapor pres- sure (the differences between vapor pressure of B100 and diesel fuel are neglected as The fuel pressure in the injection pipe was measured by means of a piezoelectric transducer, and the recorded signals were pressure referenced according to the method proposed in [53]. In fact, the piezoelectric transducers can detect rapid pressure variations but, conversely, give no information on the absolute pressure values; therefore, pressure referencing is needed if absolute pressure values are required or different situations must be compared, as in this case. Indeed, an incorrect pressure referencing may lead to errors in the injection advance and the injection pressure estimation [19]. Thus, in the following figures (Figures 7–11) the pressure values are reported as relative to the fuel’s vapor pres- sure (the differences between vapor pressure of B100 and diesel fuel are neglected as The fuel pressure in the injection pipe was measured by means of a piezoelectric transducer, and the recorded signals were pressure referenced according to the method proposed in [53]. In fact, the piezoelectric transducers can detect rapid pressure variations but, conversely, give no information on the absolute pressure values; therefore, pressure referencing is needed if absolute pressure values are required or different situations must be compared, as in this case. Indeed, an incorrect pressure referencing may lead to errors in the injection advance and the injection pressure estimation [19]. Thus, in the following figures (Figures 7–11) the pressure values are reported as relative to the fuel’s vapor pressure (the differences between vapor pressure of B100 and diesel fuel are neglected as compared to the injection pressure). 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fuels 0 50 100 150 200 250 300 350 400 450 500 15.0 17.5 20.0 22.5 25.0 27.5 30.0 1750 2000 2250 2500 2750 3000 3250 brake specific fuel consumption (g/kWh) torque (Nm) rotational speed (rpm) torque Diesel fuel torque RME BSFC Diesel fuel BSFC RME Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the tw ure 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fue Figure 4 Engine torque and BSFC as a function of the rotational speed for the two tested fuels 0 50 100 150 200 250 300 350 400 450 500 15.0 17.5 20.0 22.5 25.0 27.5 30.0 1750 2000 2250 2500 2750 3000 3250 brake specific fuel consumption (g/kWh) torque (Nm) rotational speed (rpm) torque Diesel fuel torque RME BSFC Diesel fuel BSFC RME Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. 9 of 25 10 of 26 10 of 26 Energies 2021, 14, 7535 Energies 2021, 14, x FOR Energies 2021, 14, x FOR Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. 0 20 40 60 80 100 120 140 1750 2000 2250 2500 2750 3000 3250 NOx emissions (ppm) rotational speed (rpm) Diesel fuel RME Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. 0 20 40 60 80 100 120 140 1750 2000 2250 2500 2750 3000 3250 NOx emissions (ppm) rotational speed (rpm) Diesel fuel RME Figure 5. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions While not true, this is a reasonable approximation since with injection pressures around 200 bar the error in absolute values is of the order of 1/200. 10 of 25 ng to a etected Energies 2021, 14, 7535 with the RME than with diesel. ure 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2000 rpm Figure 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load. 4, x FOR PEER REVIEW re 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2250 rpm 80 300 Figure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full load. ure 7 RME and diesel fuel injection pressures in-cylinder pressures and NHRR at 2000 rpm and at ful -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2000 rpm Figure 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load. 4, x FOR PEER REVIEW re 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at ful -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2000 rpm Figure 7. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2750 rpm 80 300 3000 rpm Figure 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load. ure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at ful -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2500 rpm Figure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load. 4, x FOR PEER REVIEW re 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2500 rpm Figure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load. x FOR PEER REVIEW ME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and a re 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load e 10. RME and diesel fuel injection pressures in-cylinder pressures and NHRR at 2750 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2750 rpm Figure 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load. re 10. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load. x FOR PEER REVIEW in-cylinder pressures ME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and a re 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load j p y p p re 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2250 rpm Figure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full load. re 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2250 rpm Figure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full load. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm an igure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full l 11 of 25 Energies 2021, 14, 7535 e 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu e 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2500 rpm Figure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load. x FOR PEER REVIEW 10. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at fu -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 2750 rpm Figure 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load. ME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and a re 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load 12 of 25 Energies 2021, 14, 7535 re 11. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full lo -10 0 10 20 30 40 50 60 70 80 0 50 100 150 200 250 300 -30 -20 -10 0 10 20 30 40 50 60 NHRR (J/ °) - in-cylinder pressure (bar) injection pressure (bar) crank angle (°) RME Diesel fuel injection pressures NHRRs in-cylinder pressures 3000 rpm Figure 11. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. E and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at f 1. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. By analyzing the injection pressure signals in a higher detail, some interes derations can be drawn regarding the injection advance and the injection durat emise, since the needle lift was not measured, the SOI was assumed to coincide ank angle position at which the fuel pressure reaches the value of the injecto ening pressure (i.e., 210 bar). Similarly, the end of injection (EOI) was assumed de with the crank angle position at which the fuel pressure reaches the value With the biodiesel, the fuel pressure recorded in the pipe just before injection was lower at all rotational speeds; nevertheless, the injection pressure was reached in advance (the black circles on the injection pressure curves of Figures 7–11 evidence the crank angular position at which the injector nozzle opening pressure is reached). 3.1. Brake Torque, Fuel Consumption, and NOx Emissions In fact, due to the lower compressibility of the biodiesel, the pressure rise produced by the pump was faster and also propagated faster toward the injectors (as a consequence of its higher sound speed) [32]. In all the investigated cases, the RME injection pressure curves seem smoother, probably because of the higher viscosity of the RME with respect to the viscosity of diesel fuel. tor nozzle closing pressure (i.e., ~150 bar). As an example, the SOIs, EOIs, and quent injection durations are reported in Figure 12 in the case of 2500 rpm. g y p y At the end of injection, cavitation occurred in the pipe. This is identifiable in the zones where the pressure curves, which had previously fallen steeply (see the pressure signals at 17◦, 19◦, 21◦, 23◦, and 26◦CA, for engine rotational speeds of 2000 rpm, 2250 rpm, 2500 rpm, 2750 rpm, and 3000 rpm, respectively), rapidly flatten and the pressure remains almost constant at the vapor pressure despite the transient nature of the phenomenon. At the same time, it is possible to observe an intense pressure wave travelling to-and-fro in the pipe. As the speed increases, the angular interval where cavitation is detected is longer with the RME than with diesel. By analyzing the injection pressure signals in a higher detail, some interesting con- siderations can be drawn regarding the injection advance and the injection duration. As a premise, since the needle lift was not measured, the SOI was assumed to coincide with the crank angle position at which the fuel pressure reaches the value of the injector noz- zle opening pressure (i.e., 210 bar). Similarly, the end of injection (EOI) was assumed to coincide with the crank angle position at which the fuel pressure reaches the value of the injector nozzle closing pressure (i.e., ~150 bar). As an example, the SOIs, EOIs, and the consequent injection durations are reported in Figure 12 in the case of 2500 rpm. q j p g p The effect of the rotational speed on the differences in the injection advance and injection duration will be commented on by analyzing Figures 13 and 14, respectively. 13 of 25 13 of 25 Energies 2021, 14, 7535 es 2021, 14, x FOR PE 300 2 Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions The effect of the rotational speed on the differences in the injection jection duration will be commented on by analyzing Figures 13 and 14, re 0 50 100 150 200 250 -20 -15 -10 -5 0 5 10 15 20 injection pressure (bar) crank angle (°) Diesel fuel Injection duration Diesel fuel Injection duration RME SOIRME SOIdiesel EOIdiesel EOIRME Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. The effect of the rotational speed on the differences in the injection a jection duration will be commented on by analyzing Figures 13 and 14, res -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 2000 2250 2500 2750 3000 residual pressure difference RME −diesel fuel (bar) injection advance difference RME −diesel fuel ( ) t ti l d ( ) injection advance residual pressure difference Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. p j jection duration will be commented on by analyzing Figures 13 and 14, respe 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the sa s a function of the engine speed and at full load. -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 2000 2250 2500 2750 3000 residual pressure difference RME −diesel fuel (bar) injection advance difference RME −diesel fuel ( ) rotational speed (rpm) injection advance residual pressure difference Figure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuels as a function of the engine speed and at full load. gure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. jection duration will be commented on by analyzing Figures 13 a Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions The effect of the rotational speed on the differences in the injection jection duration will be commented on by analyzing Figures 13 and 14, 0 50 100 150 200 250 300 -20 -15 -10 -5 0 5 10 15 20 injection pressure (bar) crank angle (°) RME Diesel fuel 2500 rpm Injection duration Diesel fuel Injection duration RME SOIRME SOIdiesel EOIdiesel EOIRME -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 residual pressure difference RME −diesel fuel (bar) injection advance difference RME −diesel fuel ( ) injection advance residual pressure difference Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. The effect of the rotational speed on the differences in the injection ad jection duration will be commented on by analyzing Figures 13 and 14, res Injection advances with the RME and the diesel fuel and residual pressure difference between the 0 50 100 150 200 250 -20 -15 -10 -5 0 5 10 15 20 injection pressure (bar) crank angle (°) Diesel fuel Injection duration Diesel fuel Injection duration RME SOIRME SOIdiesel EOIdiesel EOIRME -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 2000 2250 2500 2750 3000 residual pressure difference RME −diesel fuel (bar) injection advance difference RME −diesel fuel ( ) rotational speed (rpm) injection advance residual pressure difference gure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuels Figure 12 Injection durations with the RME and the diesel fuel at 2500 rpm and at full load 0 50 100 150 200 250 300 -20 -15 -10 -5 0 5 10 15 20 injection pressure (bar) crank angle (°) RME Diesel fuel 2500 rpm Injection duration Diesel fuel Injection duration RME SOIRME SOIdiesel EOIdiesel EOIRME Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions The effect of the rotational speed on the differences in the injection jection duration will be commented on by analyzing Figures 13 and 14, -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0 00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 residual pressure difference RME −diesel fuel (bar) injection advance difference RME −diesel fuel ( ) injection advance residual pressure difference I j i d i h h RME d h di l f l d id l diff b h -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 2000 2250 2500 2750 3000 residual pressure difference RME −diesel fuel (bar) injection advance difference RME −diesel fuel ( ) rotational speed (rpm) injection advance residual pressure difference gure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuel e in 0 -18 0.00 2000 2250 2500 2750 3000 r 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the s s a function of the engine speed and at full load. Figure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuels as a function of the engine speed and at full load. 14 of 25 14 of 25 Energies 2021, 14, 7535 PEER REVIEW i E d d l f l l d d Figure 14. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. Figure 14. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. nd diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm d, due to the higher bulk modulus, the injection with the RME is advanced of 0.27° CA at 2500 rpm and a maximum of around 1° CA at 3000 rpm. confirm the so-called biodiesel inadvertent injection advance effect, but As expected, due to the higher bulk modulus, the injection with the RME is advanced by a minimum of 0.27◦CA at 2500 rpm and a maximum of around 1◦CA at 3000 rpm. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions These findings confirm the so-called biodiesel inadvertent injection advance effect, but also evidence a nonmonotone trend with the rotational speed. The reason for this nonmonotone behavior can be explained by having a look at the variation in the rotational speed of the residual pressure differences between the two fuels. j nonmonotone trend with the rotational speed. The reason for this non- vior can be explained by having a look at the variation in the rotational dual pressure differences between the two fuels. hown in the graphs in Figure 13 the decrease in the residual pressure In fact, as shown in the graphs in Figure 13, the decrease in the residual pressure, caused by the use of the RME, which partially compensates for the advancing effect of its higher bulk modulus, has a very similar trend to that of the advance—only the data at 3000 rpm seem against the above-evidenced behavior. hown in the graphs in Figure 13, the decrease in the residual pressure, e of the RME, which partially compensates for the advancing effect of its dulus, has a very similar trend to that of the advance—only the data at against the above-evidenced behavior. reconsider the results in Figure 6. Surprisingly, it appears that the bio- t is more evident at 2500 rpm where the injection advance differences Now, let us reconsider the results in Figure 6. Surprisingly, it appears that the biodiesel NOx effect is more evident at 2500 rpm, where the injection advance differences between the two fuels are the lowest. However, as already evidenced by Figure 6, this is explainable by the higher peak of the heat release, which occurs despite the reduced advance. This is another example of the not always straightforward behavior of biodiesel as concerns the combustion analysis and NOx formation mechanisms. t is more evident at 2500 rpm, where the injection advance differences fuels are the lowest. However, as already evidenced by Figure 6, this is h hi h k f th h t l hi h d it th d d d As is visible in Figure 14, the total injection angle increases with the rotational speed for both fuels, but the injection lasts almost 3–4% longer when using RME. he higher peak of the heat release, which occurs despite the reduced ad- other example of the not always straightforward behavior of biodiesel as mbustion analysis and NOx formation mechanisms. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions in Figure 14, the total injection angle increases with the rotational speed These results contribute to explain the increase of the injection rate of the RME with re- spect to diesel fuel. Overall, the higher injection pressures and the longer injection duration cause an increase of the injected mass per cycle of RME, which in the end compensates for the reduction of the fuel LHVm, thus yielding almost the same torque values for both fuels. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics As confirmed by the positio e local minima of the in-cylinder pressure 1st derivative in the Figures 15–19, the r the biodiesel was almost the same as for the diesel fuel at 2000 rpm; then, as the onal speed increased, the SOC of the biodiesel was slightly shifted to an earlier ith the advance reaching about 0.75° CA at 3000 rpm. g p gure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full loa gure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full loa 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Fi 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full load. gure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full loa gure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full loa As the advances in the SOC were almost identical to the SOI advance 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Fi 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full load. ure 15. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics s contribute to explain the increase of the injection rate of the RME with fuel. Overall, the higher injection pressures and the longer injection du- ncrease of the injected mass per cycle of RME, which in the end compen- uction of the fuel LHVm, thus yielding almost the same torque values for As previously shown in Figures 7–11, the NHRR graphs and the general combustion characteristics are those of a classic direct injection diesel engine for both biodiesel and diesel fuel. Starting from the SOI, the HRR is slightly negative during the ignition delay period due to the cooling effect of fuel vaporization; then, in the initial phase of combustion, a rapid HRR occurs because of the combustion of the fuel that has mixed with air during the ignition delay. After this phase, the combustion continues slowly until most of fuel is Energies 2021, 14, 7535 15 of 25 15 of 25 burned; the final combustion phase is the late combustion, which continues until the end of the expansion stroke. Instead, some differences between the biodiesel and the diesel fuel occurred regarding the SOC and HRR values. As confirmed by the positions of the local minima of the in-cylinder pressure 1st derivative in the Figures 15–19, the SOC for the biodiesel was almost the same as for the diesel fuel at 2000 rpm; then, as the rotational speed increased, the SOC of the biodiesel was slightly shifted to an earlier stage, with the advance reaching about 0.75◦CA at 3000 rpm. e end of the expansion stroke. Instead, some differences between the biodiesel an esel fuel occurred regarding the SOC and HRR values. As confirmed by the positio e local minima of the in-cylinder pressure 1st derivative in the Figures 15–19, the r the biodiesel was almost the same as for the diesel fuel at 2000 rpm; then, as the onal speed increased, the SOC of the biodiesel was slightly shifted to an earlier s ith the advance reaching about 0.75° CA at 3000 rpm. esel fuel occurred regarding the SOC and HRR values. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full lo 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Fi Figure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full load. ure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full lo 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Fi 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full l igure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full load. 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full l ure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full lo 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel ure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full lo 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full load. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel 16 RME d di l f l i li d d t f i t 2250 d t f g ( ) re 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at fu Figure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full load Energies 2021, 14, 7535 16 of 25 ure peak p 16 of 25 ure peak Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full loa 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 17. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics This behavior can be interprete considering that the reduction of the chemical ignition delay due to the higher ce number of the RME is probably compensated by an increased physical ignition delay t t i ti d i t f ti A h i h 3 2 th i j ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full lo crank angle (°) Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full lo crank angle ( ) 90 2 7. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full igure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. 7. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel g y p p p ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. As the advances in the SOC were almost identical to the SOI advances, the angular ignition delays were comparable for the two fuels. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full loa 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0 15 30 45 60 75 -1 -0.5 0 0.5 1 1.5 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. As the advances in the SOC were almost identical to the SOI advances, the ang ignition delays were comparable for the two fuels. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics i 20 i i ibl h h f f li h li i d ff p g y g p g g In order to assess the effect of the variation of the injection timing on the performance and emissions, and to try to offset the biodiesel NOx effect, the standard injection timing has been modified starting from the reference value of 16◦CA before top dead center (BTDC). Starting from this reference injection condition, and based on the manufacturer’s technical indications, two other configurations were set, properly adjusting the number of shims in the injection pump: a late injection equal to 14◦CA BTDC, and an early injection equal to 18◦CA BTDC. igure 20, it is possible to note that a change of fueling has a limited effect rque. Moreover, consider that, for example, the differences in torque at he same magnitude of the measurement error, and therefore no precise e depicted. Nevertheless, in the analysis of the trends one can observe gher value is obtained with the RME by retarding the injection timing to ds with the RME with the standard SOI, and by anticipating the injection, l i i h h di l f li Observing Figure 20, it is possible to note that a change of fueling has a limited effect on the engine torque. Moreover, consider that, for example, the differences in torque at 2500 rpm are of the same magnitude of the measurement error, and therefore no precise indications can be depicted. Nevertheless, in the analysis of the trends one can observe that a slightly higher value is obtained with the RME by retarding the injection timing to +2◦CA. The trends with the RME with the standard SOI, and by anticipating the injection, are practically overlapping with the diesel fueling. erlapping with the diesel fueling. the specific fuel consumption (Figure 21), a higher influence of the SOI g can be appreciated. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics This behavior can be interpreted by considering that the reduction of the chemical ignition delay due to the higher cetane number of the RME is probably compensated by an increased physical ignition delay due ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel ure 18 RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. crank angle (°) e 18 RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at f Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load esel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. esel fuel in cylinder pressures and rate of pressure rise at 2750 rpm and at full load. As the advances in the SOC were almost identical to the SOI advances, the angular ignition delays were comparable for the two fuels. This behavior can be interpreted by considering that the reduction of the chemical ignition delay due to the higher cetane number of the RME is probably compensated by an increased physical ignition delay due to a worse atomization and mixture formation. As shown in paragraph 3.2, the injection pressure of the RME and diesel fuel were similar, and the worse mixture formation is attributable to the RME’s higher surface tension, higher flash point, and poorer volatility. 17 of 25 Energies 2021, 14, 7535 EER REVIEW Figure 19. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm and at 0 15 30 45 60 75 90 -1 -0.5 0 0.5 1 1.5 2 -40 -20 0 20 40 pressure (bar) 1st pressure derivative (bar/°) crank angle (°) 1st derivative of in−cylinder pressure RME Diesel fuel in−cylinder pressure RME Diesel fuel Figure 19. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm and at full load. gure 19. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm a Figure 19. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm and at full load values of NHRR are likely to be a consequence of the biodiesel molecular deed, as the oxygenated fuel evaporates, the oxygen bound in the fuel is After the SOC, the NHRR curves of biodiesel became steeper than those of diesel fuel, and a higher peak was reached due to an increased amount of fuel undergoing premixed combustion. This also caused higher rates of pressure rise and higher pressure peaks (Figures 15–19). eed, as the oxygenated fuel evaporates, the oxygen bound in the fuel is eased, thus enhancing the flame speed and resulting in a higher HRR. ssess the effect of the variation of the injection timing on the performance d t t t ff t th bi di l NO ff t th t d d i j ti ti i g The higher values of NHRR are likely to be a consequence of the biodiesel molecular composition; indeed, as the oxygenated fuel evaporates, the oxygen bound in the fuel is progressively released, thus enhancing the flame speed and resulting in a higher HRR. nd to try to offset the biodiesel NOx effect, the standard injection timing ed starting from the reference value of 16° CA before top dead center from this reference injection condition, and based on the manufacturer’s ions, two other configurations were set, properly adjusting the number njection pump: a late injection equal to 14° CA BTDC, and an early injec- CA BTDC. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics Specifically, by anticipating the SOI (−2° CA), the ME becomes comparable with that of the diesel fuel at all the rotational he lower LHVm of biodiesel, which entails a higher global conversion ef- oth a standard SOI and a retarding of the injection (+2° CA), the BSFC l h h l f b d l d d l h h p y pp g g As concerns the specific fuel consumption (Figure 21), a higher influence of the SOI and of the fueling can be appreciated. Specifically, by anticipating the SOI (−2◦CA), the BSFC with the RME becomes comparable with that of the diesel fuel at all the rotational speeds despite the lower LHVm of biodiesel, which entails a higher global conversion efficiency. With both a standard SOI and a retarding of the injection (+2◦CA), the BSFC increases consistently with the lower LHVm of biodiesel, and a delay in the injection shows a slightly higher performance at high rotational speeds than at lower rpms. ently with the lower LHVm of biodiesel, and a delay in the injection shows performance at high rotational speeds than at lower rpms. As concerns the emissions, the use of RME consistently reduced the concentration of CO in the exhaust at all the operative conditions, as shown in Figure 22. The highest abate- ment is obtained by anticipating the SOI by two degrees. This outcome is a consequence of Energies 2021, 14, 7535 OR PEER REVIEW 18 of 25 of 26 the higher combustion temperature and pressure, which facilitate the shift of the oxidation reactions toward a complete conversion to CO2. However, the higher oxygen content and the higher HRR with the RME grants lower CO emissions for all the injection timings. RME +2CA the higher combustion temperature and pressure, which facilitate the shift of the oxidati reactions toward a complete conversion to CO2. However, the higher oxygen content a the higher HRR with the RME grants lower CO emissions for all the injection timings. 20 Torque as a function of the rotational speed at full load for different values of the SOI and for the tw 15 17.5 20 22.5 25 27.5 30 1750 2000 2250 2500 2750 3000 3250 torque (Nm) rotational speed (rpm) RME +2CA Diesel fuel RME RME -2CA Figure 20. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics Torque as a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. e as a function of the rotational speed at full load, for different values of the SOI and for the two tested 15 17.5 20 22.5 25 27.5 30 1750 2000 2250 2500 2750 3000 3250 torque (Nm) rotational speed (rpm) RME +2CA Diesel fuel RME RME -2CA rotational speed (rpm) ent values of the SOI and fo 20 Torque as a function of the rotational speed at full load for different values of the SOI and for the tw Figure 20. Torque as a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. nction of the rotational speed at full load for different values of the SOI and for the tw a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. 20. Torque as a function of the rotational speed at full load, for different values of the SOI and for the tw 250 300 350 400 450 500 1750 2000 2250 2500 2750 3000 3 brake specific fuel consumption (g/kWh) rotational speed (rpm) RME +2CA Diesel fuel RME RME -2CA Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for dif- ferent values of the SOI and for the two tested fuels. 250 300 350 400 450 500 1750 2000 2250 2500 2750 3000 3250 brake specific fuel consumption (g/kWh) rotational speed (rpm) RME +2CA Diesel fuel RME RME -2CA Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. 20. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics diesel emissions with 16° CA of injection advance). As is visible in Figure 22, whatever the injection advance, the use of biodiesel allows reduction of the CO emissions. A further decrease can be obtained when shifting to RME by reducing the injection advance. The higher percentage decreases can be ob- ed in correspondence with the low rotational speeds. Moving toward the higher rpms, i i ff d h il i i b 2750 As is visible in Figure 22, whatever the injection advance, the use of biodiesel allows for a reduction of the CO emissions. A further decrease can be obtained when shifting to the RME by reducing the injection advance. The higher percentage decreases can be observed in correspondence with the low rotational speeds. Moving toward the higher rpms, the positive effects decrease somewhat until an inversion occurs at about 2750 rpm. As is visible in Figure 22, whatever the injection advance, the use of biodiesel allows for a reduction of the CO emissions. A further decrease can be obtained when shifting to the RME by reducing the injection advance. The higher percentage decreases can be ob- served in correspondence with the low rotational speeds. Moving toward the higher rpms, the positive effects decrease somewhat until an inversion occurs at about 2750 rpm. positive effects decrease somewhat until an inversion occurs at about 2750 rpm. Figure 23 shows the comparison of the concentrations of NOx between the diesel and RME, where the NOx effect can be clearly identified; however, the negative NOx effect ms to disappear, shifting the SOI to +2° CA (i.e., in correspondence with an angle caus- h d l i h h f h di l f l) p p p Figure 23 shows the comparison of the concentrations of NOx between the diesel and the RME, where the NOx effect can be clearly identified; however, the negative NOx effect seems to disappear, shifting the SOI to +2◦CA (i.e., in correspondence with an angle causing the trend to overlap with that of the diesel fuel). Figure 23 shows the comparison of the concentrations of NOx between the diesel and the RME, where the NOx effect can be clearly identified; however, the negative NOx effect seems to disappear, shifting the SOI to +2° CA (i.e., in correspondence with an angle caus- ing the trend to overlap with that of the diesel fuel). ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics Torque as a function of the rotational speed at full load, for different values of the SOI and for the tw 250 300 350 400 450 500 1750 2000 2250 2500 2750 3000 brake specific fuel consumption (g/kWh) RME +2CA Diesel fuel RME RME -2CA Figure 21 Brake specific fuel consumption as a function of the rotational speed at full load for dif- 250 300 350 400 450 500 1750 2000 2250 2500 2750 3000 3250 brake specific fuel consumption (g/kWh) rotational speed (rpm) RME +2CA Diesel fuel RME RME -2CA Figure 21 Brake specific fuel consumption as a function of the rotational speed at full load for different values of the SOI rotational speed (rpm) 1750 2000 2250 2500 2750 3000 rotational speed (rpm) Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for dif ferent values of the SOI and for the two tested fuels. Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. 19 of 25 19 of 25 Energies 2021, 14, 7535 re 22. CO emissions variations for the biodiesel at different values of the SOI and at full load diesel emissions with 16° CA of injection advance). 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 1750 2000 2250 2500 2750 3000 3250 CO emissions (%) rotational speed [rpm] RME +2CA Diesel RME RME -2CA Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16◦CA of injection advance). Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16° CA of injection advance). 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 1750 2000 2250 2500 2750 3000 3250 CO emissions (%) rotational speed [rpm] RME +2CA Diesel RME RME -2CA e 22. CO emissions variations for the biodiesel at different values of the SOI and at full load diesel emissions with 16° CA of injection advance). Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16◦CA of injection advance). Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load (ref. 3.4. Comments on the Results and Comparison with Literature Studies 3.4. Comments on the Results and Comparison with Literature Studies In this section, the presented performance results are commented on and compared to other similar studies available in the literature. The analysis focuses on both the combustion and the emissions. When the RME is used to fuel the studied engine, and having an in-line fuel pumping system: • the brake torque at full load is minimally affected, and the variation of the torque with the diesel oil and the RME fueling is within the accuracy of the measuring instrumentation; • the NOx emissions increase between 9% (at higher rotational speeds) and 40% (at lower rotational speeds); • the SOI is slightly advanced (of a maximum of about 0.75◦CA) and the SOC takes place earlier, accordingly; • when the fuel delivery ends, the pressure in the injection system drops rapidly, causing severe cavitation in the fuel, and the cavitation period lasts more with the RME as, due to its higher bulk modulus, the pressure drop is the steeper; • the pressure in the fuel line just before injection (i.e., the residual pressure) is lower with the RME, probably due to the combined effects of a more intense cavitation at the end of the injection and the higher fluid viscosity; • the maximum fuel pressure during the injection is only slightly higher with the RME if a correct pressure reference is taken into account, and thus the effect of the residual pressure decrease with RME is considered; • the net heat release during the premixed phase is steeper, and a higher peak of heat release is reached; • with the same pump settings, the mass of injected fuel is higher with the RME than with the mineral diesel fuel due to the increased injection duration and the slightly higher injection pressure; • the control of the fuel injection timing of the line pump can help to counterbalance the NOx effect, and with the RME, a retarding of 2◦CA on the standard timing can offset the increase of the NOx emissions so that the other performance parameters remain comparable to a standard fueling. The results show that, apart from a slight advance in the SOC caused by the advance in the SOI, the RME differs from diesel fuel for the higher HRR during the first part of the combustion. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics the trend to overlap with that of the diesel fuel). ure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load diesel emissions with 16° CA of injection advance). 0 20 40 60 80 100 120 140 1750 2000 2250 2500 2750 3000 3250 rotational speed (rpm) RME +2CA Diesel RME RME -2CA Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16° CA of injection advance). 0 20 40 60 80 100 120 140 1750 2000 2250 2500 2750 3000 3250 NOx emissions (ppm) rotational speed (rpm) RME +2CA Diesel RME RME -2CA Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16◦CA of injection advance). e 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load diesel emissions with 16° CA of injection advance). rotational speed (rpm) Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16° CA of injection advance). Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16◦CA of injection advance). Energies 2021, 14, 7535 20 of 25 20 of 25 What seems a little surprising is the reduction of NOx in the 2500–2750 rpm range when the injection of RME is anticipated. This is another example of the not-so-straightforward behavior of biodiesel as concerns NOx emissions. The factors influencing this behavior are, in effect, not independent but comprehensive and interactive, and include, besides the biodiesel properties, engine loads and also, as in this case, engine speeds to influence the final determinant for the NOx emission of biodiesel combustion temperature [31]. Unfortunately, in this case, we are not able to correlate this behavior to the heat release, as no in-cylinder pressure recordings were available for these tests. Nevertheless, we can conclude that retarding the SOI by two degrees of CA with respect to the factory settings seems to be the best compromise for the performance and emissions of the engine fueled with the RME. ut the injection lasts almost 3 ts contribute to explain the in 3.3. Combustion Characteristics Indeed, (i) the NOx effect is eliminated, (ii) the CO emissions do not increase compared to diesel combustion, (iii) the torque improves, and the global efficiency is acceptable. 3.4. Comments on the Results and Comparison with Literature Studies This causes a higher peak of heat release, a higher rise in pressure, and in-cylinder peak pressure, and thus a higher combustion temperature. Considering that the atomization of biodiesel is poorer than that of diesel fuel, this behavior is likely to be due to the oxygen bound in the fuel molecules. This is consistent with the literature results, in particular with Labeckas G. and Slavinskas S. [39] who, having tested RME on a four-cylinder, water-cooled, direct injection, naturally aspirated diesel engine, stated that “in contrast to air-borne oxygen, the fuel-based oxygen accelerates the combustion Energies 2021, 14, 7535 21 of 25 21 of 25 process from within the fuel-rich spray patterns themselves”, and added: “this advantage is especially beneficial at heavy loads”. As far as the biodiesel NOx effect is concerned, the bound oxygen has a twofold effect: boosting the thermal production of NOx and supplying the oxygen other than that which is air borne. This last mechanism of NOx formation is also consistent with the NOx emissions increase observable in common rail injection systems. Data are in agreement with researchers that have tested RME on similar engines, but different behaviors can be found in the literature when biodiesels from different feedstock are used in similar engines or when the RME is used in different engine types. In fact, in the premixed combustion phase, an increased HRR with respect to diesel fuel was also found for RME by Tsolakis A. et al. [54]; similarly, Purushothaman K. and Nagarajan G. [55] also evidenced higher HRRs for the orange oil, and Pradeep V. and Sharma R.P. [56] for the Jatropha oil-based biodiesel. On the contrary, a lower NHRR during the premixed combustion period was found by Sahoo and Das [57] for Jatropha-, Karanja-, and Polanga-based biodiesels, while Sinha and Agarwal found it for a rice bran oil-based biodiesel [58] and by Nwafor and Rice [59] found it for straight rapeseed oil. Qi et al. [60] tested a diesel engine with a neat biodiesel produced from straight soybean oil and found that, at lower engine loads, the net heat release for diesel was slightly lower than for biodiesel, but at higher engine loads, the HRR for diesel fuel was higher. Apart from the biodiesel typology and origin, the engine type may also influence the fuel injection and combustion characteristics of biodiesel. 3.4. Comments on the Results and Comparison with Literature Studies For example, Breda [61] tested RME on a bus diesel engine with an M injection system and found a lower maximum HRR and in-cylinder gas pressure when compared to diesel fuel. y g p p Table 4 summarizes the main results available in the literature, reported here for proper comparisons with the results obtained in this study. All the data refer to engines adopting a PNL injection system at their own rated full load, unless differently indicated. Table 4. Main characteristics, emissions, and performance of biodiesel-fueled engines in the prese haracteristics, emissions, and performance of biodiesel-fueled engines in the presented literature. Table 4. Main characteristics, emissions, and performance of biodiesel-fueled engines in the presented literature. Author and Reference Nr. Type of Biodiesel NOx Concentration Peak NHRR Fuel Injection System BSFC Tsolakis [54] B100—rapeseed oil +50% +20% PLN unmodified +15.0% Purushothaman [55] B100—orange oil +12.5% +33% PLN unmodified +10.5% Pradeep [56] B100—Jatropha +8% +2% PLN optimized for B100 +12.3% Sahoo [57] B100—Jatropha N/A −23% PLN unmodified N/A B100—Karanja N/A −22% N/A B100—Polanga N/A −25% N/A Nwafor [59] B100—neat rapeseed oil ~ +5% PLN unmodified— indirect injection +7% Qi [60] B100—soybean oil −1% (full load) ~ +2% (min load) ~ −5% (max load) PLN unmodified +10% (full load) Breda [61] B100—rapeseed oil +12.5% −13.3% PLN unmodified—M system +16% 4. Conclusions In this study, a comparative analysis on the use of RME and diesel fuel mixtures on a single-cylinder compression ignition diesel engine has been carried out. The engine presents an in-line mechanical fuel pumping system. This typology of fuel injection technology is still widely adopted in small scale engines and in agricultural machine engines, and, thanks to its intrinsic simplicity, is also useful to better evidence the effects of biodiesel use. In particular, the study focuses on the effect of an alternative fueling on the injection timing, the in-chamber pressure, the heat release rate, and the NOx emissions. The results of the present article, and the ones available in the literature, regarding the fuel pressure trace in the injection system allow for the conclusion that there is no general Energies 2021, 14, 7535 22 of 25 22 of 25 agreement on the effects of biodiesel on the injection pressure even in the same types of injection systems. This may be due to a noncorrect pressure referencing of the injection pressure traces. In the PLN injection systems, the pressure increase with biodiesel is indeed steeper than with diesel fuel, but the biodiesel pressure just before the injection (i.e., the residual pressure) is lower and, thus, its maximum injection pressure is only slightly greater to the level recorded with the diesel fuel. Unfortunately, even after carefully consulting the literature, it is not always possible to verify which kind of injection pump was used because most of the authors did not record such details. The fuel injection also affects the combustion heat release and the emissions. No significant variations in the engine torque are found with the variation of the fueling. The injection advance with the RME also anticipates the start of the combustion, and a higher heat release and peak in-cylinder pressure are found on the whole rotational speed range of the engine. As concerns the emissions, the higher nitrogen oxides are often found when adopting biodiesels. In the literature, there are several interpretations of the biodiesel NOx effect, and the causes of the increase of this pollutant are mainly attributed to the higher content of oxygen in the fuel, the higher combustion pressure and temperature (due to an advance in the SOC), and the higher heat release rate. 4. Conclusions This study also proposes a modification in the fuel injection advance so to counterbalance the NOx effect without affecting the other performance figures of the engine. g p g g Nevertheless, though research on the characterization of biodiesel performance in diesel engines started a long time ago, some issues have not yet been successfully addressed, and further research is still needed to better focus on them. In this respect, this study has tried to supply some considerations and remarks for a specific category of biodiesel-fueled engines with a mechanical injection system that can, however, be extended also to modern engines adopting common rail systems. Author Contributions: The authors contributed equally to each part of this work. All authors have read and agreed to the published version of the manuscript. Funding: This research was developed within the “Bio-Tract-Efficiency” (“Experimental investigation on the efficiency of agricultural machines powered with different fuels”) internal research project, grant number TN200F, and the “XENERGY” (“Collaboration project with UNIVPM in the field of fluid machines and energy systems”) research frame agreement, involving the Libera Università di Bolzano and the Università Politecnica delle Marche. Institutional Review Board Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Giorgio Tombari for his help during the experimental phase of this study. Acknowledgments: The authors would like to thank Giorgio Tombari for his help during the experimental phase of this study. Acknowledgments: The authors would like to thank Giorgio Tombari for his help during the experimental phase of this study. Nomenclature B100 pure biodiesel BMEP brake mean effective pressures BSFC brake-specific fuel consumption BTDC before top dead center BTE brake thermal efficiency CA crank angle (degrees) CI compression-ignited (engine) CO carbon monoxide CO2 carbon dioxide EASAC European Academies’ Science Advisory Council 23 of 25 Energies 2021, 14, 7535 ECU electronic control unit EOI end of injection EU European Union GHG greenhouse gases HC hydrocarbon HRR heat release rate LHVm lower heating value on a mass basis ME methyl esters MSW municipal solid waste NHRR net heat release rate NOx nitrogen oxides NREL National Renewable Energy Laboratory NY State of New York OHV on-head valve PLN pump-line-nozzle PM particulate matter RME rapeseed oil methyl ester SOC start of combustion SOI start of ignition USA United States of America References [CrossRef] Wang, M.; Nie, K.; Yun, F.; Cao, H.; Deng, L.; Wang, F.; Tan, T. 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Development of a methodology for evaluating the efficiency level of monitoring agroecosystems using big data technologies
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VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 Development of a methodology for evaluating the efficiency level of monitoring agroecosystems using big data technologies Aleksey Petrov1,*, Leonid Andreev2 and Sergey Grigorishin3 1Norilsk State Industrial Institute, 663310, 50 years of October str. 7, Norilsk, Russian Federation 2Northern Trans-Ural State Agricultural Universtity, 625003, 7, Republic st. Tyumen, Russian Federation 3Tyumen State University, 625003, 6, Volodarskogo st., Tyumen, Russian Federation 3Tyumen State University, 625003, 6, Volodarskogo st., Tyumen, Russian Federation Abstract. The article discusses the theory of monitoring agroecosystems for the effectiveness of using Big Data technologies. The relationship between the agricultural areas of the Tyumen region, the Big Data sources available in them, and the technologies for working with Big Data obtained from sources are described. The article also developed a methodology that makes it possible to assess the level of effectiveness of monitoring agroecosystems using Big Data technologies, based on the result of which a strategy for development of the region as a whole and its agroecosystems, in particular, is formed in terms of equipment with information technologies. The methodology presented in the article is formed on the basis of an engineering ontology, which in the future is able to lower the degree of the human factor in global and local monitoring of agroecosystems for the effectiveness of using Big Data technologies. 1 Introduction The monitoring and assessment of natural ecosystems primarily implies development and operation of such observation systems, on the basis of which it is possible to assess their condition. The assessment, in turn, is primarily the result of an analysis of the quantitative characteristics of ecosystems, which are dynamic, since they have the property of variability, relative to the impact of external factors (the name of the factors depends on the type of ecosystems). Agricultural ecosystems are commonly referred to as agroecosystems. Such monitoring of agroecosystems [1], on the one hand, is of great economic importance for the region [2], since, based on its result, the agroecosystem manager can develop a strategy and take measures (crop conservation, pest control, and etc.), on the other hand, it is of great ecological importance, as agroecosystems are part of larger natural ecosystems. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: darker2012@yandex.ru 2 Methods In the existing realities, monitoring of agroecosystems is not possible without the use of technologies for processing large amounts of data [3], since the agroecosystem is, first of all, dynamic, which means that the manager of the agroecosystem is interested in obtaining information about its state as quickly as possible. This, in turn, means that it is possible to develop a methodology for assessing the readiness of an agroecosystem to analyze the data obtained about its state using Big Data technologies, or in other words, a methodology for assessing the level of effectiveness of monitoring agroecosystems using Big Data technologies. This implies that if ten sensors of soil acidity are located on some hypothetical agricultural field (agroecosystem), then using Big Data technologies will be ineffective, since the data obtained can be processed in simple Microsoft Excel and there is no point in creating software for analyzing such data based on Python or another programming language. This means that there is a certain minimum required amount of data that must come from the agricultural field (agroeosystem) for use of Big Data technologies to be expedient. Moreover, even if the required amount of data is available, it is necessary to determine which of the many currently developed Big Data technologies for agriculture [4] are suitable for analysis. y In the article [5], the authors presented a table responsible for the distribution of technologies for analyzing big data, depending on the field of agriculture. For clarity, we present Table 1. Table 1. Distribution of Big Data technologies, depending on the field of agriculture. Table 1. Distribution of Big Data technologies, depending on the field of agriculture. Agriculture area Sources of Big Data Big Data technologies Weather and climate data Geospatial data; Meteorological stations; Freely available historical information Other data obtained remotely. Statistical analysis; Machine notation (K-means clustering algorithm, random/deep tree construction algorithm); GIS analysis; Distributed computing model. Livestock breeding Ground sensors; Thermal data; Optical sensors; Incoming feed sensors; Data on meat and dairy products. Neural networks; Scalable vector machines; Decision trees. Crops Freely available historical information Satellite data; Ground sensors. K-means clustering algorithm; Support Vector Machine; Fourier transform; Wavelet analysis; Land resources; Geospatial data; Freely available historical information Other data obtained remotely; Aerial survey data; K-means clustering algorithm; algorithm for constructing random/deep trees; Image processing; Vegetation index ndvi. * Corresponding author: darker2012@yandex.ru © 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/). VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 2 Methods Weeds Freely available historical information Data received from drones; Aerial survey data; Sensor placed in the fields; Digital Web Libraries. Neural networks; Logistic regression; Image processing; Soil condition; Freely available historical K-means clustering algorithm; 2 2 VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 information Ground sensors; Information posted in the database of government agencies; Humidity sensors; Optical sensors; Neural networks. Biological resistance Geospatial data; Freely available historical information Information posted in the database of government agencies; Statistical modeling; Bayesian functions; Food safety; Geospatial data; Freely available historical information Other data obtained remotely; Survey data; Growth depth sensors; Neural networks; Geospatial modeling; Statistical modeling; Image processing; Farms; Freely available historical information Optical sensors; Information posted in the database of government agencies; Meteorological stations; Social networks; Big Data Benchmarking; Web services; Mobile applications; Remote sensing; Geospatial data; Satellite data; Geospatial data; Meteorological stations; Data received from drones; Digital Web Libraries. Cloud computing with a distributed computing model; Geospatial modeling; Computer vision; Artificial intelligence; Insurance and finance; Optical sensors; Information posted in the database of government agencies; Digital web libraries; Information from private banks; Survey data; Statistical modeling; Predictive analytics; Cloud technologies. As you can see, for each area of agriculture, a source of information was selected for analysis using Big data technology and directly, the methods of analysis themselves. Of course, the areas of agriculture in Ireland, in any case, their categorization is different from the categorization of similar areas of the Tyumen region. However, the "general" idea can still be traced and "transferred" it to the realities of the Tyumen region. It would be advisable to check the adequacy of the selected methods of analysis based on the applied problem. 3 Results Currently, in the Tyumen region, there is an acute issue of Big Data study in agriculture. Therefore, using the column "Sources of Big Data", one can assess the level of preparedness of a particular area of agriculture in the region for such studies. Also, based on the direct availability of "Sources of Big Data", it is possible to build an ontological graph of interaction with Big Data technologies, which in turn will lead to a methodology for assessing agricultural areas on the effectiveness of using Big Data technologies and 3 3 VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 ommendations in which particular area of agriculture which sources to develop. For this, present Table 2. recommendations in which particular area of agriculture which sources to develop. For this, we present Table 2. present Table 2. Table 2. Availability of Big Data sources regarding agriculture in the Tyumen region. 3 Results Agriculture area Sources of Big Data Source availability Weather and climate data Geospatial data Absent Meteorological stations Present Freely available historical information Present Other data obtained remotely Absent Livestock breeding Ground sensors Present Thermal data Absent Optical sensors Absent Incoming feed sensors Present Data on meat and dairy products Present Crops Freely available historical information Present Satellite data Present Ground sensors Present Land resources Geospatial data Absent Freely available historical information Present Other data obtained remotely Present Aerial survey data Absent Weeds Freely available historical information Absent Data received from drones Absent Aerial survey data; Absent Sensor placed in the fields Absent Digital Web Libraries Absent Soil condition Freely available historical information; Present Ground sensors; Present Information posted in the database of government agencies; Present Humidity sensors; Present Optical sensors; Absent Biological resistance Geospatial data; Present Freely available historical information Absent Information posted in the database of government agencies; Present Food safety; Geospatial data; Absent Freely available historical information Absent Other data obtained remotely Present Survey data Absent Growth depth sensors Absent Farms Freely available historical information Present Optical sensors Absent Information posted in the database of government agencies Present Meteorological stations Present Social networks Present Remote sensing Geospatial data Absent Satellite data Absent Geospatial data Absent 4 VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 Meteorological stations Absent Data received from drones Absent Digital Web Libraries Absent Insurance and finance Optical sensors Absent Information posted in the database of government agencies; Present Digital web libraries Absent Information from private banks Absent Survey data Absent By adding the column "presence of a source", you can quantitatively visualize the filling of areas and display in Table 3. the Tyumen region. Agriculture area Source availability Weather and climate data 2/4 Livestock breeding 2/5 Crops 3/3 Land resources 2/4 Weeds 0/5 Soil condition 4/5 Biological resistance 2/3 Food safety 1/5 Farms 4/5 Remote sensing 0/5 Insurance and finance 1/5 So it can be seen that the most prepared area of agriculture is Crops, Soil Condition, and Farms. Based on them, it will be possible to make initial ontological graphs for creating a methodology based on engineering ontology. 4 Discussion nsider the process of creating an ontological graph based on the "Crops" area. Consider the process of creating an ontological graph based on the "Crops" area. Consider the process of creating an ontological graph based on the Crops area. Table 4. Analysis of the "Crops" area. Crops Freely available historical information K-means clustering algorithm Support Vector Machine Fourier transform Wavelet analysis Satellite data Ground sensors To begin with, you need to understand to which subdomain "Crops" which Big Data technology to implement most effectively. To begin with, you need to understand to which subdomain "Crops" which Big Data technology to implement most effectively. The K-means clustering algorithm technology consists in dividing a certain volume of observations (points) into an arbitrary number of clusters, limited by a certain standard [6]. The key feature is the presence of a centroid for each cluster and the separation of points in 5 VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 accordance with similarity. In other words, distribution into clusters occurs, depending on the location of the central point (centroid), which is obtained by averaging the positions of all points, or vice versa. After distribution into clusters, through the calculation of the distance, two or more samples are compared with each other. accordance with similarity. In other words, distribution into clusters occurs, depending on the location of the central point (centroid), which is obtained by averaging the positions of all points, or vice versa. After distribution into clusters, through the calculation of the distance, two or more samples are compared with each other. The use of this technology is correct if there are a number of points associated with a geographic location. That is, from the available "Sources of Big Data" only ground sensors in tandem with satellite or other methods of obtaining spatial information about ground sensors can be used. A source such as “Freely available historical information” is too broad concept; therefore, its rating of use for the analysis of “Crops” by Big Data technologies will be low. Support vector machine technology [7] consists in learning by precedents that take into account the cases of linear separability, optima and gaps between classes. It is noteworthy that analytics using this technology most often occurs in tasks related to linear regression, which in parallel includes the search for minimization. 4 Discussion The correct application of this technology is possible if there is a time axis, in other words, to trace the change in information over time. Among the offered "Sources of Big Data", this can be "Freely available historical information", but on condition that it can be expressed in quantitative metrics. The technology of the Fourier transform [8] consists in the comparison of one function into a complex variable, which describes the coefficients in the expansion of the original function into elementary components. In the area we are considering, such a transformation is used to process signals and represent them in the form of time series, with the possibility of displaying them in the form of a frequency spectrum. Correct application is possible in presence of constantly incoming signals, in other words, in the presence of ground sensors. Wavelet analysis technology [9] consists in the analysis of different types of frequency components of the data, most often along the "scale - time - level" chain. Most often they are used to obtain more accurate data after the Fourier transform, or to refine this transformation, since they give a more accurate binding of the quantitative features of the parameter, relative to time. Therefore, we can conclude that the wavelet analysis is only an auxiliary analysis and is used in special cases together with the Fourier transform. Based on the above, an ontological graph of the "Crops" area can be built. For construction, we use the Protégé environment [10], the resulting graph is shown in Figure 1. Fig. 1.. Ontological graph of the "Crops" area. As you can see in the presented column, "Big Data Sources" were represented in the form of "Entities" of the Protégé environment, and "Big Data technologies" in the form of "Individuals", such a solution gives us, first of all, the opportunity to trace "paired" sources for big data analysis. From an applied perspective, this means the presence of the same analysis technologies, the same software for data from different sources, but in the same Fig. 1.. Ontological graph of the "Crops" area. Fig. 1.. Ontological graph of the "Crops" area. Fig. 1.. Ontological graph of the "Crops" area. 4 Discussion As you can see in the presented column, "Big Data Sources" were represented in the form of "Entities" of the Protégé environment, and "Big Data technologies" in the form of "Individuals", such a solution gives us, first of all, the opportunity to trace "paired" sources for big data analysis. From an applied perspective, this means the presence of the same analysis technologies, the same software for data from different sources, but in the same As you can see in the presented column, "Big Data Sources" were represented in the form of "Entities" of the Protégé environment, and "Big Data technologies" in the form of "Individuals", such a solution gives us, first of all, the opportunity to trace "paired" sources for big data analysis. From an applied perspective, this means the presence of the same analysis technologies, the same software for data from different sources, but in the same 6 VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 agroecosystem. Thus, it will be easier for big data analysts to navigate when finding dependencies, obtaining additional information, making forecasts, and more. agroecosystem. Thus, it will be easier for big data analysts to navigate when finding dependencies, obtaining additional information, making forecasts, and more. In addition, in the ontological graph, you can see that such a source as "Freely available historical information" is analyzed by only one technique and does not intersect with other "Sources of Big Data". This, in turn, suggests that with development of Big Data in the region, there are two ways to go: 1. Stop this kind of source, without the need for its further development and analysis of big data. g 2. Present data in an accessible form for the analysis of other Big Data technologies. As you can understand, such ontological graphs can be built for any area of agriculture and matched for any agroecosystem. For clarity, let's expand the existing graph to include areas such as Soil Condition, Farms, and Weather and Climate Data, thereby creating a hypothetical agro-ecosystem that includes the listed areas. That is, the regions are chosen in such a way that, in fact, they are responsible for the analysis of the meta-region and cover the interests in Big Data of farms engaged in crop production. Thus, we have obtained a search ontological graph. 4 Discussion It is difficult to visualize it in full, but Protégé has search functions, by searching for the keyword "data" we see what is shown in Figure 2. Fig. 2. The result of displaying a search ontological graph by the keyword "data". Fig. 2. The result of displaying a search ontological graph by the keyword "data". On this search ontological graph, we see that all "Entities" related to the column "Sources of Big Data" and one way or another associated with data, as well as the "Individuals" and areas of agriculture attached to them, were displayed. In view of this, a kind of "coupling" is obtained, where, on the one hand, we can observe the fullness of Big Data technologies, and on the other hand, the field of agriculture. Such a visualized dependence and work with the resulting ontology can give a specialist the following information: 1. Understanding of the readiness of the agricultural sector for Big Data technologies. derstanding of the readiness of the agricultural sector for Big Data technologies. g g g g 2. Understanding of the fullness of the agricultural field with Big Data sources. 3. What sources of Big Data in what area of agriculture needs to be brought in to rease the efficiency of using Big Data technologies. 4. What Big Data technologies to use in what sources. 5. What "paired" Big Data sources can be used to capture the largest number of areas of agriculture for analysis. 7 VVRD 2021 BIO Web of Conferences 43, 0 (2022) 3012 https://doi.org/10.1051/bioconf/20224303012 6. What Big Data technologies can be used, depending on the type of agricultural enterprise. As a result, it turns out that the very methodology for assessing the level of effectiveness of monitoring agroecosystems using Big Data technologies is, first of all, in the correctness of work with the resulting ontology. In order to translate the methodology directly into a quantitative metric, that is, to be able to "put down" each agroecosystem its own "score" from one to ten points, it is necessary to use such Protege functionality as "Data properties" and "Object properties". "Data properties" in Protégé are literal properties and can be attached to "Entities", so it becomes possible to represent "Entities" as a list of property names and associated quantitative metrics. 5 Conclusion As a summary of this article, a number of the following conclusions can be made:  The method of monitoring agroecosystems to assess the level of efficiency of using Big Data technologies allows visualizing the lagging and leading areas to form an effective development strategy.  The agroecosystem manager shall guide not only the selection of Big Data technologies for analyzing information about the agroecosystem, but also focus on the available data sources about it.  The methodology can be improved through use of engineering ontologies and Protégé software, which can internally evaluate technologies for agro-ecosystems and search for hidden knowledge, excluding the human factor. To improve the methodology in the future, it is necessary to compare different agroecosystems for the effectiveness of using Big Data technologies and analyze the results. 4 Discussion As a result, each "Entities" can be represented by a strictly defined numerical value, which can be changed only if additional information is added to the ontology. "Object properties" in Protégé are a property pointer to "Entities". The pointer, in turn, has two key properties, namely the address allocated for a specific "Entities" and the connection with a specific type of "Entities". Having filled in "Data properties" and "Object properties" for each "Entities", we will be able to quantify each area of agriculture, in other words, present them in the form of a line histogram, at Figure 3. Fig. 3. Linear histogram for assessing the level of effectiveness of monitoring agroecosystems using Big Data technologies. Fig. 3. Linear histogram for assessing the level of effectiveness of monitoring agroecosystems using Big Data technologies. From this diagram, we can immediately observe the lagging areas of agriculture, namely the areas "Remote sensing" and "Weeds". This is due to the fact that such regions are not considered separately in the Tyumen region. However, it is still logical that in order to increase the efficiency of using Big Data technologies, a quantitative increase is necessary, first of all, “Sources of Big Data”. By itself, such a distribution was seen already at the stage of adding the column "presence of a source" and was displayed in Table 3. However, it is the addition of quantitative metrics for “Data properties” and “Object properties” to the ontology that will allow us to adjust the source metrics for the agroecosystem and see a more accurate picture of “the effectiveness of using Big Data technologies” at a more detailed level for a specialist. 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